Thesis
Tracing back the early evolution of ParaHox genes and the ancestral neurogenic function of Gsx/Anthox2 in the developing sea anemone, Nematostella vectensis
QUIQUAND, Manon
Abstract
The Hox/ParaHox genes are chromosomally clustered genes encoding transcription factors that regulate neurogenesis in developing bilaterians. They emerged in early eumetazoan evolution, being already expressed in cnidarians, a prebilaterian phylum where the early steps of neurogenesis can be traced back. We first performed a systematic phylogenetic reconstruction of the Hox/ParaHox families and proposed that the ProtoHox gene most likely resembled the Gsx/Pdx genes. We then made use of the sea anemone (Nematostella) cnidarian model system to investigate the developmental role of Gsx. Thanks to neuronal markers that we identified to monitor neurogenesis in developing Nematostella, we could show in loss of function assays that Gsx/Anthox2 is likely involved in nerve net formation. Moreover Gsx/Anthox2 upstream sequences drive expression in apical neurons, suggesting that they contain a neurogenic element to be identified. Our result provide advances about the genetic circuitry involved in the differentiation of neurons in early animal evolution.
Reference
QUIQUAND, Manon. Tracing back the early evolution of ParaHox genes and the ancestral neurogenic function of Gsx/Anthox2 in the developing sea anemone, Nematostella vectensis. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4101
URN : urn:nbn:ch:unige-46644 DOI : 10.13097/archive-ouverte/unige:4664
Available at: http://archive-ouverte.unige.ch/unige:4664
Disclaimer: layout of this document may differ from the published version.
1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES
Département de Zoologie et Biologie Animale Dr. Brigitte GALLIOT
Tracing back the early evolution of ParaHox genes and the ancestral neurogenic function of Gsx/Anthox2 in the developing sea anemone, Nematostella vectensis
THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par Manon QUIQUAND de Nemours (France)
Thèse N°4101 Genève Atelier d’impression ReproMail 2009
REMERCIEMENTS
Ce travail a été réalisé au département de Zoologie et Biologie Animale de l’Université de Genève et financé par le Fonds National Suisse de la Recherche Scientifique et le Département de l’Instruction Publique de la République et du Canton de Genève. Introduction À l’Université Pierre et Marie Curie, entre Paris, Villefranche-sur-Mer et Montpellier, je remercie ceux qui m’ont transmis leur passion, m’ont donné l’envie de réaliser une thèse, et surtout ceux qui m’y ont aidé. Méthodes Je tiens à présenter toute ma reconnaissance à Brigitte Galliot, ma directrice de thèse, qui m’a encadrée pendant plus de cinq années et m’a donné l’opportunité de réaliser ce travail. Merci Brigitte pour votre disponibilité, vos conseils et votre soutien qui furent au rendez-vous. Je remercie les membres actuels et passés du laboratoire, ceux qui sont devenus plus que des collègues. Aussi merci aux membres des autres laboratoires du département qui se comportent souvent comme si je faisais partie du leur. Plus particulièrement merci aux Dr. Juan Montoya et Dr. Cedric Berney pour leurs conseils en phylogénie. Résultats Je remercie les membres de mon comité de thèse pour leur temps et l’intérêt qu’ils ont porté à mon manuscrit: Dr. Evelyn Houliston, Dr. Jean-Stéphane Joly et Prof. Ivan Rodriguez. Discussion Je remercie mes ami(e)s pour les moments partagés, ceux avec qui j’ai travaillé pendant cette phase de rédaction, ceux qui m’ont aidée, ceux qui ont fini par tolérer qu’une thèse à l’Université de Genève dure souvent plus de trois ans. Enfin je remercie ma famille, particulièrement ma mère et mon père, à lui je voudrais dédier cette thèse. Conclusion Belle experience !! Perspective Continuons l’aventure…
ACKNOWLEDGEMENTS
This work was carried out at the Department of Zoology and Animal Biology, University of Geneva. Financal support for this work was provided by le Fonds National Suisse de la Recherche Scientifique and le Département de l’Instruction Publique de la République et du Canton de Genève. I am grateful to Brigitte Galliot for giving me the oppotunity to conduct my thesis project in her laboratory. I would like to thank her for support during these years. I would like to thank all past and present members of the laboratory for their help and the stimulating environment that they create everyday : Marijana Miljkovic-Licina, Simona Chera, Renaud de Rosa, Gaspare Benenati, Yvan Wenger, Wanda Buzgariu, Luiza Ghila, Anne Forget, Josefina Lascano, Pierre Heuze, Han Petry, Lisbeth Muster, Kevin Dobretz, Virginie Voeffray ;-), Janina Karpinsky, Fadi Hamdan, Philippe Jean. Thank you also to the far or close neighbouring of the department. Thank you to my friends and my family. Futhermore, I thank the members of my thesis comittee for their time and interest : Dr. Evelyn Houliston, Dr. Jean-Stéphane Joly and Prof. Ivan Rodriguez.
i INDEX
REMERCIEMENTS...... I ACKNOWLEDGEMENTS ...... I INDEX ...... II RESUME...... 1 SUMMARY ...... 5 INTRODUCTION ...... 7 Le phylum des cnidaires...... 8 I.1.1. Un phylum ancestral ...... 8 I.1.2. Diversité et cycle de vie chez les cnidaires...... 10 I.1.3. Développement embryonnaire des cnidaires...... 12 I.1.4. Organisation générale des cnidaires ...... 13 I.1.4.1 Axe(s) de symétrie chez les cnidaires...... 13 I.1.4.2 Organisation des tissus à partir de deux feuillets: l’ectoderme, l’endoderme, (mésoderme)...... 13 I.1.4.3. Organisation cellulaire de l’hydre ...... 14 I.1.4.4. Le système nerveux des cnidaires...... 15 Les neurones...... 15 Les cnidocytes...... 16 La transmission synaptique...... 18 I.1.5. Nematostella vectensis : un nouveau modèle...... 19 I.1.5.1. Mode et cycle de vie de Nematostella...... 20 I.1.5.2. Développement embryonnaire de Nematostella...... 21 I.1.5.3. Organisation anatomique de Nematostella...... 22 I.1.5.4. Système nerveux de Nematostella ...... 23 I.1.6. Les cnidaires comme système modèle...... 24 I.1.6.1. Les différents modèles de cnidaires utilisés...... 24 I.1.6.2. Avantages et inconvénients du modèle anthozoaire Nematostella...... 25 I.1.6.3. Outils génétiques disponibles chez les cnidaires...... 26 I.1.7. Conservation des gènes et des voies de signalisation...... 27 Les gènes à homéoboîte ...... 28 I.2.1. Les mutations homéotiques et les gènes à homéoboîte...... 28 I.2.2. La classe ANTP ...... 29 I.2.3. Rôle des gènes Hox dans la neurogénèse...... 30 I.2.3.1. Organisation des gènes Hox en complexes...... 30 I.2.3.2. La colinéarité spatiale et temporelle et la prévalence postérieure...... 32 I.2.3.3. Les gènes Hox fonctionnent comme des gènes sélecteurs...... 33 I.2.3.4. Fonction au cours de la mise en place du système nerveux...... 34 I.2.4. Rôle des gènes ParaHox...... 35 I.2.4.1. Phylogénie des gènes ParaHox...... 35 I.2.4.2. Gènes ParaHox en complexes et notion de complexe « ProtoHOX » ancestral ...... 36 I.2.4.3. Fonction des gènes ParaHox au cours du développement...... 38 I.2.5. Les gènes Hox/ParaHox chez les cnidaires...... 39 I.2.5.1. Familles Hox/ParaHox représentées chez les cnidaires et leur liaison génétique.....39 I.2.5.2. Patron d’expression des gènes Hox chez les cnidaires...... 40 I.2.6. Approche évolution-développement ...... 41
ii La neurogénèse : une innovation clé au cours de l’évolution ...... 42 I.3.1. Inversion de l’axe dorso-ventral ...... 42 I.3.2. Développement du patron neuronal chez les bilatériens...... 43 I.3.2.1. L’induction neurale : la cascade de signalisation BMP...... 44 La cascade de signalisation BMP...... 44 Patron d’expression des BMP et de leurs antagonistes...... 44 Ectoderme versus neuroectoderme ...... 45 I.3.2.2. Les gènes d’identité neurale : vnd/Nkx2.2, ind/Gsh, msh/Msx1/2 ...... 46 Patron d’expression et fonction ...... 46 Régulation par la cascade BMP ...... 46 Dominance ventrale ...... 47 I.3.2.3. Spécification spatiale et temporelle du destin nerveux...... 48 I.3.3. La neurogénèse chez les cnidaires : acteurs génétiques conservés ...... 49 Considération évolutive ...... 50 Objectifs du travail...... 51 RESULTATS ...... 55 II.1. CHAPITRE 1 : Phylogénie des gènes Hox et Parahox...... 55 II.2. CHAPITRE 2 : Analyse d’un régulateur précoce de la neurogénèse chez Nematostella vectentsis, Anthox2...... 89 II.3. CHAPITRE 3 : Evolution de la neurogénèse...... 125 DISCUSSION ...... 151 Organisation ancestrale des gènes Hox et ParaHox ...... 151 III.1.1. Une méthode originale pour reconstruire l’évolution des gènes Hox et ParaHox...... 151 III.1.2. Duplication en tandem d’un complexe ProtoHOX versus duplications multiples de gènes de type Hox...... 154 III.1.3. Les gènes ParaHox ont-ils un mode d’expression conservé au cours de l’évolution ?.. 155 Rôle des gènes ParaHox dans l’apparition de nouveaux types cellulaires...... 156 Conclusions ...... 158 Perspectives concernant les cnidaires en tant que modèles de développement et d’évolution...... 160 REFERENCES BIBLIOGRAPHIQUES...... 163 ANNEXES...... 175 Cartes des plamides / Plasmid maps...... 175 Séquences des gènes et amorces / Gene sequences and primers ...... 275
CURRICULUM VITAE……………………………………………………………………... 285
iii
iv RESUME
Les cnidaires (anémone de mer, hydre, méduse) représentent un phylum ancestral, groupe frère des bilatériens. Ils sont vraisemblablement les premiers avec les cténophores à utiliser un système nerveux différencié présentant une organisation à la fois diffuse et centralisée chez les cnidaires. Ils représentent donc un modèle de choix pour l’étude des gènes et des mécanismes de régulation liés à l’apparition de la neurogénèse.
Les processus neurogéniques chez les bilatériens impliquent différentes classes de facteurs de transcription, comme les gènes des familles bHLH et à homéoboîte, et reposent sur des cascades génétiques hautement conservées. Nombre de ces acteurs sont également retrouvés chez les cnidaires. Tandis que certains sont apparus chez l’ancêtre commun aux eumétazoaires comme par exemple les gènes à homéoboîte des familles Hox et ParaHox d’autres sont apparus plus tôt chez l’ancêtre commun aux métazoaires et sont donc retrouvés chez les éponges, qui bien que possèdant des cellules sensorielles ne présentent pas encore de neurones.
Ce travail s’appuie sur l’hypothèse selon laquelle la comparaison des familles de gènes et des cascades de régulation qui régissent la neurogénèse chez les cnidaires et les bilatériens pourraient fournir des arguments permettant de mettre en place des scénarios évolutifs. Les questions que nous avons posées au cours de ce travail sont donc les suivantes : Comment les familles de gène impliquées dans la neurogénèse se sont-elles diversifiées? Est-il possible d’identifier des acteurs neurogéniques ancestraux et comment les réseaux génétiques et les modes de régulation liés à la neurogénèse ont-ils évolué? Nous avons utilisé trois approches différentes pour apporter des éléments de réponses. Tout d’abord, au moyen d’analyses phylogénétiques, nous avons porté notre attention sur l’histoire évolutive des familles Hox et ParaHox depuis leur apparition chez les eumétazoaires. Nous avons ensuite mis en place une approche fonctionnelle en nous focalisant sur le rôle du gène ParaHox Gsx/Anthox2 au cours du développement de Nematostella, représentant un acteur potentiel ancestral de la neurogénèse. Enfin dans un travail synthétique, nous avons recensé les acteurs connus pour leur fonction dans la neurogénèse chez les bilatériens qui sont également représentés chez les cnidaires.
Pour reconstruire l’histoire évolutive des familles de gènes Hox (PG1, PG2, PG3, PG4-9) et ParaHox (Gsx, Pdx, Cdx), toutes les séquences de cnidaires connues pour êtres relatés aux familles Hox et ParaHox ont été confrontées aux familles Hox et
1 ParaHox de nombreux phyla bilatériens au travers d’analyses phylogénétiques. Notre premier résultat fut d’identifier des séquences hautement dérivées qui se sont avérées perturbatrices du message phylogénétique. Nous avons donc mis au point une méthode permettant de tester la solidité des nœuds des familles et de mettre en évidence les relations phylogénétiques qui existent entre elles. Cette approche a permis de caractériser pour la première fois des gènes de cnidaires appartenant à la famille ParaHox Pdx impliquant donc que ces organismes possèdent en réalité les trois familles ParaHox, Gsx, Pdx, Cdx. De plus, de nombreuses séquences de cnidaires forment des groupes isolés spécifiques à ce phylum, soit orphelins, soit plus probablement relatés aux familles postérieures. Par ailleurs notre méthode a révélé que les familles Hox étaient moins bien conservées que les familles ParaHox au sein des eumétazoaires. Enfin, nous montrons que les familles ParaHox Gsx et Pdx forment un groupe monophylétique avec les familles Hox PG2 et PG3 à l’exclusion de PG1. Ces résultats nous ont mené à l’hypothèse d’un complexe ProtoHOX à trois gènes contenant les paralogues Gsx/PG2, Pdx/PG3, Cdx/PG9, qui aurait pu donner après une duplication en tandem les complexes HOX et ParaHOX primitifs présents chez l’ancêtre commun aux eumétazoaires.
La seconde partie de ce travail est consacrée à l’étude ciblée du gène ParaHox Gsx/Anthox2 chez Nematostella vectensis (anémone de mer). Ce gène ParaHox, représente un acteur potentiel de la mise en place du système nerveux, partagé par les cnidaires et les bilatériens, chez qui la structure, l’expression et la fonction sont très conservées. Son expression a été détectée au cours du développement de Nematostella dans des cellules reconnues comme des précurseurs neuronaux et dans des neurones différenciés. Nous avons suivi une approche fonctionnelle consistant tout d’abord à sélectionner des marqueurs cellulaires reflétant le développement neuronal normal chez Nematostella. Grâce à l’injection de morpholinos au stade une cellule nous avons pu montrer qu’Anthox2 était un régulateur précoce de la neurogénèse. Enfin, nous avons mis au point une technique d’injection de gènes rapporteurs pour disséquer la régulation de ce gène. Ceci nous a permis d’observer l’expression d’un transgène portant 3 kb de séquences amonts du gène Anthox2, dans des cellules dont la forme et la localisation sont évocatrices des cellules neuronales. Nous avons également localisé une région portant un potentiel élément répresseur.
Dans la troisième partie, nous faisons une synthèse sur la mise en place du système nerveux chez les cnidaires au niveau cellulaire ainsi que sur les cascades de signalisation et les facteurs de transcription dont l’expression et la fonction chez les cnidaires suggèrent une implication dans la neurogénèse, comme chez les bilatériens.
2 Nous montrons que les neurones et les nématocytes, cellules qui composent le système nerveux des cnidaires, partagent un certain nombre de gènes régulateurs suggérant que ces cellules pourraient descendre de la même cellule ancestrale. La combinaison de ces résultats nous conduira à discuter de l’évolution des gènes ParaHox, des types neuronaux et des différentes étapes de la neurogénèse.
3
4 SUMMARY
Cnidarians (sea anemones, hydras, medusaes), sister group to bilaterians, are considered as an ancestral phylum. They are likely the first with Ctenophora to use a differentiated nervous system. In cnidarians this organization is both diffused and centralized. Thus, they represent a model of choice to study genes and regulation mechanisms link to neurogenesis appearance.
Bilaterians neurogenic processes involve different classes of transcription factors as bHLH and homeobox genes and rely on genetic cascades highly conserved. Many of those actors are found in cnidarians as well. While some of them appeared in the common ancestor of eumetazoans as homeobox genes belonging to Hox and ParaHox families, others appeared earlier in the common ancestor of metazoans as they are found in sponges, that do not present neurons but already sensory cells.
This work relies on the hypothesis that the comparisons of gene families and regulation cascades, which govern neurogenesis in both cnidarians and bilaterians could provide evidences allowing to propose evolutive scenarios. The following questions were addressed in this work: How gene families involved in neurogenesis were diversified ? Is it possible to identify ancestral neurogenic actors and how genetic regulatory networks and regulation mechanisms evolved? We used three different approaches to provide answer elements. First, by using phylogenetic analysis, we focused on the evolutive history of Hox and ParaHox families since their appearance in eumetazoans. Then, though a functional analysis, we focused on the role of the ParaHox Gsx/Anthox2 gene in developing Nematostella. This gene is a putative ancestral actor involved in neurogenesis. At last though a synthetic work, we made a census of actors known for their function in neurogenesis in bilateriens and that are also present in cnidarians.
To reconstruct the evolutive history of Hox (PG1, PG2, PG3, PG4-9) and ParaHox (Gsx, Pdx, Cdx) families, all cnidarian sequences related to Hox and ParaHox families were compared to Hox and ParaHox sequences from numerous bilaterian phyla through phylogenetic analyses. The first result identified highly derived sequences that blurred the phylogenetic signal. Therefore we set up a method to test robustness of node families and to highlight phylogenetic relations that exist between them. By this way, we characterized for the first time cnidarian genes belonging to the ParaHox Pdx family meaning that in fact cnidarians possess the three ParaHox gene families, Gsx, Pdx, Cdx. In addition, many cnidarian sequences appeared as isolated groups specific
5 to this phylum either orphans or the most likely related to posterior families. Moreover, our method revealed that cnidarian Hox families were less conserved than ParaHox ones. At last, we showed that Gsx and Pdx ParaHox families branched as monophyletic groups with the PG2 and PG3 Hox families excluding the PG1 one. From those results we proposed a model of a three genes ProtoHOX cluster bearing Gsx/PG2, Pdx/PG3, Cdx/PG9 paralog genes. The primitif HOX and ParaHOX cluster existing in the commun ancestor of eumatazoan arosed after tandem duplication of the ProtoHOX cluster.
The second part of this work consisted in the specific study of the Gsx/Anthox2 ParaHox gene in Nematostella (sea anemone). This ParaHox gene is a putative actor involved in the nervous system development and shared by cnidarians and bilaterians, where the structure, the expression and the function are sonserved. Its expression was detected during Nematostella development in cells recognized as neuronal precursors or differentiated neurons. We followed a functional approach consisting first in the selection of cellular markers reflecting the normal neuronal development in Nematostella. Thanks to the morpholino injections at the one cell stage we showed that Anthox2 was a precocious regulator of the neurogenesis. At last, we set up the reporter gene injections in Nematostella to dissect the regulation of this gene. That allowed us to observed the expression of a transgene carrying 3 kb of the upstream sequences of the Anthox2 gene, in cells whose the shape and the localization evoked neuronal cells. We also charaterized a region carrying a putative repressor element.
In the third part, we did a synthesis of the cellular processes taking place in cnidarian neurogenesis as well as the cascades of signalisation and transcription factors, whose expression and function in cnidarians suggest that they are implicated in neurogenesis, as in bilaterians. We showed that neurons and nematocytes, cells that composed the nervous system in cnidarians, shared many regulatory genes suggesting that they could come from a common ancestral cell. Taken together those results will lead us to discuss ParaHox genes and neuronal types evolution as well as the different steps of neurogenesis.
6 INTRODUCTION
Les processus neurogéniques reposent sur des mécanismes qui impliquent des cascades génétiques et des facteurs de transcription conservés chez les bilatériens. Les familles de facteurs de transcription qui interviennent dans la neurogénèse sont diversifiées et comprennent des homéoprotéines, des protéines basic-helix-loop-helix (bHLH – basique-hélice-boucle-hélice), zinc finger (doigt de zinc), Sox, Fox, basic leucin zipper (b-ZIP – basique-fermeture leucine) ou encore Runx. L’étude de ces gènes chez les cnidaires et en particulier la comparaison de ces gènes et de leur fonction avec ceux présents chez les bilatériens, offre la possibilité d’élucider leur diversification génétique qui a eu lieu précocement au cours de l’évolution ainsi que la fonction ancestrale de ces familles de gènes. Les gènes Hox et ParaHox (Gsx, Pdx, Cdx) codent pour des homéoprotéines, dont les séquences sont hautement affiliées et très conservées au cours de l’évolution. Chez les bilatériens, les gènes Hox participent à la détermination de la polarité antéro-postérieure embryonnaire, particulièrement du cerveau postérieur en développement. Le gène Gsx est impliqué dans la mise en place du patron dorso-ventral du système nerveux, dans la prolifération et la détemination des précurseurs neuronaux. Les cnidaires sont les premiers animaux du règne animal avec un système nerveux différencié leur permettant d’avoir des comportements actifs. Par ailleurs, ils sont aussi les premiers à posséder des gènes Hox et ParaHox. Ils représentent donc un modèle particulièrement informatif pour retracer les premières étapes qui ont permis l’apparition de la neurogénèse et de mettre en évidence les acteurs génétiques clés qui ont mené à cette innovation dans le règne animal.
Nous avons utilisé des représentants du phylum des cnidaires pour répondre à trois questions. 1) Quelle est l’origine des gènes Hox et ParaHox ? (Résultats-Chapitre 1) (Quiquand et al., 2009). 2) Le gène ParaHox Anthox2/Gsx a-t-il un rôle ancestral dans les mécanismes de neurogénèse ? (Résultats-Chapitre 2). 3) Quels acteurs clés de la neurogénèse connus des bilatériens sont représentés chez les cnidaires ? (Résultats- Chapitre 3) (Galliot et al., 2009).
Dans cette introduction, nous verrons pourquoi les cnidaires sont des systèmes modèles intéressants pour étudier divers mécanismes développementaux qui dépassent leur propre phylum. Nous décrirons l’aspect fonctionnel des gènes Hox et ParaHox chez les bilatériens en nous focalisant d’avantage sur leur implication dans la mise en place du système nerveux. Nous nous pencherons ensuite sur l’origine de ces
7 gènes à travers le phylum des cnidaires. Enfin, nous décrirons les mécanismes intervenant précocement au cours de la mise en place des territoires nerveux chez les bilatériens et nous verrons quels acteurs sont également connus chez les cnidaires.
Le phylum des cnidaires
Les cnidaires actuels sont les repésentants d’un phylum ancestral qui a divergé avant l’apparition des bilatériens. Après avoir relaté leur grande diversité morphologique et de mode de vie, nous regarderons plus précisement la manière dont ces organismes sont organisés avec une attention plus particulière pour l’anthozoaire Nematostella vectensis. Nous regarderons les différents modèles cnidaires utilisés et nous intéresserons aux différents outils de biologie moléculaire déjà disponibles permettant de disséquer les mécanismes développementaux. Bien que moins complexes que les bilatériens, les cnidaires possèdent néanmoins de nombreux gènes et cascades de régulations connus chez les bilatériens.
I.1.1. Un phylum ancestral
Les métazoaires forment un groupe monophylétique comprenant tous les descendants de l’ancêtre commun aux éponges et aux eumétazoaires (Medina et al., 2001). La plus grande majorité des représentants de cet embranchement appartient au phylum des bilatériens dont l’ancêtre commun possédait une symétrie bilatérale. Les cnidaires (du grec knide= ortie et du latin aria= qui ressemble, comme) comprenant les anémones de mer, les coraux, l’hydre ainsi que les méduses et les cténophores (Ctenophora, du grec ktenos, « peigne » et phorein, « porter) sont les deux phyla d’eumétazoaires n’appartenant pas aux bilatériens regroupés sous le nom de Radiata. Les manuels de zoologie distinguent les radiaires des bilatériens en comparant les axes de leur corps, leur symétrie et le nombre de couches tissulaires germinales. Tandis que les bilatériens sont des organismes triploblastiques, c’est-à- dire dérivant de trois couches tissulaires embryologiques (endoderme, ectoderme et mésoderme), présentant une symétrie bilatérale, avec deux axes principaux de polarité (antéro-postérieur et dorso-ventral), les radiaires sont des organismes à symétrie radiaire, diploblastiques, dérivant de deux couches tissulaires (endoderme et ectoderme) et ne possédant qu’un seul axe de polarité (oral-aboral). Nous verrons plus loin que cette définition est pour le moins simpliste (voir I.1.4.1-2, I.1.5.3.).
Des données fossiles indiquent que le phylum des cnidaires est apparu très tôt au cours de l’évolution (Chen et al., 2000; Chen et al., 2002; Cartwright et al., 2007), avant l’explosion Cambrienne (-543 millions d’années). Le modèle de l’horloge
8 moléculaire estime par ailleurs l’émergence des eumetazoaires à environ -725 millions d’années et celle des bilatériens à environ -700 millions d’années (Peterson et al., 2008). Les cnidaires sont donc apparus il y a environ 700 millions d’années, avant l’émergence des bilatériens (protostomiens et deutérostomiens) (Figure 1).
Figure 1: Phylogénie du monde animal modifié à partir de (Adoutte et al., 1999). La position des cnidaires et des cténaires par rapport aux bilatériens est incertaine. Les organismes bilatériens sont divisés en trois branches principales : les ecdysozoaires (invertébrés à mues comme les arthropodes ou les nématodes), les lophotrochozoaires (invertébrés sans mues comme les mollusques ou les annélides) et les deutérostomiens incluant les chordés, les hémichordés et les échinodermes (Mieko Mizutani and Bier, 2008). Les eumétazoaires regroupent les cnidaires, les cténophores et les bilatériens. Les placozoaires ne sont pas représentés sur cet arbre. Une étude phylogénétique récente les place en position basale des eumétazoaires, avant le séparation cnidaires-bilatériens mais après la divergence des éponges (Srivastava et al., 2008). Schéma pris de (Miller et al., 2005).
Cette position phylogénétique, comme groupe frère aux bilatériens, fait des cnidaires un modèle de choix pour des études comparatives visant à reconstruire l’histoire évolutive (Collins, 1998; Medina et al., 2001). L’extrapolation la plus exacte possible de l’organisation et des mécanismes de développement existant chez l’ancêtre commun aux cnidaires et aux bilatériens est un élément indispensable, nécessaire pour reconstruire les changements évolutifs fondateurs dans l’histoire des
9 eumétazoaires. Ils précèdent l’élaboration d’organismes de plus en plus complexes présentant des innovations spécifiques aux bilatériens comme des modifications de l’organisation du plan du corps (Martindale et al., 2002). Un groupe externe aux bilatériens est essentiel pour comprendre la condition ancestrale, la diversité et la complexité des organismes à symétrie bilatérale (Miller and Ball, 2000). L’étude des cnidaires devrait permettre d’identifier les caractères anatomiques ou génomiques déjà présents chez l’ancêtre commun des eumétazoaires. De plus, dans le cadre de la biologie du développement, les informations apportées par ce phylum sont uniques puisque ces animaux se développent de manière sexuée et asexuée, régénèrent et maintiennent par ailleurs constamment leurs conditions homéostatiques (Galliot and Schmid, 2002; Holstein et al., 2003; Darling et al., 2005; Reitzel et al., 2007) (voir I.1.2., I.1.5.1.).
Pour le premier travail consacré aux gènes Hox et ParaHox (II.1. Chapitre 1), nous avons utilisé les cnidaires comme modèle de la condition existante chez l’ancêtre commun aux eumétazoaires, pour retracer les étapes évolutives de ces gènes, jusqu’à la condition bilatérienne. Le deuxième travail consacré à l’étude spécifique du gène ParaHox Anthox2/Gsx chez Nematostella (II.2. Chapitre 2) cherche à retracer la fonction ancestrale de ce gène chez les cnidaires, donc lors de l’apparition de la neurogénèse. Dans la troisième partie, relative à l’origine de la neurogénèse (II.3. Chapitre 3), les cnidaires sont là encore utilisés comme représentants de l’état primitif qui existait lors de la mise en place du premier système nerveux chez les animaux.
I.1.2. Diversité et cycle de vie chez les cnidaires
Les cnidaires se distribuent en deux classes : les anthozoaires, exclusivement polypes (anémone de mer, corail), et les médusozoaires qui alternent la forme méduse et la forme polype au cours de leur cycle de vie et auraient inventé la forme méduse (Collins, 2002; Marques and Collins, 2004). Parmi les médusozoaires, on distingue classiquement les hydrozoaires (hydre, méduses de type Clytia, Cladonema – avec yeux -, Podocoryne), les scyphozoaires (méduses de type Aurelia) et les cubozoaires (méduses de type Tripedalia). Des données moléculaires indiquent que les anthozoaires sont apparus les premiers (Bridge et al., 1995; Odorico and Miller, 1997). Ils sont supposés avoir divergé des autres cnidaires il y a environ 575 millions d’années (Chen et al., 2002; Peterson and Butterfield, 2005) (Figure 2).
10
Figure 2: Relations phylogénétiques au sein du phylum des cnidaires. Les anthozoaires sont les seuls représentants de ce groupe à ne posséder qu’un seul stade : le stade polype. Les hydrozoaires, les cubozaoires et les scyphozoaires ont un cycle de vie qui alterne entre un stade polype fixé et un stade méduse libre. L’importance de chacun des stades varie suivant le groupe considéré. Schéma pris de (Miller et al., 2005).
Composés d’environ 11 000 espèces (Figure 3), les cnidaires sont pour 99% d’entre eux des organismes marins. Cependant certaines espèces, comme l’hydre ou la méduse Craspedacusta sowerbyii (deux hydrozoaires), vivent en eau douce (Bouillon, 1994). Leurs cycles de vie et donc leurs modes de reproduction sont complexes et variés. Classiquement, ils alternent entre un stade polype et un stade méduse. Le polype fixé peut être solitaire ou colonial et bourgeonne par reproduction asexuée des méduses. Ces dernières vivent sous forme pélagique et accomplissent la reproduction sexuée externe. La larve nageuse produite après fécondation, appelée planula, se posera sur le substrat pour se métamorphoser en polype (Figure 4). Et ainsi de suite…
Figure 3 : Diversité des cnidaires. A) Le corail rouge et B) l’anémone de mer sont des anthozoaires. C) L’hydre est un hydrozoaire. D) Méduse cubozoaire. E) Méduse scyphozoaire.
11
Figure 4 : Cycle de vie complet d’un hydrozoaire (Clytia hemisphaerica) avec une alternance entre deux phases : méduse et polype. A) Stade méduse. B) Cycle de vie (Tardent, 1978). C) Stade polype.
Il existe cependant des alternatives à ce schéma classique. Chaque classe de cnidaires est définie par l’importance relative de chacun des stades de vie. Les deux stades de vie sont souvent capables d’accomplir des cycles de reproduction asexuée (bourgeonnement et régénération). Certaines espèces vivent uniquement sous l’une des deux formes. Chez les hydrozoaires et les cubozaoires, les deux stades sont la plupart du temps représentés (sauf chez l’hydre par exemple) et de prévalence similaire. L’hydre est un cas particulier puisqu’elle a perdu le stade méduse et présente d’étonnantes capacités de régénération. Chez les scyphozoaires, le stade méduse est prédominant, le polype bourgeonne des méduses par strobilation (division segmentée transversale le long de l’axe au stade polype). Les anthozoaires vivent uniquement à l’état de polype et la reproduction sexuée s’effectue directement à partir de celui-ci. Il relâche des gamètes dans l’eau. Ces polypes qui peuvent pour certains se multiplier aussi par fission possèdent en plus des capacités de régénération.
Les nouveaux gènes rapportés dans la première partie (II.1. Chapitre 1) ont tous été isolés à partir d’hydrozoaires à cycle complet (Clytia, Turritopsis, Cladonema). Par ailleurs, le modèle principal d’étude qui a été utilisé lors de cette thèse (Nematostella) (II.2. Chapitre 2) appartient au groupe des anthozoaires. Comme tous les anthozoaires, cette anémone ne vit que sous forme de polype et se reproduit de manière sexuée.
I.1.3. Développement embryonnaire des cnidaires
Les œufs pondus par les cnidaires possèdent déjà une polarité qui se définit par la position du pronoyau femelle, l’emplacement des globules polaires et parfois par la distribution de composants cytoplasmiques et de symbiontes. Il s’agit de la polarité animale-végétale. La fertilisation a lieu lors de la deuxième division de maturation. C’est au pôle animal que s’initie le premier sillon de clivage qui deviendra par la suite
12 la partie orale de la planula. Les modalités de clivages et de gastrulation sont très variables chez les cnidaires même au sein d’une espèce (références voir (Fritzenwanker et al., 2007).
I.1.4. Organisation générale des cnidaires
I.1.4.1 Axe(s) de symétrie chez les cnidaires
La méduse et le polype s’organisent autour d’un axe oral-aboral unique et présentent une symétrie radiaire. Les cnidaires ne possèdent qu’un seul orifice, relié à une cavité gastrovasculaire, faisant office à la fois de bouche et d’anus (Figure 5A). Cette extrémité orale entourée de tentacules est appelée l’hypostome chez l’hydre. L’extrémité opposée est définie comme le pôle aboral et il s’agit du pied chez le polype. D’une manière étonnante, c’est en fait l’extrémité antérieure de la planula, définie par la direction de la nage de celle-ci, qui se fixe au substrat pour donner, après la métamorphose, le pied du polype. Par la suite, l’extrémité postérieure de la larve génèrera la bouche.
Nous allons voir que la morphologie larvaire peut être regardée comme présentant une symétrie bilatérale à deux axes (voir I.1.5.3. axe primaire et secondaire de la larve de Nematostella) (Finnerty et al., 2004). Par analogie, il est donc également possible d’extrapoler ces deux axes sur le polype. Cependant le nombre d’axes définissant le plan du corps des cnidaires est à l’heure actuelle toujours un débat. Combien et lesquels ?
I.1.4.2 Organisation des tissus à partir de deux feuillets: l’ectoderme, l’endoderme, (mésoderme)
Classiquement les cnidaires sont définis comme des organismes diploblastiques c’est-à-dire constitués de deux feuillets embryonnaires, l’ectoderme et l’endoderme, séparés par la mésoglée (Bode, 1996) (Figure 5B). Ils sont les premiers animaux à arborer un système neuromusculaire différencié ainsi que des organes sensoriels (Kozmik et al., 2003; Nordstrom et al., 2003; Seipel et al., 2004; Hwang et al., 2007). Cependant des évidences morphologiques et moléculaires (voir I.1.7.) conduisent à reconsidérer le statut des cnidaires comme des animaux triploblastiques (Seipel and Schmid, 2005, 2006) oú l’état diploblastique du polype d’hydrozoaire serait considéré comme un caractère dérivé. En effet, de nombreux cnidaires possèdent des muscles striés qui pourraient correspondre à des dérivés mésodermiques (Seipel and Schmid, 2006).
13 I.1.4.3. Organisation cellulaire de l’hydre
Figure 5: Organisation anatomique et cellulaire de l’hydre. A) Coupe transversale schématique d’une hydre. Ne sont pas représentées les cellules de la lignée interstitielle. B) Coupe transversale schématique dans les deux couches cellulaires qui constituent les parois du corps de l’hydre. Le positionnement des cellules de la lignée interstitielle dans les interstices des cellules épithéliales est représenté. Les différents types cellulaires de la lignée interstitielle sont schématisés. Pour les correspondances voir le schéma C. Les processus musculaires des cellules épithéliales de chacune des deux couches cellulaires, qui s’allongent de manière adjacente à la mésoglée, ne sont pas représentés sur ce schéma. C) Les voies de différentiation des trois classes de produits somatiques. Les cellules interstitielles incluent les cellules souches (stem cells) ainsi que les cellules déjà engagées dans une voie déterminée (commited cells). Les produits intermédiaires de différentiation incluent les cellules en division et en cours de différentiation. Schémas pris de (Bode, 1996). C’est chez l’hydre que l’organisation et le lignage cellulaire sont les mieux documentés. Chacun des feuillets se compose uniquement d’une couche cellulaire : les cellules myoépithéliales ectodermales en contact avec l’extérieur et les cellules myoépithéliales endodermales qui tapissent la cavité gastrique de l’animal. Ces deux types de cellules dérivent respectivement des cellules souches épithéliales
14 ectodermales et endodermales. Les autres types cellulaires composent la lignée des cellules interstitielles et se localisent dans les interstices des cellules épithéliales de l’ectoderme et de l’endoderme (Figure 5B). Elles dérivent des cellules souches interstitielles (Figure 5C). L’hydre possède donc trois lignées de cellules souches (les cellules souches épithéliales ectodermales et endodermales et les cellules souches interstitielles).
Les cellules souches épithéliales de l’hydre accomplissent, en plus, des fonctions physiologiques. Les cellules épithéliales ectodermales ont une fonction de protection et d’osmorégulation quant aux cellules épithéliales endodermales, elles sont impliquées dans les processus digestifs. Les cellules souches interstitielles forment une population de cellules multipotentes à l’origine de trois classes différentes de produits somatiques, cellules sécrétrices (cellules glandulaires et muqueuses), neurones (sensoriels et ganglionaires), cnidocytes ainsi que des gamètes. Les cellules engagées dans la voie de différentiation de l’une des trois classes de produits somatiques subissent tout d’abord un ou plusieurs cycles de division cellulaire amplifiant ainsi le nombre de cellules différenciées produites à terme (Bode, 1996) (Figure 5C).
I.1.4.4. Le système nerveux des cnidaires
Le système nerveux des cnidaires est constitué de deux réseaux nerveux diffus, en association avec l’endoderme ou l’ectoderme. Il est composé de neurones et de cellules mécanoréceptrices appelées les cnidocytes (Marlow et al., 2009).
Les neurones
Les neurones de cnidaires sont divisés en deux catégories : les cellules ganglionaires (grandes, bipolaires ou multipolaires) et les cellules sensorielles (généralement multipolaires et ciliées) (Bode, 1996) qui se distribuent entre les cellules éptihéliales des deux couches cellulaires de l’animal constituant ainsi un système nerveux diffus. Les cellules sensorielles sont en contact avec l’environnement (Westfall et al., 2002). Comme chez les bilatériens, les cellules nerveuses de cnidaires sont des cellules excitables généralement ramifiées qui génèrent et propagent des potentiels d’action. Cependant les neurones de cnidaires sont dépourvus des cellules gliales qui accompagnent les neurones des bilatériens. De plus, les cnidaires ne présentent pas de différentiation dans la ramification de leurs cellules nerveuses en dendrites (réception du signal) et axones (propagation du signal) comme chez les
15 bilatériens. Tous les prolongements nerveux des cnidaires sont appelés neurites. Ils conduisent des messages nerveux dans les deux directions (Anderson, 1985).
Chez certaines espèces d’hydres, il existe une concentration plus importante de neurones à la base de la bouche arrangée en un anneau nerveux (Figure 6). Cet arrangement, de part sa structure et sa localisation est proposé comme étant un système nerveux central primitif (Koizumi, 2007). En comparaison, les méduses d’hydrozoaires possèdent deux anneaux nerveux faisant le tour de l’ombrelle, un anneau interne, moteur, et un anneau externe, sensoriel (Grimmelikhuijzen et al., 1989).
Figure 6 : Anneau nerveux chez Hydra oligactis observé en immunomarquage avec l’anticorps anti-RFamide. A) Vue de côté de la tête montrant l’hypostome et la partie basale des tentacules. A l’apex de l’hypostome se trouve la bouche. B) Vue du dessus de l’hypostome qui montre la bouche au centre entourée de nombreuses cellules épidermiques sensorielles. L’anneau nerveux entourre la bouche. Échelles : A) 200 µm, B) 100 µm. Phtographies adaptées de (Koizumi et al., 1992).
Les cnidocytes
Les cnidocytes, organite explosif servant à la défense, la locomation et la nutrition de l’animal, sont des cellules urticantes spécifiques aux cnidaires et sont à l’origine du nom de ce phylum. Hautement différenciées, elles contiennent une capsule appelée cnidocyste dont la nature morphologique permet de classifier les différents types de cnidocytes en trois groupes structuraux différents : les nématocytes, les spirocytes et les ptychocytes (Kass-Simon and Scappaticci, 2002). Ils sont considérés comme des
16 cellules neurosensorielles ciliées répondant à des stimuli externes, ils sont responsables de la mécanoréception et de la chémoréception. Ces cellules sont intégrées au sein du système nerveux par l’intermédiaire de synapses (Westfall, 2004). Chez l’hydre, elles dérivent des cellules interstitielles tout comme les neurones (Bode, 1996) (Figure 5C).
La complexité nerveuse des cnidaires peut s’illustrer au travers de l’organisation cellulaire d’un tentacule d’anémone de mer. Les cellules sensorielles sont orientées perpendiculairement au plexus nerveux et aux cellules musculaires de l’épiderme. Elles sont dispersées parmis de nombreux spirocytes et nématocytes. Les cellules ganglionaires sont situées au-dessus ou dans le plexus nerveux qui longe les fibres musculaires basales (Westfall et al., 2002) (Figure 7).
Figure 7: Aiptasia pallida. Section d’un tentacule représentant une cellule sensorielle allongée (S) orientée perpendiculairement à une cellule ganglionaire (G) située au-dessus du plexus nerveux (N) et longitudinal aux cellules musculaires (M). De nombreux spirocystes (SP) et quelques nématocystes (NS) sont situés perpendiculairement au plexus nerveux. La mésoglée acellulaire sépare l’épiderme épais du gastroderme (GA) fin et du lumen (L) du tentacule creux. Échelle: 3 µm. Photographie prise de (Westfall et al., 2002)
17 Par ailleurs, les cnidaires possèdent une grande variété de structures sensorielles comme les rhopalies ou les statocystes impliquée dans la photoréception et l’équilibration.
La transmission synaptique
Chez les anémones, les projections des neurones sensoriels peuvent établir des synapses avec les spirocytes, les cellules musculaires et les cellules ganglionaires. Leur rôle est de coordonner un signal arrivant des cellules ou structures sensorielles avec un signal de sortie dirigé vers une cellule cible (épithéliomusculaire par exemple) (Westfall et al., 2002; Marlow et al., 2009). Les projections des cellules ganglionaires établissent des synapses avec les cellules sensorielles, les spirocytes, les cellules musculaires et avec d’autres neurones. Les neurones sensoriels et ganglionaires fonctionnent donc comme des motoneurones. Lorsqu’une cellule sensorielle forme une synapse directement avec un spirocyte la cascade de transmission est à deux cellules. En revanche, lorsqu’une cellule ganglionaire fait l’intermédiaire entre une cellule sensorielle et une cellule effectrice alors la cascade de transmission est à trois cellules (Westfall et al., 2002) (Figure 8).
Figure 8: Schéma hypothétique indiquant des cascades de transmission synaptique à deux ou trois cellules dans l’épiderme des tentacules de l’anémone de mer. Les cellules sensorielles forment des synpases directement sur des spirocystes et/ou des cellules musculaires pour former une cascade de transmission à deux cellules. Une cascade à trois cellules est définie quand une cellule ganglionaire fait l’intermédiaire entre des cellules sensorielles et des cellules effectrices. Schéma tiré de (Westfall et al., 2002).
18 La transmission synaptique s’opère par l’intermédiaire de synapses à la fois chimiques et électriques via des jonctions gap (Satterlie and Spencer, 1987) où des potentiels post-synaptiques excitateurs et inhibiteurs ont pu être enregistrés (Anderson and Spencer, 1989). La neurotransmission synaptique s’effectue grâce à des transmetteurs rapides (acetylcholine, glutamate, GABA, glycine) ou lents (catecholamine, sérotonine) et des neuropeptides (RFamide, RWamide par exemple) (Kass-Simon and Pierobon, 2007).
Ainsi les cnidaires sont capables de percevoir des informations sensorielles. Celles-ci sont à l’origine de mouvements coordonnés des tentacules et du corps, permettant la capture de nourriture ainsi que l’activité de l’ombrelle de la méduse nécessaire à ses déplacements. En tant que cellules mécanoréceptrices, les nématocytes entre en contact avec la proie par le biais de leur cnidocil. La décharge d’un venin toxique sur la proie va provoquer une réponse de nutrition de l’animal qui se traduit par le mouvement des tentacules vers la bouche qui s’ouvre. Le peptide glutathion agit comme un activateur du comportement de nutrition.
La mise en place du système nerveux diffus de l’hydre est bien élucidée au niveau cellulaire (Grimmelikhuijzen et al., 1989; Koizumi et al., 1990). Cependant la connaissance des mécanismes génétiques qui régulent son développement est encore relativement sommaire. De plus, les contextes de neurogénèse chez les cnidaires sont pour le moins complexes puisque le phénomène doit prendre place au cours du développement embryonaire, au cours du bourgeonement en enfin lors de la régénération.
I.1.5. Nematostella vectensis : un nouveau modèle
Depuis quelques années, l’anémone de mer Nematostella vectensis est devenue un modèle prisé, en particulier pour l’étude de l’embryogenèse et du développement larvaire. Cet anthozoaire, appartenant à l’ordre des Actiniaires, se place phylogénétiquement comme groupe frère aux médusozoaires (Bridge et al., 1995; Odorico and Miller, 1997). Si le stade méduse est une invention des médusozoaires, alors le stade polype unique de Nematostella représenterait au mieux la condition primitive des cnidaires (Collins et al., 2006). Cette position phylogénétique particulière relative aux autres cnidaires, en fait un modèle de choix pour les approches évolutives au sein de ce phylum (Darling et al., 2005).
19 I.1.5.1. Mode et cycle de vie de Nematostella
Figure 9 : Différents stades de développement représentatifs de la reproduction sexuée de Nematostella. A) Œuf au stade une cellule avant les premières divisions. Les œufs sont expulsés dans une large masse gélatineuse B) Embryon en division. C) Photographie prise dans (Darling et al., 2005). Larve planula nageuse. La flèche indique la direction de la nage (touffe apicale vers le bas). D) Jeune polype nouvellement métamorphosé. E) Animal adulte ayant atteint la maturité sexuelle. L’astérisque montre le pôle oral.
Figure 10 : Reproduction asexuée chez Nematostella. A) La régénération suivant une blessure représente un mode de reproduction asexué. Cependant, le rôle joué par ce mode de reproduction dans les populations naturelles est inconnu. B) Le mode de reproduction asexué le plus utilisé chez Nematostella est la fission transverse: la contraction du corps de l’animal conduit finalement à la séparation de la partie aborale qui par la suite régénère la structure de la tête. C) D’une manière alternative (mais ceci arrive moins fréquemment), la reproduction asexuée peut survenir par le biais d’une inversion de polarité où une nouvelle tête se forme, typiquement à la partie aborale de l’anémone et l’animal se divise en deux en son milieu et régénère alors un nouveau pied. Photographies prises dans (Darling et al., 2005).
Nematostella est un animal solitaire vivant en eaux saumâtres. Carnivore, elle capture du plancton. Elle se distribue principalement le long des côtes pacifiques et atlantiques du nord de l’Amérique. Les sexes sont séparés mais indistinguables
20 morphologiquement. Les œufs sont pondus dans une masse gélatineuse par dizaines voir par centaines. Ces derniers sont larges (environ 240 µm). La fécondation est externe. Après la fécondation, une larve planula ciliée nageuse se forme. Environ sept jours après la fertilisation, la larve se métamorphose en un polype juvénile dont la bouche se trouve au centre de quatre tentacules (Hand and Uhlinger, 1991, 1992; Kraus and Technau, 2006). La maturité sexuelle s’acquiert au bout d’environ quatre mois (Figure 9).
En plus du cycle de reproduction sexuée décrit ci-dessus, Nematostella possède également des capacités de reproduction asexuée: la fission (Hand and Uhlinger, 1995), l’inversion de polarité et la régénération (Reitzel et al., 2007) (Figure 10).
I.1.5.2. Développement embryonnaire de Nematostella
La fertilisation externe est suivie par des divisions cellulaires synchrones dont le patron de clivage est variable d’un embryon à l’autre. Il en résulte, au stade 32-64 cellules, une coeloblastula creuse organisée. En corrélation avec les cycles de division cellulaire, celle-ci subit quatre à cinq cycles de mouvements d’invagination et d’évagination. Ce processus prend fin environ quatre heures avant que les divisions cellulaires cessent d’être synchrone (13 heures après la fertilisation). Des cils se forment sur l’embryon qui commence la gastrulation environ 20 heures après la fertilisation (Figure 11).
Le pôle animal de l’œuf correspond à la fois au site du premier sillon de clivage, au côté concave de la blastula et donc aux sites d’invagination, au blastopore de la gastrula et au pôle oral du polype. Cet hémisphère contient une activité organisatrice nécessaire à la formation d’un polype normal (Fritzenwanker et al., 2007).
Figure 11 : Stade du développement embryonnaire de Nematostella. Schéma pris de (Fritzenwanker et al., 2007).
21 I.1.5.3. Organisation anatomique de Nematostella
Chez la larve, le pôle antérieur arbore une touffe, nommée « touffe apicale » dirigée dans la direction de la nage de l’animal (Figure 12A). Le blastopore, situé au pôle postérieur, deviendra la bouche de l’animal adulte. L’axe primaire du corps de la larve correspond à l’axe apical-blastopore. Comme tous les polypes de cnidaires, la structure du corps de Nematostella se compose de deux épithéliums: un ectoderme externe et un endoderme interne, séparés par la mésoglée acellulaire. Le pharynx connecte la bouche à l’intestin. Il correspond à une invagination de l’ectoderme au pôle oral. L’axe secondaire, appelé l’axe directif, traverse le pharynx à angle droit avec l’axe primaire (Finnerty et al., 2004) (Figure 18).
Figure 12 : Organisation anatomique de la larve et du polype adulte de Nematostella. A) Larve au stade de planula. B) Polype adulte. A-B) Coupes longitudinales à travers l’axe oral- aboral. C) Section dans la région du pharynx. Schémas pris de (Finnerty et al., 2004).
Le polype juvénile présente une structure endodermique particulière appelée les mésentéries. Au nombre de deux après la métamorphose, elles divisent l’intestin et augmentent sa surface permettant la production et le stockage des gamètes dans des poches (Figure 12B). L’animal adulte a la forme d’un tube de 5 – 10 cm de long (Putnam et al., 2007). Le pharynx s’attache à la structure du corps par le biais de huit mésentéries endodermiques. Chacune d’entre elles porte un muscle rétracteur (Finnerty et al., 2004) (Figure 12C). L’extrémité orale s’ouvre sur une bouche entourée d’une vingtaine de tentacules. La région aborale est fermée (Putnam et al., 2007) (Figure 12).
22 I.1.5.4. Système nerveux de Nematostella
Figure 13: Anatomie du système nerveux de Nematostella au stade polype et planula. A) Demi-coupe longitudinale d’un stade polype montrant la morphologie neuronale. Les anneaux nerveux oral et pharyngial sont surlignés en violet. B) Demi-coupe longitudinale d’un stade stade planula montrant la morphologie neuronale. C-H) Représentation schématique de la morphologie individuelle des neurones et des cnidocytes : trois ganglions, deux cnidocytes et deux cellules de type sensoriel sont représentés. Shémas modifiés de (Marlow et al., 2009).
Comme tous les cnidaires, Nematostella possède un réseau nerveux diffus. La différentiation nerveuse s’effectue dans la totalité du corps de l’animal et s’initie avant la fin de la gastrulation. Contrairement à beaucoup de métazoaires, elle prend place aussi bien dans l’ectoderme que dans l’endoderme. Les cellules neuronales sont localisées en différents domaines des deux couches cellulaires et sont produites à des stades spécifiques du développement de l’anémone. Elles se répartissent en différents territoires distribués le long de l’axe oral-aboral. Ces territoires subissent des modulations à la métamorphose. La larve présente une région neuronale, située au pôle antérieur, représentée par l’organe sensoriel apical portant la touffe apicale. La
23 formation de cet organe est sous le contôle du facteur de signalisation FGF (Fibroblast growth factor) (Rentzsch et al., 2008). Le réseau nerveux diffus du polype quant à lui est plus dense au niveau des anneaux nerveux oral et pharyngial où les mésentéries sont innervées ainsi que dans l’extrémité des tentacules (Figure 13).
Le réseau neuronal de Nematostella se compose de cellules ectodermiques sensorielles et effectrices et de cellules ganglionaires endodermales multipolaires (Grimmelikhuijzen et al., 2002; Koizumi, 2002). Il est constitué de multiples classes de cellules neuronales et de cnidocytes, qui se caractérisent par leur morphologie et par l’expression de neuropeptides et de neurotransmetteurs (Marlow et al., 2009). Néanmoins certaines cellules morphologiquement identiques n’expriment pas toujours les mêmes combinaisons de neurotransmetteurs. Le réseau nerveux n’est donc pas homogène, mais possède des cellules avec des potentiels multifonctionnels, distribuées dans les deux couches tissulaires de l’animal et dépendantes d’un patron moléculaire bien défini (Figure 13).
Marlow et al proposent qu’une ou deux populations de cellules souches localisées en des domaines déterminés (ectodermal ou alors ectodermal et endodermal) puissent produire des précurseurs qui migreraient et se positionneraient par la suite dans les régions neuronales spécifiques de la larve et du polype (Marlow et al., 2009).
I.1.6. Les cnidaires comme système modèle
L’utilisation de ce phylum comme système modèle vise à répondre à un certain nombre de problématiques : comprendre l’émergence, l’élaboration et le déploiement des cascades génétiques qui contrôlent la spécification des axes (Gauchat et al., 2000; Hobmayer et al., 2000; Yanze et al., 2001; Ball et al., 2004; Finnerty et al., 2004; Lee et al., 2006; Rentzsch et al., 2006), la mise en place des patrons d’expression selon les axes de polarité (Gauchat et al., 2000; Hobmayer et al., 2000; Yanze et al., 2001; Ball et al., 2004; Finnerty et al., 2004; Lee et al., 2006; Rentzsch et al., 2006), la transdifférenciation du tissu musculaire strié (Schmid et al., 1998; Technau and Scholz, 2003) le développement des yeux (Kozmik et al., 2003; Stierwald et al., 2004) ou encore l’alloimmunité (Frank et al., 2001).
I.1.6.1. Les différents modèles de cnidaires utilisés
L’hydre (hydrozoaire) découverte en 1740 par Abraham Trembley, est un modèle historique utilisé entre autre pour l’étude de la régénération dès les années 50 (Sturtevant et al., 1951). Cette espèce est néanmoins un représentant des hydrozoaires hautement dérivé (Collins et al., 2000). En effet, la plupart des
24 hydrozoaires sont marins et possédent un stade méduse alors que l’hydre est un animal d’eau douce qui ne vit qu’à l’état de polype (Steele, 2002). De plus, la reproduction sexuée chez l’hydre est rare et imprévisible en laboratoire, il est donc difficile d’en étudier le développement embryonnaire. L’embryogenèse de l’hydre implique en plus une période de dormance (Martin et al., 1997). D’autres hydrozoaires sont utilisés en biologie du développement en particulier Podocoryne (Momose and Schmid, 2006) ou Clytia (Momose and Houliston, 2007). Les modèles d’anthozoaires sont principalement Acropora (corail) (de Jong et al., 2006; Shinzato et al., 2008) et Nematostella qui représente le modèle le plus utilisé maintenant avec l’hydre.
I.1.6.2. Avantages et inconvénients du modèle anthozoaire Nematostella
Nematostella (anthozoaire) est un nouveau modèle en pleine émergence ces dernières années (Hand and Uhlinger, 1992; Darling et al., 2005). Cette anémone présente de nombreux avantages pratiques. Les cultures se maintiennent très facilement en laboratoire et la ponte peut être induite une fois par semaine dans des conditions de nourriture, de lumière et de température données. Ainsi les embryons sont disponibles toute l’année (Hand and Uhlinger, 1991, 1992; Fritzenwanker and Technau, 2002). Les œufs sont pondus en grand nombre et leur taille les rend facilement manipulables après retrait de la gélatine (Putnam et al., 2007). Le temps de génération de cet organisme est relativement court. Par ailleurs, Nematostella possède des modalités de développement asexué qui permettent d’étudier l’implication des gènes du développement dans des contextes diversifiés (Burton and Finnerty, 2009). Grâce à la contribution du laboratoire d’Uli Technau nous avons pu développer et maintenir une culture de Nematostella pendant 5 années à Genève. Nous avons profité des avantages liés à l’induction de la ponte pour mener à bien notre approche fonctionnelle du gène Gsx/Anthox2 au cours du développement embryonnaire de l’animal (II.2. Chapitre 2). D’un point de vue génomique, Nematostella possède un génome relativement compact (environ 450 millions de paires de bases contre environ 1100 chez l’hydre) désormais disponible et annoté (Putnam et al., 2007) dans lequel la plupart des familles de gènes impliquées dans les grandes cascades de signalisation et les processus de développement sont représentées (voir I.1.7., I.2.5., I.3.3.). Ces caractéristiques font de Nematostella un modèle d’étude offrant la possibilité de disséquer des mécanismes fondamentaux représentatifs de l’état primitif des eumétazoaires.
Nematostella présente donc de nombreuses qualités pratiques mais comme tout nouveau modèle, les études descriptives font encore défaults. Il existe maintenant
25 trois études détaillées de son développement embryonnaire rapportées ci-dessus (voir I.1.5.2.) dont deux réalisée au moyen de la microscopie électronique (Kraus and Technau, 2006; Fritzenwanker et al., 2007; Magie et al., 2007). L’anatomie et le développement de son système nerveux a fait l’objet de peu de publications. L’une, également rapportée ci-dessus (voir I.1.5.4), a décrit à l’aide de marqueurs moléculaires les différents types neuronaux (Marlow et al., 2009). L’autre utilise une approche fonctionnelle pour montrer la fonction de FGF dans le développement de la structure sensorielle apicale de la planula (Rentzsch et al., 2008). Mais il n’existe pas encore d’étude approfondie décrivant les différents types cellulaires constituant Nematostella et les marqueurs cellulaires sont encore peu nombreux. Nous avons mis au point quelques marqueurs de cellules neuronales afin d’analyser le développement du système nerveux dans les premiers jours de développement de l’animal (II.2. Chapitre 2).
I.1.6.3. Outils génétiques disponibles chez les cnidaires
Des outils génétiques et moléculaires de plus en plus nombreux sont disponibles chez les cnidaires. Ainsi, les génomes de Nematostella (Sullivan et al., 2006; Putnam et al., 2007) et celui d’ Hydra magnipapillata sont maintenant séquencés. En outre, différentes techniques permettant la dissection moléculaire des mécanismes de développement émergent désormais. L’approche hétérologue, d’un intérêt limité pour l’analyse des processus biologiques en place chez les cnidaires, a été mise en œuvre il y a quelques années (Hayward et al., 2002; Kozmik et al., 2003; Rentzsch et al., 2006). Plus récemment, des techniques permettant des études fonctionnelles en système homologue ont été mises au point. Ainsi des expériences de perte de fonction sont réalisables par le biais de l’ARN interférence (Chera et al., 2006) ou de l’injection de morpholinos (Magie et al., 2007; Momose and Houliston, 2007; Rentzsch et al., 2008). De plus, après la mise en place de techniques d’expression transitoire de constructions exprimant des protéines rapportrices (Bottger et al., 2002; Miljkovic et al., 2002), il existe désormais des lignées d’hydres transgéniques (Wittlieb et al., 2006; Khalturin et al., 2007). Ces avancées techniques offrent des possibilités d’analyses plus précises des mécanismes développementaux chez les cnidaires, pendant longtemps étudiés seulement à partir de patrons d’expression ou d’analyses phylogénétiques de séquences de familles de gènes conservés. Une partie des cinq années de thèse a été consacrée à la mise au point de la technique d’injection de gènes rapporteurs chez Nematostella permettant l’observation in vivo de l’expression des gènes et l’étude de leur régulation (II.2. Chapitre 2).
26 I.1.7. Conservation des gènes et des voies de signalisation
L’analyse des gènes clonés chez les différentes espèces de cnidaires, des ESTs (Expressed Sequence Tag ou marqueur de séquence exprimée) d’Acropora ainsi que des génomes de Nematostella et de l’hydre a révélé que les six grandes voies de signalisation cellulaire, régulatrices du développement chez les bilatériens, sont représentées dans le phylum des cnidaires. En effet, des acteurs appartenant aux cascades Wnt (Kusserow et al., 2005), Transforming Growth Factor! (TGF!), nuclear receptor (Grasso et al., 2001), Notch, Hedgehog et Ras-mitogen-activated protein kinase 1 (MAPK) ont pu être caractérisés (Steele, 2002; Miller et al., 2005; Technau et al., 2005; Putnam et al., 2007). Par ailleurs, il existe également chez les cnidaires d’autres gènes clés impliqués dans les processus cellulaires des bilatériens comme la neurogénèse (voir I.3.3. et II.3 Chapitre 3), la transmission synaptique, l’adhésion et les jonctions cellulaires, la contraction musculaire et l’apoptose. Enfin de nombreux facteurs de transcription fondamentaux chez les bilatériens sont également retrouvés (homéoboîte, b-HLH, Sox, T-box, Fox, Zic, Gli, mothers-against- decapentaplegic SMAD) (voir I.2.5., I.3.3. et II.3 Chapitre 3). D’une manière intéressante, les gènes impliqués dans la détermination du mésoderme chez les bilatériens sont présents chez les cnidaires originellement considérés comme des organismes diploblastiques (Technau and Bode, 1999; Spring et al., 2000; Spring et al., 2002; Scholz and Technau, 2003; Fritzenwanker et al., 2004; Martindale et al., 2004). Ces données impliquent qu’une complexité génétique préalablement considérée comme récente dans l’évolution animale est apparue tôt au cours de l’évolution.
Chez l’éponge Amphimedon, des gènes appartenant aux cascades de régulation Hedgehog, Wnt et TGF! sont présents et exprimés au cours du développement (Nichols et al., 2006; Adamska et al., 2007a; Adamska et al., 2007b). Par ailleurs, l’analyse de son génome a mis en évidence des facteurs de transcription des familles homéoboîte, bHLH, Sox, T-box et Fox (Larroux et al., 2008). Cependant les gènes à homéoboîtes des familles Hox et ParaHox n’ont pas été caractérisés.
La comparaison des génomes d’Amphimedon et de Nematostella menée par Larroux et al. a révélé que le nombre de représentants appartenant à chacune des familles de facteurs de transcription était significativement plus important chez l’anémone. C’est donc après la divergence des éponges, chez l’ancêtre commun aux eumétazoaires qu’ une importante expansion génétique s’est produite aboutissant à une grande diversité de gènes (Larroux et al., 2008). Cette expansion génétique s’illustre par des
27 évènements de duplication et de diversification comme par exemple pour les 11 familles de gènes Wnt décrites chez Nematostella (Kusserow et al., 2005). D’une manière surprenante, les génomes de cnidaires révèlent donc une considérable complexité. De plus, les classes de gènes et leurs transcrits représentés dans le génome de Nematostella sont plus similaires à celles des vertébrés que ne le sont ceux de la mouche ou du nématode, dont les génomes ont évolués de manière rapide (Putnam et al., 2007). Ces considérations montrent que d’un point de vue moléculaire les cnidaires ont à leur disposition la grande majorité des outils utilisés par les bilatériens. Il est donc vraisemblable que des innovations spécifiques aux bilatériens liées à l’augmentation de la complexité des modes de régulation soient due à l’évolution des modules régulateurs en cis ou à des changements dans la structure chromatidienne (Hinman et al., 2003; Romano and Wray, 2003; Ruvinsky and Ruvkun, 2003) plus qu’à une augmentation du nombre de gènes liés au développement.
Les gènes à homéoboîte
Les gènes à homéoboîte incluent de nombreux acteurs actifs dans le développement du cerveau chez les vertébrés (Holland and Takahashi, 2005). Dans cette partie, nous allons nous intéresser plus particulièrement à la famille Hox et à l’implication de ces gènes lors du développement du système nerveux des bilatériens (Hirth and Reichert, 1999). Les gènes ParaHox, groupe frère des gènes Hox, sont probablement apparus suite à un événement de duplication d’un complexe ancestral ProtoHOX (Brooke et al., 1998). Les cnidaires contiennent dans leurs génomes des gènes Hox et ParaHox, démontrant que l’origine de ces familles est antérieure à l’apparition des bilatériens (Murtha et al., 1991; Schummer et al., 1992).
I.2.1. Les mutations homéotiques et les gènes à homéoboîte
Chez la drosophile, les gènes homéotiques déterminent le plan d’organisation, c’est-à- dire la place des organes les uns par rapport aux autres et par rapport aux axes de polarité. Une mutation de ce type de gène entraîne la substitution de l’identité d’un organe dans une région donnée par l’identité d’un autre organe caractéristique d’une autre position du corps. On parle alors de mutation homéotique. Le concept d’homéose date de plus d’un siècle. Inventé par Bateson, il caractérise à cette époque des variants naturels qui présentent des phénotypes homéotiques (Bateson, 1894). Les gènes homéotiques (HOM) ont été mis en évidence chez la drosophile, tout d’abord génétiquement (Lewis, 1978; Gehring, 1987) puis au niveau moléculaire par le
28 groupe de Walter Gehring à Bâle (McGinnis et al., 1984b) et le groupe de Matt Scott aux USA (Scott and Weiner, 1984). Au nombre de huit, ces gènes se regroupent en deux complexes distincts sur le chromosome trois : les complexes Antennapedia (Ant- C) et Bithorax (BX-C). Les gènes homéotiques sont pour la plupart des gènes à homéoboîte qui présentent une région de 180 nucléotides codant pour un domaine de liaison à l’ADN appelé l’homéodomaine de 60 acides aminés (Gehring, 1987; Banerjee-Basu and Baxevanis, 2001). Les produits de ces gènes, retrouvés à la fois chez les animaux, les plantes et les champignons (Burglin, 1994), agissent comme des facteurs de transcription : ils se lient spécifiquement à l’ADN par leur homéodomaine au motif caractéristique en hélice-tour-hélice (helix-turn-helix) (Gehring et al., 1990). Chez les vertébrés, des gènes codant pour un homéodomaine orthologue à ceux des complexes Ant-C et BX-C de type Antennapedia ont été identifiés : au cours du développement, ces gènes s’expriment et agissent dans des segments spécifiques le long de l’axe antéro-posterieur comme chez l’embryon de mouche (Duboule and Dolle, 1989) (Figure 14). Ces gènes sont définis comme les gènes Hox.
Une étude menée par Ryan et al. montre que Nematostella possède plus de protéines à homéodomaines que la drosophile (131 versus 97). Ce qui implique que l’hypothèse liant complexité et nombre de gènes n’est pas si triviale (Ryan et al., 2006).
Les protéines à homéodomaine sont divisées en 10 classes : ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS et ZF (références voir (Ryan et al., 2006). Les classes ANTP et PRD sont les plus grandes et sont reconnues comme des groupes frères. Toutes les classes ont été mises en évidence chez les cnidaires à l’exception des classes PROS et ZF. Les classes ANTP, PRD, LIM, POU, SINE et TALE avaient déjà subi une importante diversification avant la divergence des cnidaires (Miles and Miller, 1992; Schummer et al., 1992; Naito et al., 1993; Kuhn et al., 1996; Finnerty and Martindale, 1997, 1999; Galliot et al., 1999; Kuhn et al., 1999; Gauchat et al., 2000; Miller et al., 2000; Bebenek et al., 2004; Ryan et al., 2006).
I.2.2. La classe ANTP
La classe ANTP tire son nom du gène Hox Antennapedia (Antp) de drosophile porté par le complexe homéotique ANT-C. Elle se subdivise en deux sous-classes : HOXL (pour HOX-like) comprenant entre autre les gènes Hox et ParaHox et NKL (NK-like) (Holland et al., 2007). Suivant les classifications, la classe HOXL comprend 14 ou 15 familles et la classe NKL 21 ou 23 (Ryan et al., 2006; Holland et al., 2007).
29 Les données accumulées ces dernières années indiquent que 10 familles HOXL sont représentées chez les cnidaires (Gsx, Pdx, Cdx, Hox1, Hox2, Mox, Evx, Exex, Gbx, Rough) (Chourrout et al., 2006; Ryan et al., 2006; Putnam et al., 2007; Ryan et al., 2007; Chiori et al., 2009; Quiquand et al., 2009) aisni que 16 familles NK (Dlx, Hhex, Hmx, Lbx, Msx, Nk-1, Nk-2, Nk-3, Nk-6, Nk-7, Tlx, Emx, Emxlx, Hlx, Mslx, Vax) (Ryan et al., 2006).
Parmi les gènes appartenant à la classe ANTP, nous allons examiner plus particulièrement ceux des familles Hox et ParaHox. D’une part, nous en avons étudié la phylogénie en détail (II.1. Chapitre 1) et d’autre part, ils régulent de nombreux aspects du développement en particulier la mise en place du système nerveux chez les bilatériens (Weiss et al., 1998; Hirth and Reichert, 1999; Toresson et al., 2000; Kriks et al., 2005).
I.2.3. Rôle des gènes Hox dans la neurogénèse
I.2.3.1. Organisation des gènes Hox en complexes
Les gènes Hox codent pour des facteurs de transcription. Ils sont apparus après de multiples évènements de duplication à partir d’un complexe HOX ancestral aboutissant à une organisation en complexe de gènes paralogues (PG) le long du chromosome. Chez la souris, on trouve quatre complexes (A-D) (Duboule and Dolle, 1989; Favier and Dolle, 1997) localisés sur des régions chromosomiques différentes (Kappen et al., 1989) portant en tout 39 gènes Hox classés en 13 familles de gènes paralogues (Scott, 1992). En conséquence, les gènes paralogues d’une même famille sont localisés à la même position relative dans leur complexe respectif. Des analyses phylogénétiques montrent que les familles Hox se divisent en quatre sous-familles de gènes, affiliées d’un point de vu évolutif : les gènes « antérieurs » (PG1 et PG2 chez les chordés, lab et pb chez les insectes), les gènes du groupe 3 (PG3 chez les chordés, zen/PG3 chez les insectes), les gènes « médians » (PG4 à PG8 chez les chordés, Dfd à abd-A chez les insectes) et les gènes « postérieurs » (PG9 à PG13 chez les chordés, Abd-B chez les insectes) (Brooke et al., 1998; Banerjee-Basu and Baxevanis, 2001) (Figure 14). Il existe jusqu’à 15 familles de gènes paralogues chez l’amphioxus (céphalochordé) (Holland et al., 2008).
30
Figure 14: Organisation génomique et colinéarité spatiale des gènes homéotiques (HOM) de drosophile et des gènes Hox de souris. Les shémas des complexes Ant-C et BX- C de drosophile, des quatre complexes HOX de drosophile et d’un complexe hypothétique ancestral sont représentés avec leur possible relation phylogénétique. Chaque gène est marqué d’un carré de couleur. Les domaines d’expression successifs des gènes HOM/Hox selon l’axe antéro-postérieur sont shématisés dans un embryon de drosophile (partie haute) ainsi que dans le système nerveux central et les prévertèbres d’un embryon de souris en mi- gestation (partie basse). Le chevauchement partiel des transcrits des gènes HOM dans les segments thoraciques et abdominaux de l’embryon de drosophile sont indiqués. En revanche, le chevauchement des transcrits des gènes Hox dans les régions postérieures de l’embryon de souris ne sont pas représentés. Ainsi, chaque couleur représente le domaine d’expression le plus antérieur d’une famille de gène donnée. Abbréviations des gènes HOM : lab (labial); pb (proboscipedia); Dfd (deformed); Scr (sex combs reduced); Antp (antennapedia); Ubx (ultrabithorax); abd-A (abdominal-A); Abd-B (abdominal-B). Schéma tiré de (Favier and Dolle, 1997). L’organisation génomique de ces gènes en complexe est conservée parmi les ecdysozoaires (Ferrier and Akam, 1996; Ruvkun and Hobert, 1998; Devenport et al., 2000; Powers et al., 2000; Brown et al., 2002), les lophotrochozoaires (Kmita-Cunisse et al., 1998) et les deutérostomes (Acampora et al., 1989; Duboule and Dolle, 1989;
31 Garcia-Fernandez and Holland, 1994; Amores et al., 1998; Martinez et al., 1999; Kim et al., 2000; Minguillon et al., 2005). Le nombre de complexes HOX est variable des invertébrés aux vertébrés.
L’organisation des gènes Hox en complexe semble donc être une caractéristique conservée ayant subit une importante pression de sélection et qui pourrait refléter l’importance de la disposition de ces gènes les uns par rapport aux autres, nécessaire à l’obtention d’une régulation correcte (Mann, 1997; Duboule, 2007). Cependant, dans certains phyla de bilatériens (urochordé, nématode, arthropode, échinoderme, plathelminthe) il existe des espèces chez lesquelles le complexe HOX s’est scindé (Ferrier and Holland, 2002; Edvardsen et al., 2005; Pierce et al., 2005; Cameron et al., 2006). Enfin, il existe encore de nombreuses espèces, notamment chez les lophotrochozoaires, pour lesquelles des fragments de gènes Hox ont pu être isolés, mais où l’organisation en complexe n’a pas été démontrée (Bayascas et al., 1997; de Rosa et al., 1999; Orii et al., 1999; Nogi and Watanabe, 2001). Les premiers gènes Hox de cnidaires ont été isolés chez Sarsia (hydrozoaire) dans les années 90 (Murtha et al., 1991). Depuis de nombreux autres ont été caractérisés dans le phylum des cnidaires et l’étude de leur liaison génétique est rendue plus facile depuis le séquençage des génomes de Nematostella (Sullivan et al., 2006; Putnam et al., 2007) et d’Hydra magnipapillata (voir I.2.5.). En revanche aucun gène Hox n’a été caractérisé dans le phylum des spongiaires. Les gènes Hox sont donc très conservés au cours de l’évolution depuis les cnidaires jusqu’au bilatériens, mais leur organisation en complexe n’a pas forcément été conservée. L’étude du nombre et de l’organisation des gènes Hox chez les cnidaires permet d’inférer l’histoire évolutive de ces gènes. C’est la démarche que nous avons suivie dans le chapitre 1 (II.1.).
I.2.3.2. La colinéarité spatiale et temporelle et la prévalence postérieure
Dans les complexes HOX de vertébrés, tous les gènes sont transcrits à partir du même brin d’ADN. Ainsi chaque complexe est défini par une orientation générale 5’ vers 3’ correspondant à la direction de la transcription. Le nombre et l’organisation de ces complexes sont très similaires d’un vertébré à un autre (Izpisua-Belmonte et al., 1991; van der Hoeven et al., 1996). Chez la souris et chez la drosophile, ces gènes sont exprimés dans des domaines spécifiques, parfois chevauchants, le long de l’axe antéro-postérieur (Gaunt, 1988; Dolle and Duboule, 1993; Duboule and Morata, 1994; Krumlauf, 1994) suivant une colinéarité spatiale (Lewis, 1978; Duboule and Dolle, 1989; Graham et al., 1989): la position des gènes Hox le long du chromosome reflète leur domaine d’expression antérieur le long de l’axe antéro-postérieur au sein d’un
32 même tissu (système nerveux central et périphérique, dérivés mésodermiques, système digestif et urogénital). Ainsi les gènes localisés le plus en 3’ s’expriment le plus antérieurement alors que les gènes les plus en 5’ s’expriment le plus postérieurement (Figure 14). Cette propriété est conservée chez la souris, le poulet et la drosophile (Sundin and Eichele, 1992; Gaunt, 1994).
Chez les vertébrés, la colinéarité spatiale est associée à une colinéarité temporelle. L’activation successive des gènes Hox au cours de développement embryonnaire reflète l’ordre de ces gènes le long du complexe. Ainsi, les gènes situés à l’extrémité 3’ sont activés plus précocement au cours du développement embryonnaire que les gènes situés en 5’. Il s’en suit une activation séquentielle des gènes adjacents situés plus en 5’ (Dolle et al., 1989; Deschamps and van Nes, 2005). Une autre caractéristique des gènes Hox chez les vertébrés est qu’ils répondent à la règle de la prévalence posterieure. Les gènes postérieurs inhibent les gènes antérieurs dans les régions où ils se chevauchent. C’est ainsi que les gènes Hox agissent dans la limite antérieure de leur domaine d’expression (Duboule and Morata, 1994; Mann, 1997). Le rôle des gènes Hox fait l’objet de nombreuses recherches, la plupart utilisant la souris et la drosophile comme système modèle où de nombreuses lignées transgéniques pour les gènes Hox sont en effet disponibles (Wellik and Capecchi, 2003; Tarchini and Duboule, 2006; Tschopp et al., 2009). Des études fonctionnelles portant sur ces gènes dans des phyla plus ancestraux comme les cnidaires apparaissent comme un prérequis pour mettre en évidence les modes de régulation ancestraux conservés chez les bilatériens.
I.2.3.3. Les gènes Hox fonctionnent comme des gènes sélecteurs
Chez les organismes à symétrie bilatérale, les gènes Hox apparaissent comme les régulateurs clés spécifiant la diversité régionale morphologique le long de l’axe antéro-postérieur (Pearson et al., 2005) et ceci chez des espèces animales présentant des aspects morphologiques très différents.
En tant que facteurs de transcription, l’expression des gènes Hox est gouvernée par les gènes activateurs et agissent eux-mêmes comme gènes sélecteurs. Leurs fonctions dépendent donc de l’activation de différents groupes de gènes en aval (Hueber and Lohmann, 2008). Ceux-ci peuvent aussi bien être d’autres facteurs de transcription comme d’autres gènes à homéoboîte, des acteurs appartenant à des cascades de signalisation ou pour la plupart directement des gènes réalisateurs conduisant des fonctions cellulaires (prolifération, apoptose, différenciation, polarité
33 cellulaire, croissance cellulaire) (Foronda et al., 2008; Hueber and Lohmann, 2008; Rogulja-Ortmann and Technau, 2008).
En contrôlant directement des gènes réalisateurs, les gènes Hox fournissent une identité unique aux cellules dans les différentes régions du corps des animaux où ils s’expriment. Leur contribution aux grandes voies de signalisation les place par ailleurs comme faisant partie intégrante du réseau de gènes qui contribue aux modifications de structures homologues et à la création de nouveaux organes (Foronda et al.,
2008).
I.2.3.4. Fonction au cours de la mise en place du système nerveux
Les gènes Hox participent à la mise en place du système nerveux. Cette fonction est conservée au cours de l’évolution et ils participent à l’établissement d’une identité neuronale régionalisée (Gavalas et al., 1998; Hirth et al., 1998; Studer et al., 1998). Les gènes Hox s’expriment d’une manière similaire, dans un ordre défini selon l’axe antéro-postérieur, dans le système nerveux en développement des insectes et des vertébrés (Figure 15). Toutefois, la fonction des gènes Hox lors de la mise en place du cerveau des vertébrés se restreint à la partie postérieure.
Figure 15: Shéma simplifié comparatif des domaines d’expression des gènes Hox dans le système nerveux central de la drosophile et de la souris. a) Domaines d’expression des gènes homéotiques du complexe Antennapedia et Bithorax dans le système nerveux central de la drosophile : lab (labial), pb (proboscipedia), Dfd (Deformed), Scr (Sex combs reduced), Antp (Antennapedia), Ubx (Ultrabithorax), abdominal-A (abd- A) et Abdominal-B (Abd-B). b) Expression des gènes homéotiques Hoxb-1, Hoxb-2, Hoxb-3, Hoxb-4, Hoxb-5, Hoxb-6, Hoxb- 7, Hoxb-8 et Hoxb-9 dans le système nerveux central de l’embryon de souris. Abbreviations: b1, protocerebrum; b2, deutocerebrum; b3, tritocerebrum; s1, mandibular neuromere; s2, maxillary neuromere; s3, labial neuromere; T, telencephalon; D, diencephalon; M, mesencephalon; 1–8, rhombomeres 1–8; wt, wild type. Schéma modifié d’après (Hirth and Reichert, 1999).
34 Lors du développement du cerveau et de la chorde nerveuse ventral de la drosophile, les gènes Hox s’expriment dans des domaines particuliers. Souvent, la limite antérieure de leur domaine d’expression coïncide avec les frontières des compartiments neuromériques (Hirth and Reichert, 1999). Chez les vertébrés, les gènes Hox sont exprimés avant la formation des rhombomères dans le cerveau postérieur et dans la moelle épinière du système nerveux en développement. Au cours de l’embryogénèse, les domaines d’expression se restreignent progressivement en des localisations plus spécifiques. Les gènes Hox orthologues partagent donc une expression et une fonction similaire chez les bilatériens soutenant l’idée d’une origine commune du système nerveux central (Lichtneckert and Reichert, 2005).
La régionalisation postérieure du cerveau par les gènes Hox s’accompagne d’une régionalisation du cerveau antérieur par les gènes otd/Otx et ems/Emx (nommés gènes gap chez la drosophile) tandis que la régionalisation du cerveau moyen repose sur les gènes Pax2/5/8. Ce plan de base d’une régionalisation en trois parties du cerveau embryonnaire a été rapporté chez les vertébrés, mais également chez les urochordés, les hémichordés et les arthropodes (Wada et al., 1998; Holland and Holland, 1999; Wada and Satoh, 2001; Hirth et al., 2003; Lowe et al., 2003). Ce mode d’organisation était donc vraisemblablement déjà présent chez l’ancêtre commun des bilatériens.
I.2.4. Rôle des gènes ParaHox
I.2.4.1. Phylogénie des gènes ParaHox
Les séquences des gènes ParaHox sont hautement similaires à celles des gènes Hox, mais aucuns d’entre eux ne fait partie des complexes HOX. Brooke et al. ont montré que les trois familles de gènes ParaHox i.e Gsx/Gsh, Xlox/Pdx et Cdx ont des affiliations phylogénétiques différentes avec les sous-familles de gènes Hox: respectivement avec les gènes « antérieurs », les gènes du groupe 3 et les gènes « postérieurs ». Notre analyse phylogénétique suggère un autre point de vue concernant l’origine et la descendance des gènes Hox et ParaHox comme il sera discuté plus loin (II.1. Chapitre 1). Les trois gènes ParaHox ont pu être identifiés pour la première fois chez l’amphioxus où on les trouve en une seule copie (Brooke et al., 1998). Chez le nématode, il n’existe que le gène Cdx (Ruvkun and Hobert, 1998) et la drosophile ne possède pas le gène Pdx (Mlodzik et al., 1988; Weiss et al., 1998). En revanche, les trois gènes ParaHox ont pu être identifiés chez des siponcles, des annélides et un mollusque (Ferrier and Holland, 2001a; Frobius and Seaver, 2006).
35 L’absence de la série complète des gènes ParaHox chez la drosophile et le nématode représenterait donc un caractère dérivé secondaire à ces protostomes en particulier. Chez les tétrapodes, il existe deux copies du gène Gsx (Gsh1 et Gsh2), une simple copie du gène Pdx (Xlox/Pdx) et trois gènes Cdx (Cdx1, Cdx2 et Cdx4) (Coulier et al., 2000; Pollard and Holland, 2000; Ferrier et al., 2005; Mulley et al., 2006; Illes et al., 2009).
I.2.4.2. Gènes ParaHox en complexes et notion de complexe « ProtoHOX » ancestral
Chez l’amphioxus les gènes ParaHox s’organisent, comme les gènes Hox, en un complexe (Brooke et al., 1998). Les gènes Gsx et Xlox sont adjacents et orientés dans le même sens, suivis par le gène Cdx orienté dans le sens opposé (Ferrier et al., 2005). Chez les tétrapodes, il n’existe qu’un complexe ParaHOX, rapporté chez le xénope, le marsupial, la souris et l’homme comprenant les gènes Gsh1, Pdx et Cdx2 (Coulier et al., 2000; Furlong and Mulley, 2008; Illes et al., 2009). L’organisation génomique les orientations transcriptionelles sont conservées. D’une manière intéressante, la taille des complexes varie considérablement entre les groupes, mais l’éloignement relatif des gènes les uns par rapport aux autres est conservé (Mulley et al., 2006; Furlong et al., 2007; Illes et al., 2009) (Figure 16).
Figure 16: Diagramme des complexes ParaHOX de l’amphioxus, de la souris, de l’humain et du xénope (pas dessinés à la même échelle). Les flèches indiquent l’orientation de la transcription des gènes. Le diagramme du complexe de xénope montre la structure des introns et des exons. Schéma tiré de (Illes et al., 2009).
Chez les poissons, on retrouve un complexe ParaHOX chez les actinoptérygiens à l’exception des téléostéens (Mulley et al., 2006; Furlong et al., 2007). Chez les urochordés et un échinoderme, les gènes ParaHox ne sont pas liés génétiquement
36 (Ferrier and Holland, 2002; Wada et al., 2003; Arnone et al., 2006; Furlong and Mulley, 2008).
L’affiliation des gènes ParaHox à des familles de gènes Hox ainsi que leur organisation en complexe suggère que les gènes Hox et ParaHox sont apparu suite à un évènement de duplication d’un complexe « ProtoHox » ancestral (Figure 17). Chacun des complexes résultant aurait par la suite subi des évènements de duplication et de pertes de gènes pour donner la configuration actuelle des complexes HOX et ParaHOX (Brooke et al., 1998).
Figure 17 : Origine des complexes HOX et ParaHOX déduite des liaisons génétiques entre les gènes et d’analyses phylogénétiques. Les complexes HOX et ParaHOX ont évolué à partir de deux complexes jumeaux générés par duplication. Les flèches horizontales indiquent la polarité de la colinéarité spatiale. (A, antérieure ; P, postérieur). Schéma pris de (Brooke et al., 1998).
De nombreuses tentatives cherchent à inférer la constitution originale du complexe « ProtoHOX » (Kourakis and Martindale, 2000; Garcia-Fernandez, 2005b; Chourrout et al., 2006). Nous proposons dans ce travail de thèse une hypothèse qui concilie les différentes données disponibles à ce jour (II.1. Chapitre 1). Les différentes copies des gènes ParaHox qui existent chez les tétrapodes sont apparues suite à des duplications d’un complexe ParaHOX ancestral. L’existence d’un seul complexe ParaHOX et d’un nombre variable d’orthologues dans chacune des familles implique que des pertes de gènes sont par la suite survenues. Les gènes qui ne se trouvent pas en complexe sont isolés sur des complexes dégénérés (Pollard and Holland, 2000; Ferrier et al., 2005; Illes et al., 2009).
37 I.2.4.3. Fonction des gènes ParaHox au cours du développement
L’étude de la fonction des trois gènes ParaHox chez les vertébrés et les invertébrés montre un rôle dans les processus de développement. Cependant ces gènes semblent plus particulièrement impliqués dans la spécification des tissus et des types cellulaires que dans la détermination des axes. Par exemple le gène Gsx est impliqué dans la spécification de l’identité neuronale (voir I.3.2.2) et joue par ailleurs un rôle dans la mise en place du cerveau et du système nerveux (Szucsik et al., 1997; Toresson and Campbell, 2001; Kriks et al., 2005). Son expression a été rapportée à deux reprises dans des dérivés endodermaux (Rosanas-Urgell et al., 2005; Illes et al., 2009) et ne semble pas conservée au cours de l’évolution. Il pourrait donc s’agir d’une fonction dérivée. Une vue plus détaillée des patrons d’expression des orthologues des gènes Gsx dans le règne animal est discutée dans le chapitre 2 (II.2.). L’analyse fonctionnelle de ce gène chez les bilatériens n’a pour le moment été mennée qu’au travers de lignées mutantes de souris (Li et al., 1996; Szucsik et al., 1997) et de drosophile (Weiss et al., 1998). Le travail de doctorat de Marijana Miljkovic-Licina, dans notre laboratoire, a montré l’implication de ce gène dans la mise en place du système nerveux chez l’hydre (Miljkovic-Licina et al., 2007) (voir I.3.3.). Dans le chapitre 2 (II.2.) nous avons étudié la fonction de l’orthologue de Gsx chez Nematostella (Anthox2) dans la mise en place de son système nerveux en développement.
Pdx s’exprime dans la région centrale de l’intestin des vertébrés et des invertébrés (Wright et al., 1989; Wysocka-Diller et al., 1995; Offield et al., 1996; Brooke et al., 1998; Frobius and Seaver, 2006) et est impliqué dans le développement du pancréas et du duodenum rostral des vertébrés (Jonsson et al., 1994; Offield et al., 1996). Quant aux gènes Cdx, ils s’expriment dans l’intestin et le neuroectoderme postérieurs chez les vertébrés, les arthropodes et l’amphioxus (Duprey et al., 1988; Gamer and Wright, 1993; Marom et al., 1997; Brooke et al., 1998; Guo et al., 2004). Chez les vertébrés, les différentes copies du gène spécifient par ailleurs l’identité axiale du squelette (Subramanian et al., 1995), suggérant un fonction dans la mise en place des régions postérieures de l’embryon.
Plus spécifiquement chez l’amphioxus, Gsx s’exprime dans la vésicule cérébrale, Pdx s’exprime transitoirement dans le tube neural et dans l’intestin présomptif et Cdx dans le tube neural et l’intestin postérieurs (Brooke et al., 1998). Brooke et al. proposent que les gènes ParaHox suivent, comme les gènes Hox, la règle de la colinéarité spatiale dans le tube neural et dans l’intestin. De plus, ces gènes sont activés dans un
38 ordre défini en commençant par le gène Cdx et en finissant par le gène Gsx. Ils pourraient donc être également régulés selon une colinéarité temporelle (Osborne et al., 2009).
I.2.5. Les gènes Hox/ParaHox chez les cnidaires
I.2.5.1. Familles Hox/ParaHox représentées chez les cnidaires et leur liaison génétique
Aujourd’hui, les analyses phylogénétiques tirent avantage du nombre grandissant de séquences disponibles provenant de cnidaires (Kortschak et al., 2003; Sullivan et al., 2006; Chiori et al., 2009) ou encore de gènes clonés par pêche ciblée au moyen d’amorces dégénérées. La combinaison de ces analyses a permis de mettre en évidence des gènes appartenant sans ambiguïté aux familles Hox antérieures PG1 et PG2 et aux familles ParaHox Gsx et Cdx. Par ailleurs, plusieurs séquences sont proposées comme affiliées au groupe PG9 et une séquence de Nematostella (Xlox/Cdx) est proposée comme affiliée à la fois aux familles ParaHox Pdx et Cdx (Schierwater et al., 1991; Schummer et al., 1992; Finnerty and Martindale, 1997; Galliot et al., 1999; Gauchat et al., 2000; Hayward et al., 2001; Yanze et al., 2001; Finnerty et al., 2004; Chourrout et al., 2006; Ryan et al., 2006; Ryan et al., 2007; Chiori et al., 2009). Dans l’analyse phylogénétique présentée dans le chapitre 1 (II.1.) nous avons pu consolider certaines hypothèses et trancher des incertitudes.
Chez Nematostella, sept gènes Hox et deux gènes ParaHox sont recensés : deux représentants de la famille Hox antérieure PG1 (Anthox6-HoxA et Anthox6a-HoxB), trois représentants de la famille Hox antérieure PG2 (Anthox7-HoxC, Anthox8-HoxDa, Anthox8a-HoxDb) et deux proposés comme affiliés à la famille Hox postérieure PG9. Les gènes de chacune de ces familles, représentées chez Nematostella par au moins deux représentants, sont très probablement issus d’évènements de duplication (Ryan et al., 2007). Par ailleurs, Nematostella possède également deux gènes ParaHox, Anthox2 affilié à la famille Gsx et Xlox/Cdx. Parmi ces gènes, Anthox6, Anthox8, Anthox8a, Anthox7 se trouvent sur un complexe de sept gènes au total (Rough-HlxB9- Anthox6-Evx-Anthox8b-Anthox8a-Anthox7) et les deux gènes ParaHox se trouvent également liés (Chourrout et al., 2006). Chez Hydra magnipapillata, six gènes Hox (Hoxa, Hoxb, Hoxc1, Hoxc2, Hoxc3, Hoxd), et un gène ParaHox (Gsx/cnox2) sont recensés et seulement deux des gènes Hox sont liés génétiquement (Hoxc3-Hoxc1) (Chourrout et al., 2006).
39 I.2.5.2. Patron d’expression des gènes Hox chez les cnidaires
Figure 18: Expression des gènes Hox et TGF! chez Nematostella . A) Homologie provisoire des gènes Hox de Nematostella basée sur des analyses phylogénétiques d’homéodomaines. Les paralogues de vertébrés sont numérotés de 1 à 13. Les paralogues d’arthropode sont nommés avec la terminologie de la drosophile (lab, labial; pb, proboscipedia; zen, zerknullt; Dfd, Deformed; scr, sex combs reduced; ftz, fushi tarazu; Antp, Antennapedia; Ubx, Ultrabithorax; abd-A, abdominalA; et AbdB, Abdominal. B) Patron d’expression des gènes le long de l’axe oral-aboral et l’axe directionel. La composition de la couche germinale est montrée en section longitudinale. Pour simplifier la représentation de l’axe du corps, le pharynx est dessiné éversé et les mésentéries ne sont pas représentées. Les domaines d’expression des cinq gènes Hox s’étendent sur la quasi-totalité de l’axe oral-aboral. Anthox1a, Anthox7, Anthox8 sont restreints d’un seul coté de l’axe directionel. De même que les deux gènes de la famille TGF!, Dpp et GDF5-like. Ici ne sont représentés que les domaines d’expression asymétrique de chacun des gènes. Dpp est exprimé dans l’ectoderme pharyngial sur le coté de l’axe directionnel à l’opposé du domaine d’expression d’Anthox1a, Anthox7 et Anthox8. GDF5- like s’exprime dans l’endoderme du même coté qu’Anthox1a, Anthox7 et Anthox8. Schémas pris de (Finnerty et al., 2004).
Des analyses de patrons d’expression réalisés chez Nematostella, ont montré que les gènes Hox putatifs étaient exprimés sur la totalité de l’axe oral-aboral dans des domaines distincts c’est-à-dire le pharynx, la colonne du corps et l’extrémité aborale subdivisant ainsi l’axe primaire en régions distinctes pendant l’embryogenèse et le développement larvaire. Cinq d’entre eux (Anthox7, Anthox8, Anthox8a, Anthox6a, Anthox1a) présentent de plus une expression différentielle le long de l’axe secondaire (Figure 18). Ces données suggèrent l’importance de ces gènes au cours du développement embryonnaire dans la mise en place des axes primaires et secondaires du corps de Nematostella (Finnerty et al., 2004; Ryan et al., 2007). De plus, si l’on corrèle les domaines d’expression des gènes Hox de Nematostella avec la position de leur groupe paralogue dans les complexes connus (trans-colinéarité) (Duboule, 2007), alors le gène Anthox6/antérieur s’exprime au pôle oral du polype et
40 le gène Anthox1/postérieur s’exprime au pôle aboral. Ce patron d’expression évoque une colinéarité spatiale (Finnerty et al., 2004).
En ce qui concerne les gènes ParaHox, Gsx/Anthox2 est exprimé au futur pôle orale chez la larve et dans les tentacules du polype métamorphosé dans des cellules suggérées comme étant des progéniteurs neuronaux et Xlox/Cdx s’exprime au stade planula et polype le long de la ligne médiane ventrale (relativement à l’axe secondaire). D ‘une manière intéressante, chez Eleutheria les gènes ParaHox Cnox4- Ed (relaté à la famille Cdx) et Cnox2-Ed (relaté à la famille Gsx) sont exprimé da manière opposée dans le polype (Jakob and Schierwater, 2007).
L’existence d’une corrélation entre le patron d’expression des gènes Hox et ParaHox avec la position axiale chez les cnidaires, selon une colinéarité, est un sujet de controverse. Les études phylogénétiques et de patron d’expression ont poussé Kamm et al. à soutenir l’hypothèse que le système Hox serait une innovation bilatérienne (Kamm et al., 2006). Les analyses faites par Ryan et al. chez Nematostella montrent que les gènes Hox sont exprimés dans des territoires distincts le long des axes primaires et secondaires suggérant qu’ils pourraient jouer un rôle dans la mise en place du patron des axes du corps. Ces auteurs soutiennent l’idée de « vrais » gènes Hox et ParaHox ainsi que d’un « code Hox » rudimentaire chez l’ancêtre commun des cnidaires et des bilatériens (Finnerty et al., 2004; Ryan et al., 2007). Des expériences fonctionnelles sont nécessaires pour comprendre si les gènes Hox sont indispensables à la mise en place des axes de Nematostella et plus généralement des cnidaires.
I.2.6. Approche évolution-développement
La mise en évidence des gènes Hox d’abord chez la drosophile puis chez les vertébrés (McGinnis et al., 1984a; Duboule and Dolle, 1989; Akam, 1995) a montré qu’il existe des mécanismes développementaux communs partagés entre des phyla éloignés phylogénétiquement et qui possédent des plans d’organisation très différents (McGinnis et al., 1984a; Duboule and Dolle, 1989; Slack et al., 1993; Akam, 1995; Gilbert et al., 1996; Rokas et al., 2005). De cette découverte a émergé une nouvelle discipline appelée l’ «evo-devo » pour Evolutionary developmental biology (Hall, 1992). Par le biais de la génomique, de la phylogénie, de la biologie moléculaire et de la génétique, l’evo-devo vise à comparer les processus moléculaires impliqués dans les modes de développement animaux (mise en place des axes dorso-ventral, antéro- postérieur, neurogénèse) afin d’inférer des mécanismes ancestraux communs à tous les organismes, donc fondamentaux et présents chez leur ancêtre commun.
41 La neurogénèse : une innovation clé au cours de l’évolution
L’évolution du règne animal a été marquée par une innovation fondamentale : l’acquisition des cellules neurosensorielles. Organisé en un système nerveux diffus chez les cnidaires, le tissu formé par ces cellules est plus complexe chez les bilatériens où il forme un système nerveux centralisé. La fonction du système nerveux est de percevoir et de relayer des informations provenant de l’environnement. Le réseau nerveux a pour fonction de transmettre des informations perçues par les cellules sensorielles à des cellules spécifiques comme les cellules musculaires par exemple. Il n’est pas encore clairement déterminé si les différents types de systèmes nerveux ont une origine unique. Une étude comparative des caractéristiques cellulaires et moléculaires du système nerveux, de son fonctionnement et de son développement chez les cnidaires et les bilatériens est nécessaire à la compréhension de son origine et de son évolution. Les caractères partagés entre les cnidaires et les bilatériens sont alors considérés comme ancestraux (Kortschak et al., 2003). Dans la revue présentée dans le chapitre 3 (II.3.), nous nous sommes attachés à recenser les acteurs fonctionnant lors de la neurogénèse chez les bilatériens, retrouvés actifs également chez les cnidaires. Par ailleurs dans le chapitre 2 (II.2.) nous nous sommes intéressés plus spécifiquement à l’un de ces régulateurs clés chez Nematostella.
I.3.1. Inversion de l’axe dorso-ventral
La différence fondamentale entre le système nerveux central des vertébrés et celui des invertébrés est sa position selon l’axe dorso-ventral. Chez les vertébrés, les urochordés et les céphalochordés, le système nerveux est dorsal tandis qu’il est ventral chez les invertébrés (arthropodes, annélides, mollusques). De ces observations a découlé l’hypothèse d’une acquisition indépendante du système nerveux chez les invertébrés et les vertébrés. Cependant, Geoffrey St-Hilaire suggère en 1822 que l’axe dorso-ventral aurait pu s’inverser au cours de l’évolution (Geoffroy St-Hilaire, 1822) (Figure 19). Cette hypothèse fut confortée par des données génétiques et moléculaires qui montrent que les processus de développement clés qui gouvernent la neurogénèse (prolifération, régionalisation, et spécification) sont conservés entre insectes et vertébrés et gouvernés par des gènes orthologues (Arendt and Nubler-Jung, 1999; Reichert and Simeone, 1999; Sprecher and Reichert, 2003). Ces observations favorisent l’hypothèse d’un ancêtre commun aux bilatériens pourvu d’un système nerveux organisé (De Robertis and Sasai, 1996).
42
Figure 19 : Organisation générale de l’Urbilateria. L’Urbilateria correspond à un animal archétype, dernier ancêtre commun des protostomes et des deutérostomes. Il est représenté sur cette image comme un animal benthique arborant des yeux, un système nerveux, de petit appendice et un blastopore ouvert. L’endoderme est représenté en rouge, le système nerveux central en bleu foncé et l’ectoderme en bleu clair. Schéma pris dans (De Robertis, 2008).
I.3.2. Développement du patron neuronal chez les bilatériens
Les neurones et les cellules gliales (astrocytes et olygodendrocytes) qui composent le système nerveux centralisé des bilatériens sont générés à partir de progéniteurs multipotents dans le tube nerveux et leur mise en place se fait d’une manière contrôlée dans le temps et dans l’espace. Elle repose sur l’activité de cascades de signalisation et de différentes classes de facteurs qui contrôlent la détermination du territoire nerveux ainsi que la différentiation des cellules progénitrices et la spécification de leur identité.
Deux exemples d’anatomie moléculaire comparée sont classiquement étudiés en ce qui concerne la mise en place du système nerveux chez les embryons de bilatériens. D’une part, l’induction neurale controlée par la cascade de signalisation BMP (Bone Morphogenetic Proteins) résulte en une partition de l’ectoderme embryonnaire en un domaine neural et un domaine épidermique le long de l’axe dorso-ventral (Mieko Mizutani and Bier, 2008). D’autre part, les gènes d’identité neurale (dont le gène ParaHox Gsx fait partie) appartenant à la famille des gènes à homéoboîte définissent trois régions non-chevauchantes au sein du neuroectoderme qui se répartissent le long de l’axe dorso-ventral (Cornell and Ohlen, 2000). La cascade de signalisation BMP est également impliquée dans la sous-division du neuroectoderme
43 (Mizutani et al., 2006). Suite à la spécification des territoires, différentes classes de facteurs de transcription incluant les familles à homéodomaines et bHLH (protéines proneurales) agissent selon un code moléculaire pour diriger la différentiation et la spécification des cellules nerveuses (Guillemot, 2007).
I.3.2.1. L’induction neurale : la cascade de signalisation BMP
La cascade de signalisation BMP
Les protéines BMP2 et 4 de vertébrés (DPP chez la drosophile) sont des ligands extracellulaires diffusibles sécrétés, appartenant à la famille des Transforming Growth Factors (TGF!). Sous forme d’homo- ou d’hétérodimères, ils se lient et activent des récepteurs tétramériques de type sérine-thréonine kinase, présents à la surface de la membrane cellulaire. Ensuite, des protéines de type SMAD chez les vertébrés (MAD chez la drosophile) subissent des phénomènes de phosphorylation leur permettant de se grouper en complexe pour entrer dans le noyau et altérer l’expression des gènes. Pendant qu’un complexe dimérique active l’expression des gènes épidermiques, dans le même temps, un complexe trimérique réprime l’expression des gènes neuronaux. Cette voie de signalisation est modulée par des inhibitions intra- et extracellulaires (antagonistes des protéines BMPs).
Patron d’expression des BMP et de leurs antagonistes
Figure 20: Conservation du réseau de signalisation Chordin-BMP. Bien que les réseaux de signalisation Chordin-BMP soient conservés, il y a eu un phénomène d’inversion des axes dorso-ventral entre la drosophile et le xénope. A) Chez le xénope, Chordin est exprimé du côté dorsal et BMP4 à l’opposé coté ventral (image Hojoon X. Lee). B) Chez la drosophile, Dpp est exprimé dans la partie dorsale de l’embryon (bleu) et Sog est ventral (marron) dans l’ectoderme (image Ethan Bier. C) Réseau de protéines sécrétées et conservées à l’origine de la mise en place de l’axe dorso-ventral chez le xénope et la drosophile. Schéma tiré de (De Robertis, 2008).
44 BMP4 chez le xénope et Dpp, son orthologue chez la drosophile, définissent l’ectoderme épidermique et sont exprimés avec des patrons inversés dans l’embryon. Il en est de même pour leur antagoniste Chordin (CHD) et Short gastrulation (Sog) dont l’expression est complémentaire à BMP et Dpp respectivement et qui sont à l’origine de la détermination du territoire nerveux (De Robertis and Sasai, 1996) (Figure 20). Ces données corroborent l’hypothèse d’une inversion des axes au cours de l’évolution.
Ectoderme versus neuroectoderme
L’expression exclusive des protéines BMPs dans l’ectoderme active l’expression des gènes ectodermaux et réprime l’expression des gènes neuronaux (De Robertis and Sasai, 1996). Dans le futur territoire neuronal, les antagonistes de la voie BMP (chordin-CHD chez les vertébrés et Short gastrulation-SOG chez la drosophile) se lient aux ligands en empêchant leur accès aux récepteurs. Ainsi, les cellules contenant les protéines antagonistes des BMPs (par expression ou par diffusion) voient les gènes neuronaux s’exprimer et adoptent un destin neuronal correspondant à un état par défaut (Figure 21).
Figure 21: L’induction neurale chez la mouche et les vertébrés. Dans l’épiderme embryonnaire, le signal Decapentaplegic (DPP) chez la mouche (bone morphogenetic protein 4 (BMP4) chez les vertebrés) active l’expression des gènes épidermiques et réprime l’expression des gènes neuraux. DPP (BMP4) peut diffuser ventralement dans le neuroectoderme, mais son autoactivation est empêchée par des antagonistes extracellulaires (Short gastrulation-SOG chez la mouche, chordin-CHD chez les vertébrés) ou par des répresseurs intracellulaires. Ainsi l’inactivité de DPP (BMP) dans ce domaine entraîne les cellules à se développer en un tissu neuroectodermique correspondant à leur état par défaut. Schéma pris dans (Mieko Mizutani and Bier, 2008).
45 I.3.2.2. Les gènes d’identité neurale : vnd/Nkx2.2, ind/Gsh, msh/Msx1/2
Patron d’expression et fonction
La mise en place des cellules souches neuronales chez la drosophile, appelées neuroblastes, correspond à l’étape d’initiation du développment du système nerveux embryonnaire. Les différents types de neuroblastes se répartissent en trois colonnes distinctes le long de l’axe dorso-ventral du neuroectoderme. Elles sont définies par l’expression de trois facteurs de transcription différents codés par les gènes d’identité neurale. Ceux-ci, ventral nervous system defective (vnd)/NK2 transcription factor related (Nkx2.2), intermediate neuroblasts defective (ind)/ genomic screen homeobox (Gsh), and muscle segment homeobox (msh)/MSH homeobox (Msx), appartiennent à la famille des gènes à homéoboîte. Chez les bilatériens, leurs patrons d’expression sont conservés relativement les uns par rapport aux autres et permettent la subdivision du système nerveux en trois domaines le long de l’axe dorso- ventral. Ainsi, suivant l’induction neurale, le gène vnd/Nkx2.2 s’exprime ventralement dans les cellules adjacentes à la ligne médiane du système nerveux central, ind/Gsh s’exprime dans le domaine intermédiaire et le gène msh/Msx s’exprime dans le domaine dorsale (Figure 22) (Arendt and Nubler-Jung, 1999; Cornell and Ohlen, 2000). Chez la drosophile, ces gènes sont appelés gènes d’identité neurale car ils sont nécessaires à la détermination du destin neuronal des cellules (Isshiki et al., 1997; McDonald et al., 1998; Weiss et al., 1998). Ils donnent naissance à trois rangées primaires de neuroblastes qui se différencieront en autant de progéniteurs neuronaux (Mieko Mizutani and Bier, 2008).
Régulation par la cascade BMP
Mieko Mizutani et al. ont montré que chez la drosophile de faibles quantités de BMP diffusent vers le neuroectoderme et contribuent à partitionner ce territoire en trois domaines. Cette diffusion réprime les gènes neuronaux d’une manière dose- dépendante. Ainsi les gènes neuronaux les plus sensibles à la répression par la voie BMP s’expriment dans les régions les plus ventrales du neuroectoderme (vnd et ind), le plus loin de la source de BMP épidermique, tandis que les gènes qui y sont moins sensibles peuvent s’exprimer plus proche de la source dans des régions plus dorsales du neuroectoderme (msh). Ainsi un seuil de diffusion des protéines BMP vers le neuroectoderme contribue à l’organisation de l’axe nerveux chez la drosophile en réprimant les gènes d’identité neurale. Ce phénomène conduit à la mise en place du patron d’expression de ces gènes (Mizutani et al., 2006). Néanmoins, les quantités de
46 BMP qui diffusent dans cette région ne sont pas suffisantes à l’induction des gènes épidermiques (Biehs et al., 1996) (Figure 22).
Figure 22 : Position dorso-ventral des domaines d’expression des gènes d’identité neurale. A) Représentation schématique d’une section du tube nerveux de souris. C) Représentation schématique d’une section du neuroectoderme de drosophile. En bleu, les domaines d’expression de Nkx2.1 (souris) et vnd (drosophile), en vert les domaines d’expression de Gsh et Pax6 (souris) et ind (drosophile) en rouge les domaines d’expression de Msx (souris) et msh (drosophile). B) Seulement la moitié du corps est représentée pour les vertébrés et les arthropodes dans la section schématique dorso-ventral. Pour chacun des groupes animaux, le côté dorsal est situé vers le haut. Le produit de sécrétion Dpp/BMP4 forme un gradient dorso-ventral inversé chez les vertébrés par rapport à la drosophile. Ils sont antagonisés par Sog/Chordin dans la région de l’embryon qui adoptera un potentiel neurogénique. Cette région est par la suite divisée par une série de gènes à homéoboîte en trois domaines neurogéniques : médiant (vnd/Nkx2), intermédiaire (ind/Gsh) et latéral (msh/Msx). Schémas tirés de (Lichtneckert and Reichert, 2005; Mieko Mizutani and Bier, 2008).
Dominance ventrale
Des expériences de perte de fonction et de dérégulation d’expression chez la drosophile ont montré que ces gènes se régulent entre eux. Ainsi les gènes exprimés ventralement inhibent les gènes exprimés plus dorsalement (McDonald et al., 1998; Weiss et al., 1998; Cowden and Levine, 2003) (Figure 23). Ces mécanismes de répression sembleraient conservés chez les vertébrés (McMahon, 2000; Muhr et al., 2001; Jacob and Briscoe, 2003).
Ainsi, le patron d’expression des gènes d’identité neuronale est soumis à une double régulation aboutissant à la mise en place de territoires cellulaires définis, adjacents
47 mais non-chevauchants : d’une part, l’activité inhibitrice de la cascade BMP qui est dose-dépendante, d’autre part, l’activité inhibitrice que les gènes d’identité neuronale exercent les uns sur les autres. Le même type d’activité organisatrice est rapporté dans la plaque neurale du poulet. Les cascades de signalisation BMP joueraient donc un rôle ancestral dans la mise en place du patron d’expression des gènes dans le neuroectoderme (Mizutani et al., 2006).
Figure 23 : A) Diagramme montrant l’expression de DPP dans l’ectoderme dorsal d’un embryon de mouche et l’expression des gènes d’identité neurale ventral nervous defective (vnd), intermediate neuroblasts defective (ind) et muscle segment homeobox (msh) dans le neuroectoderme.Il existe une cascade de régulation entre les gènes d’identité neurale ou vnd inhibe l’expression de ind et msh et ind inhibe l’expression de msh. B) Expression de Dpp (jaune), msh (rouge), ind (vert), et vnd (bleu) au stade blastoderme de l’embryon de mouche. Schémas et photographies tirés de (Mieko Mizutani and Bier, 2008).
I.3.2.3. Spécification spatiale et temporelle du destin nerveux
Différentes classes de facteurs de transcription spécifie le destin neuronal des cellules :
Les protéines progénitrices agissent sur le destin des progéniteurs neuronaux en division. Elles incluent les facteurs à homéodomaine des familles Pax, Nkx, Irx ainsi que Olig2 de la famille des bHLH, également reponsables de la mise en place du patron dorso-ventral et antéro-postérieur, ainsi que d’autres facteurs comme les protéines à homéodomaine de la famille LIM. Après cette étape, les progéniteurs multipotents ne peuvent produire qu’un seul type neuronal primaire (neurones ou astrocytes ou oligodendrocytes). Les protéines proneurales de la famille des bHLH (comme Mash1 (Asl1), Neurogenin 1-3, Math1 (Atoh1)) initient le programme de neurogénèse. Leur expression entraîne la sortie du cycle cellulaire des progéniteurs et la différentiation neuronale. La casacade de signalisation Notch est alors activée dans les progéniteurs adjacents. La famille des bHLH participe également au programme de différentiation neuronal dans les cellules post-mitotiques et des facteurs de transcription comme NeuroM (Neurod4) et NeuroD /Neurod1) sont activés par les protéines proneurales. Les protéines à homéodomaines comme Hb9 (Mnx1),
48 Mbh1 (Barhl2), Brn3 (Pou4f1) sont exprimées dans les cellules neurales post- mitotiques et contribuent au programme de différentiation du sous-type neuronal. Enfin des protéines inhibitrices des familles HLH (Id) et bHLH (Hes) ont des activités anti-neurogéniques et anti-oligodendrogéniques et inhibent les protéines proneurales et l’expression des gènes proneuraux. Au cours de leur différentiation les progéniteurs neuronaux migrent en dehors de la zone progénitrice vers des zones différenciées du tube neural où ils initient leur programme de différentiation final (Guillemot, 2007).
I.3.3. La neurogénèse chez les cnidaires : acteurs génétiques conservés
Les orthologues des gènes de bilatériens impliqués dans la mise en place de l’axe dorso-ventral sont exprimés de manière assymétrique chez les cnidaires généralement regardés comme des organismes à symétrie radiaire (Hayward et al., 2002; Finnerty et al., 2004; Matus et al., 2006a; Matus et al., 2006b; Rentzsch et al., 2006; Ryan et al., 2007). L’orthologue du gène Dpp de la famille des TGF! s’exprime de manière asymétrique le long de l’axe secondaire des embryons de Nematostella et d’Acropora ; en marge du blastopore. Cette expression asymétrique se retrouve également dans la larve de Nematostella oú Dpp est localisé dans l’ectoderme longeant le pharynx (Hayward et al., 2002; Finnerty et al., 2004) (voir I.2.5.2. Figure 18). L’expression asymétrique commune au cours du développement de deux anthozoaires, comparable à celle observée chez les bilatériens, suggère donc un rôle ancestral de Dpp dans la formation des axes. Cependant chez Nematostella, l’orthologue de BMP/Dpp colocalise avec Chordin/Sog (Matus et al., 2006b), antagoniste de Dpp chez la drosophile.
Des orthologues de gènes d’identité neuronale ont également été caractérisés chez les cnidaires. Le gène Gsh/ind a pu être identifié dans trois classes de cnidaires (Schierwater et al., 2002; Ball et al., 2004; Chiori et al., 2009), Msx/msh a été cloné chez deux espèces d’hydre et Acropora (Schummer et al., 1992; Gauchat et al., 2000; de Jong et al., 2006) et Nk2/vnd existe chez Nematostella et Acropora (de Jong et al., 2006). Le patron d’expression de ces trois gènes a été rapporté chez Acropora, ainsi que d’autres gènes impliqués dans la détermination des axes chez les bilatériens (Otx/otd, Emx/ems, Pax-3/7). Les gènes d’identité neuronales sont tous exprimés dans l’ectoderme dans des domaines restreints le long de l’axe oral-aboral, mais il est difficile de tirer des homologies entre les domaines d’expression de ces gènes chez les cnidaires et chez les bilatériens. Par ailleurs, la cascade d’inhibition agissant entre eux chez les bilatériens ne semble pas exister chez Acropora puisque que certains de ces gènes ont des domaines d’expression chevauchants. Il est donc difficile de mettre
49 en relation l’axe unique oral-aboral des cnidaires avec l’un de ceux existant chez les bilatériens (de Jong et al., 2006).
Si les axes de polarité des cnidaires ne sont pas homologues à ceux des bilatériens, alors l’étude des patrons d’expression des gènes fonctionnant lors de la mise en place du patron nerveux (Hox, BMP/DPP, gènes d’identité neurale) ne peuvent être révélateurs d’homologies. En revanche, le problème de l’origine commune du système nerveux chez les eumétazoaires peut être abordée différement à un niveau cellulaire. Par exemple, l’analyse du patron d’expression des orthologues de Gsx chez différents cnidaires suggère qu’ils s’expriment dans des précurseurs neuronaux ou dans des cellules nerveuses différenciées au cours du développement embryonnaire ou chez l’animal adulte (Hayward et al., 2001; Finnerty et al., 2003; Miljkovic-Licina et al., 2007; Ryan et al., 2007; Chiori et al., 2009). Une étude précise menée au niveau cellulaire dans notre laboratoire a montré que le gène Cnox2/Gsx s’exprime dans les précurseurs neuronaux ainsi que dans les nématoblastes et les neurones différenciés de l’hydre (Miljkovic-Licina et al., 2007). Par ailleurs Cnox2/Gsx parrait nécessaire au maintien du système nerveux apical et pourrait être un acteur précoce, nécessaire à sa restauration lors de la régénération (Miljkovic-Licina et al., 2007). Le gène Gsx semblerait donc avoir un rôle conservé des cnidaires aux bilatériens dans la détermination de la lignée neuronale. Nous avons voulu étudier sa fonction et sa régulation au cours du développement sexué chez les cnidaires (II.2. Chapitre 2). La seule autre étude fonctionnelle portant sur la mise en place des structures sensorielles chez Nematostella a montré que le gène FGF est nécessaire à la mise en place de l’organe sensoriel apicale de la larve (Rentzsch et al., 2008).
Considération évolutive
Les données rapportées illustrent la complexité du phylum des cnidaires. L’ancêtre commun aux cnidaires et aux bilatériens possédait probablement déjà tous les outils génétiques essentiels utilisés aujourd’hui aussi par les organismes complexes (Caroll et al., 2001). Après la divergence des cnidaires et des bilatériens, ces deux embranchements ont évolué indépendamment en utilisant un héritage génétique commun. La radiation des cnidaires a néanmoins été moins considérable que celle des bilatériens en terme de diversité et de variété morphologique du plan du corps (Darling et al., 2005). La grande variété de forme animale est attribuée à l’activité restreinte de différents gènes dans des goupes particuliers de cellules (Foronda et al., 2008).
50 L’étude des mécanismes contrôlant la mise en place du système nerveux dans différents phyla montre que des gènes orthologues s’expriment de manière similaire et ont des fonctions identiques chez des organismes éloignés phylogénétiquement. Des mécanismes génétiques conservés agissent dans la régionalisation du cerveau pendant son développement et suggèrent donc une origine monophylétique du cerveau chez les bilatériens (Lichtneckert and Reichert, 2005). L’ancêtre urbilateria utilisait déjà un système nerveux central complexe (Arendt and Nubler-Jung, 1996; De Robertis and Sasai, 1996; Hirth and Reichert, 1999). On retrouve par ailleurs certains de ces gènes chez les cnidaires où ils semblent également jouer un rôle dans la détermination de la lignée cellulaire neuronale comme le gène Gsx/cnox2 par exemple (Hayward et al., 2001; Miljkovic-Licina et al., 2007).
Objectifs du travail
Ce travail de doctorat s’inscrit dans une démarche de compréhension des processus évolutifs au travers de l’analyse des mécanismes développementaux. Nous avons cherché à mettre en évidence les gènes et les mécanismes acteurs de la neurogénèse, conservés au cours de l’évolution. Pour cela nous avons utilisé des espèces de cnidaires qui fournissent par leur position phylogénétique un modèle, qui en comparaison avec les bilatériens, permet d’inférer la condition ancestrale des eumétazoaires.
De nombreuses familles de gènes intervenant dans la mise en place du système nerveux chez les bilatériens sont en effet représentées chez les cnidaires. Les gènes Hox et ParaHox sont connus pour leurs rôles au cours du développement des bilatériens, en particulier dans la mise en place du système nerveux. Plus spécifiquement, le gène Gsx fonctionne dans la mise en place précoce des territoires neuronaux. Des analyses fonctionnelles et des patrons d’expression suggèrent l’implication de ce gène dans la lignée cellulaire neuronale chez les cnidaires également. Nous avons cherché à apporter une contribution, par le biais de trois approches différentes, pour répondre à trois questions essentielles (déjà exposées en introduction):
Quelle est l’histoire évolutive des gènes Hox et ParaHox ? (Résultats-Chapitre 1) (Quiquand et al., 2009).
La reconstruction de l’histoire évolutive des familles Hox et ParaHox a nécessité une approche phylogénétique mettant en confrontation des séquences provenant de nombreux phyla animaux. Nous avons mené cette étude alors que le génome de
51 Nematostella devenait accessible et que l’inventaire de ses séquences Hox/ParaHox donnait lieu à diverses publications (Chourrout et al., 2006; Ryan et al., 2006; Ryan et al., 2007). La totalité des séquences Hox/ParaHox de cnidaires disponibles à ce moment-là ont été intégrées dans notre analyse. Certaines séquences de cnidaires sont très dérivées, ce qui rend leur affiliation aux familles de bilatériens parfois difficile. De plus, quand ces séquences dérivées sont intégrées au jeu de données, elles ont tendance à brouiller le message phylogénétique. Pour faire face à ce problème, nous avons mis au point une méthode permettant de tester la solidité des nœuds des familles de gènes. En faisant varier systématiquement les jeux de données, avec ou sans les séquences que nous avions caractérisées comme perturbatrices, nous avons pu tirer le message phylogénétique le plus soutenu. Par ailleurs, nous avons examiné les affiliations des familles de gènes entre elles dans le but de déterminer leur origine. Puisque les supports à ces nœuds sont souvent bas, nous avons regardé les fréquences d’apparition des évènements d’affiliation des familles les unes par rapport aux autres. Cette analyse nous a permis de proposer un scénario relatif à l’histoire évolutive des complexes HOX et ParaHOX et d’inférer la composition ancestrale du complexe ProtoHOX chez l’ancêtre commun aux eumétazoaires. Notre hypothèse doit être confrontée avec celles faites par d’autres auteurs sur la composition de ce complexe ancestral (Chourrout et al., 2006; Ryan et al., 2007; Chiori et al., 2009).
La reconstitution du complexe ancestral ProtoHOX est un travail important permettant de répondre à différentes questions :
- Quels sont les gènes Hox et ParaHox qui sont ancestraux dans le règne animal et lesquels sont des innovations bilatériennes ?
- Comment se sont diversifiés les gènes Hox et ParaHox c’est-à-dire quelle est leur histoire évolutive?
- Quelle relation existe-t-il entre l’organisation des complexes HOX/ParaHOX et les différents plans d’organisation ?
Le gène ParaHox Anthox2/Gsx est-il un régulateur précoce de la neurogénèse lors du développement embryonnaire des cnidaires? (Résultats-Chapitre 2)
Chez les bilatériens, le gène Gsx (Gsh chez la souris, ind chez la drosophile) est impliqué dans la mise en place du patron dorso-ventral du système nerveux (Hsieh-Li et al., 1995; Szucsik et al., 1997; Weiss et al., 1998; Cornell and Ohlen, 2000; Toresson and Campbell, 2001; De Robertis, 2008; Mieko Mizutani and Bier, 2008) et
52 définit l’identité neuronale des cellules nerveuses (McDonald et al., 1998; Weiss et al., 1998; Kriks et al., 2005; Mizuguchi et al., 2006). Les orthologues du gène Gsx sont très bien conservés au cours de l’évolution. De plus, les analyses du patron d’expression de ce gène (Hayward et al., 2001; Finnerty et al., 2003; Ryan et al., 2007; Chiori et al., 2009) ainsi qu’une étude fonctionnelle menée dans notre laboratoire (Miljkovic-Licina et al., 2007) montrent que chez les cnidaires également, l’orthologue de Gsx est exprimé dans la lignée cellulaire neuronale et est essentiel à la mise en place du réseau nerveux pendant la régénération de l’hydre ainsi qu’à son maintien dans les conditions homéostatiques. Nous avons tiré parti des nombreux avantages fournis par Nematostella, en particulier l’accessibilité à la reproduction sexuée, pour étudier la fonction du gène Anthox2/Gsx dans la mise en place de son système nerveux au cours du développement. Plusieurs outils génétiques, dont certains ont été mis au point par notre laboratoire pendant mon travail de doctorat, ont permis d’étudier la fonction et la régulation d’Anthox2/Gsx (injection de morpholinos et de gènes rapporteurs).
L’étude du gène Gsx dans un contexte évolutif permet d’aborder les questions suivantes :
- Quelle est sa fonction dans la mise en place du système nerveux pendant le développement embryonnaire des cnidaires ?
- Cette fonction est-elle conservée au cours de l’évolution ?
- Gsx représente-t-il un acteur précoce agissant sur la différenciation des précurseurs neuronaux et responsable de la destinée des cellules nerveuses ?
Quels sont les molécules signalisatrices et les facteurs de transcription jouants des rôles clés au cours du développement neuronal des bilatériens qui sont des candidats potentiels de la neurogénèse chez les cnidaires ? (Résultats-Chapitre 3) (Galliot et al., 2009).
Cette question plus globale a constitué un travail de synthèse des différentes données déjà publiées chez les cnidaires et a été mis en valeur dans une revue.
Le système nerveux des bilatériens se met en place par le biais de différentes cascades de signalisation et par l’activité de différentes familles de facteurs de transcription. Les cnidaires et les cténophores sont les premiers animaux du règne animal à présenter un système nerveux différencié. D’une manière étonnante, les
53 cnidaires présentent déjà dans leur génome, la plupart des acteurs intervenant dans la mise en place du système nerveux chez les bilatériens. La revue met en évidence les mécanismes cellulaires de la neurogénèse chez les cnidaires. Par ailleurs elle recence les gènes candidats responsable de la mise en place du système nerveux, leur patron d’expression et leur rôle potentiel dans ce phylum ancestral. Plusieurs questions sont sous-jacentes à ce travail :
- Quels sont les acteurs moléculaires fondamentaux à l’origine de la neurogénèse ?
- Ces acteurs et leurs fonctions sont-ils conservés au cours de l’évolution ?
- L’apparition de la neurogénèse dans le monde animal est-elle un événement unique ou de convergence ?
54 RESULTATS
II.1. CHAPITRE 1 : Phylogénie des gènes Hox et Parahox
Les bilatériens possèdent des gènes Hox et des gènes ParaHox. Phylogénétiquement très proches, ces deux familles de gènes sont chacunes arrangées en complexes dans le génome de divers bilatériens et dérivent vraisemblablement de la duplication d’un complexe commun ancestral : le complexe ProtoHox (Brooke et al., 1998)
Depuis le séquençage du génome de l’éponge Amphimedon (Larroux et al., 2007) il apparaît certainement que les gènes Hox et ParaHox sont apparus dans la lignée des eumétazoaires puisque les spongiaires en sont dépourvus. Les premiers gènes Hox et ParaHox de cnidaires ont été caractérisés chez des hydrozoaires (Murtha et al., 1991; Schummer et al., 1992). Ainsi, la duplication du complexe ProtoHox aurait eu lieu avant la divergence des cnidaires et des bilatériens. Des gènes non-Hox déjà présents chez les éponges seraient à l’origine du complexe ancestral (Larroux et al., 2007).
La recherche exhaustive des familles de gènes Hox et ParaHox chez les cnidaires ainsi que l’analyse comparative de leur organisation génomique en comparaison avec les bilatériens, représente donc le moyen actuel de spéculer sur la constitution du complexe ProtoHOX avant sa duplication. Par ailleurs, la confrontation des séquences Hox et ParaHox d’eumétazoaires, au travers d’analyses phylogénétiques, donne des indications sur les étapes de diversification qui ont pris place à partir du complexe ProtoHOX. Quelles familles Hox et ParaHox sont les plus proches phylogénétiquement et dérivent d’un ancêtre commun? L’apport de notre travail a consisté d’une part à intégrer dans des analyses phylogénétiques toutes les séquences Hox et ParaHox de cnidaires disponibles ainsi que de nouvelles séquences identifiées chez trois hydrozoaires, mettant en évidence avec certitude une nouvelle famille de gène ParaHox chez les cnidaires ; d’autre part il s’est agi de mettre au point une méthode permettant de valider, de réfuter ou de trancher parmi certaines hypothèses préexistantes. Ainsi, nous avons pu tester la solidité des nœuds des familles de gènes et les relations phylogénétiques qui existent entre elles. Les résultats obtenus nous ont permis de proposer un modèle robuste de l’histoire évolutive des gènes Hox et ParaHox, particulièrement détaillé au niveau des étapes précoces suivant la duplication du complexe ProtoHOX.
55 De plus, la connaissance du répertoire des gènes Hox et ParaHox des cnidaires représente un intérêt fondamental en biologie du développement étant donné le rôle indiscutable de ces gènes au cours de l’embryogénèse des bilatériens. Un pas évolutif a été franchi au passage d’animaux à symétrie radiaire au système nerveux diffus aux animaux à symétrie bilatérale au système nerveux centralisé. Quelles innovations génétiques ont permis de mettre en place ces organisations ? Les cnidaires détiennent dans leur génome les pièces d’un puzzle à reconstruire.
56 Developmental Biology 328 (2009) 173–187
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Developmental Biology
journal homepage: www.elsevier.com/developmentalbiology
Perspective More constraint on ParaHox than Hox gene families in early metazoan evolution
Manon Quiquand a, Nathalie Yanze b, Jürgen Schmich c, Volker Schmid b, Brigitte Galliot a,⁎,1, Stefano Piraino c,1 a Department of Zoology and Animal Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneve 4, Switzerland b Institute of Zoology, Biocenter/Pharmacenter, Basel University, Switzerland c Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, 73100 Lecce, Italy article info a b s t r a c t
Article history: Hox and ParaHox (H/P) genes belong to evolutionary-sister clusters that arose through duplication of a Received for publication 19 June 2008 ProtoHOX cluster early in animal evolution. In contrast to bilaterians, cnidarians express, beside PG1, PG2 and Revised 14 January 2009 Gsx orthologs, numerous Hox-related genes with unclear origin. We characterized from marine hydrozoans Accepted 14 January 2009 three novel Hox-related genes expressed at medusa and polyp stages, which include a Pdx/Xlox ParaHox Available online 27 January 2009 ortholog induced 1 day later than Gsx during embryonic development. To reconstruct H/P genes' early fi Keywords: evolution, we performed multiple systematic comparative phylogenetic analyses, which identi ed derived Hox/ParaHox genes sequences that blur the phylogenetic picture, recorded dramatically different evolutionary rates between ProtoHox gene ParaHox and Hox in cnidarians and showed the unexpected grouping of [Gsx–Pdx/Xlox–PG2–PG3] families Pdx/Xlox ortholog in a single metagroup distinct from PG1. We propose a novel more parsimonious evolutionary scenario Cnidarians whereby H/P genes originated from a [Gsx–Pdx/Xlox–PG2–PG3]-related ProtoHox gene, the «posterior» and Hydrozoans «anterior» H/P genes appearing secondarily. The ProtoHOX cluster would have contained the three Gsx/PG2, Phylogenetic scoring Pdx/PG3, Cdx/PG9 paralogs and produced through tandem duplication the primordial HOX and ParaHOX Metagrouping of gene families clusters in the Cnidaria–Bilateria ancestor. The stronger constraint on cnidarian ParaHox genes suggests that Clytia the primary function of pre-bilaterian H/P genes was to drive cellular evolutionary novelties such as Turritopsis fi Cladonema neurogenesis rather than axis speci cation. © 2009 Elsevier Inc. All rights reserved.
Introduction distribute in several classes according to their embryonic expression pattern: anterior (PG1, PG2, PG3), central (PG4–PG8) and posterior The ANTP-class Hox genes encode transcription factors that act as (PG9–PG13/14/15). Moreover, in vertebrates the physical arrange- vectorial driving systems for patterning the animal body axes during ment of Hox genes on the chromosome also drives their progressive early development. They arose via repeated gene duplication events temporal activation during development, the expression of the most that led to the formation of multiple evolutionarily-conserved gene anterior genes preceding that of more posterior genes, a property families in bilaterians, with structural features conserved from named temporal colinearity (Kmita and Duboule, 2003). Invertebrate protostomes to deuterostomes, namely their 60 amino acid long genomes usually contain a single cluster with a number of PGs varying DNA-binding domain (named homeodomain, HD) and a clustered among taxa, although in some phyla as nematodes and tunicates, the chromosomal organization. As a result Hox gene families form cluster got disintegrated (Ferrier and Holland, 2002; Seo et al., 2004). paralogous groups (PGs) distributed in a conserved order along the In contrast, tetrapod genomes typically contain four clusters as the cluster indicating that this organization was already present in the HOX cluster underwent several rounds of duplication in the common bilaterian ancestor (McGinnis and Krumlauf, 1992). Moreover these vertebrate ancestor (Wagner et al., 2003; Duboule, 2007). multigenic complexes obey to the spatial colinearity rule whereby The ParaHOX cluster is the evolutionary sister of the HOX cluster during early development the spatial domain of activity of one given but much simpler as it is unique and contains only three genes, Gsx/ gene along the anterior–posterior body axis in a given cell layer Ind, Pdx/Xlox (also named IPF-1, insulin promoter factor 1 in (neural tube, mesodermal derivatives) is related to its specific position vertebrates) and Cdx/Cad, initially described as related to anterior, within the cluster. Hence the most 3′ genes along the cluster are PG3 and posterior genes respectively (Brooke et al., 1998). Phyloge- expressed anteriorly, the most 5′ genes posteriorly, and the PGs netic analyses indicate that the HOX and ParaHOX clusters originated from an ancestral ProtoHOX cluster by segmental tandem duplication (Garcia-Fernandez, 2005b). Moreover, the close relationship of Hox ⁎ Corresponding author. Fax: +41 22 379 33 40. E-mail address: [email protected] (B. Galliot). and ParaHox (H/P) genes strongly suggested that the hypothetical 1 These authors equally contributed to this work. ProtoHOX cluster arose by repeated cis-duplications from a founder
0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.01.022 174 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187
ProtoHox gene, itself originating from the duplication of a non-Hox Gauchat et al., 2000; Hayward et al., 2001; Yanze et al., 2001), whereas ANTP-class gene possibly related to evx and mox (Brooke et al., 1998; Cnox4 from Eleutheria (hydrozoan) was proposed as a Cdx/cad homolog Gauchat et al., 2000; Garcia-Fernandez, 2005b). (Gauchat et al., 2000; Kamm et al., 2006). Hence the identification in Cnidarians, whose origin predated the diversification of Bilateria, cnidarians of Hox (PG1, PG2) and ParaHox (Gsx, Cdx-like) orthologs are organized along an oral–aboral axis, differentiate a neuro-muscular (Fig. 1B) indicated that the tandem segmental duplication of the system as well as sensory organs, and therefore provide a unique ancestral ProtoHOX cluster predated the Cnidaria–Bilateria divergence evolutionary position to trace the core developmental processes at (Gauchat et al., 2000). However, the Cnox4/Cdx grouping is signifi- work in early eumetazoans as apical/anterior patterning (Bode et al., cantly supported only in small datasets with few “posterior” bilaterian 1999; Galliot and Miller, 2000), axis specification (Gauchat et al., 2000; H/P sequences (Kamm et al., 2006). Therefore, as for posterior Hox Hobmayer et al., 2000; Yanze et al., 2001; Ball et al., 2004; Finnerty et genes, a clear correlation between cnidarian and “posterior” bilaterian al., 2004; Lee et al., 2006; Rentzsch et al., 2006) or eye development ParaHox genes is missing. Concerning the “central” Pdx/Xlox ParaHox (Kozmik et al., 2003; Stierwald et al., 2004). Moreover, cnidarians are gene, cnidarian orthologs were never identified to date although a HD complex animals, one class living exclusively as polyps (anthozoans, distantly related to both Pdx/Xlox and Cdx families (therefore named i.e. coral, sea anemone), whereas the three other classes (hydrozoans, Xlox/Cdx) has been found within the Nematostella genome (Chourrout cubozoans, scyphozoans), collectively named medusozoans, include in et al., 2006). most species a parental medusa stage (Fig. 1A). Morphological and The recent genomic analyses indicate a fragmented chromosomal molecular evidences have recently depicted a new scenario on the organization of the cnidarian H/P genes. In the coral Acropora formosa, evolutionary position of cnidarians, which are lately considered as a physical linkage was first found between the PG1-related gene Antp triploblastic (Seipel and Schmid, 2005, 2006) and possibly bilaterian and the evx homolog (Miller and Miles, 1993). More recently, studies animals (Finnerty et al., 2004). using in silico genomic walks in Nematostella confirmed the existence To unravel the composition of the ProtoHOX cluster at the time of of a linkage between eve, PG1 and PG2 Hox genes (Chourrout et al., its duplication and the evolutionary steps leading to the extant H/P 2006; Kamm et al., 2006). They also showed the clustering of Gsx and complement, numerous ANTP-class homeobox genes were identified Xlox/Cdx and the frequent repetitive duplication of homeogenes, from evolutionarily-distant cnidarian species and insights were those multiple copies remaining clustered in most cases. In Hydra, obtained from comparative genomic studies of Porifera, Cnidaria molecular analyses failed to find any clustering between PG1 and PG9- and higher Metazoa (Finnerty and Martindale, 1999; Gauchat et al., like genes over a 250 kb scale (Gauchat et al., 2000), a result 2000; Finnerty et al., 2004; Chourrout et al., 2006; Kamm et al., 2006; confirmed by genomic analyses performed in Hydra and Eleutheria, Ryan et al., 2007; Larroux et al., 2007). Whereas phylogenetic analyses which revealed clustering of H/P genes only when duplicated clearly identified the cnidarian ANTP-class genes belonging to non- (Chourrout et al., 2006; Kamm et al., 2006). Hox families (Gauchat et al., 2000; Kamm and Schierwater, 2006; To provide a more complete picture of the early evolution of H/P Ryan et al., 2006), the case of cnidarian H/P gene families still remains genes, three hydrozoan species that present a full life cycle (alternating ambiguous. The Evx/Mox families, proposed to represent the closest between the polyp and medusa stages) were screened for H/P genes: ancestors to ProtoHox genes (Gauchat et al., 2000; Minguillon and Turritopsis dohrnii (Td), an anthomedusa with an outstanding potential Garcia-Fernandez, 2003), were both identified in anthozoans and for life cycle reversal and morph rejuvenation (Piraino et al., 1996, hydrozoans. But the cnidarian Hox-like genes (see Table 1) show a less 2004), Cladonema radiatum (Cr), an anthomedusa currently used as a supported affiliation with any of the bilaterian Hox PGs (Gauchat et al., model for lens eye differentiation (Stierwald et al., 2004; Suga et al., 2000; Yanze et al., 2001; Kamm et al., 2006; Chourrout et al., 2006; 2008) and Clytia hemisphaerica (Ch), a leptomedusa, whose early Ryan et al., 2007). For example, several anthozoan and hydrozoan development is amenable to functional dissection (Momose and genes display some signature residues of the bilaterian PG1 family Houliston, 2007). Five novel H/P sequences were obtained, represent- suggesting a common origin (Fig. S2). Similarly, three Nematostella ing three distinct gene families, the Pdx/Xlox ParaHox family and two genes with no counterpart in Medusozoa appear as PG2-related genes. orphan cnidarian Hox-related families, CnoxA and CnoxC. We used More controversial is the situation of “posterior-like” genes that these new sequences to reconsider the phylogeny of the H/P genes. As despite several PG9 signature residues (Fig. S2), exhibit a weak the size and the composition of the sampling was shown to dramatically grouping with PG9 genes, not confirmed when the dataset and/or the influence the phylogenetic reconstruction (Wallberg et al., 2004), a methodology were modified (Fig. 1B). systematic approach was followed to test the robustness of the nodes Among the ParaHox genes, the cnox2/anthox2 genes isolated from that define the H/P families within three distinct samplings, each of hydrozoan, scyphozoan and anthozoan species were unambiguously them being submitted to variations of their content. Thanks to this recognized as Gsx/Ind homologs (Finnerty and Martindale, 1999; strategy, we were able i) to identify some cnidarian and bilaterian
Fig. 1. Introductory schemes. (A) Scheme depicting the four distinct classes that compose the Cnidaria and its phylogenetic position. (B) Outline of recorded paralogous Hox and ParaHox gene families in extant Cnidaria and Bilateria. Empty boxes in cnidarians correspond to intermediate gene families not identified so far. (?) Homology between cnidarian and bilaterian posterior (post) or Cdx homeodomains was proposed on some shared signature residues but their grouping is weakly supported in phylogenetic analyses. M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 175
Table 1 Cnidarian genes representative of the Pre-Hox, ProtoHox, ParaHox and Hox gene families in anthozoans and medusozoans
Cnidarian H/P families Anthozoa Medusozoaa Comments Hydrozoa Scyphozoa Pre-Hox Evx/Eve Evx Nv, Eve Af Evx Ssp Nd Highly conserved family Mox MoxA Nv, MoxB Nv, MoxC Nv, Cnox5 Hm=Mox Hm Nd Highly conserved family, 04 copies in Nematostella MoxD Nv ProtoHox-related? CnoxA Cnox2 Pc, CnoxA Td, CnoxA Cr Unclear status: either orphan (datasetB), or grouping with Mox (datasetA), Gsx (datasetC) Hydrozoan-specific? ParaHox Gsx Anthox2 Nv=Gsx, Cnox2 Am, Cnox2 Cv, Cnox2 Hv, Cnox2 Ed, Scox2 Cx Divergence of the Gsx family between Anthozoa, Medusozoa Cnox2 Ssp, Gsx Pc, Gsx/Cnox2 Hys and Bilateria Pdx/Xlox XloxCdx-Nv, Antp-Nv Pdx Td, Pdx Ch Nd Highly conserved in Hydrozoa, highly derived in Nematostella Cdx Nd Cnox4 Ed Nd Unclear status: grouping either Cdx (small datasets only) or with CnoxA (all datasets) Hox PG1 Anthox6 Nv=HoxA, Anthox6 Ms, Cnox1 Hv=Cnox4 Hm, Cnox1 Cv, Nd Divergence of the PG1 gene family between Anthozoa, AntpC Af (HoxB Nv=NVHD060) Cnox5 Ed, Cnox1 Pc Hydrozoa and Bilateria PG2 Anthox7 Nv=HoxC, Anthox8a Nd Nd Duplicated in Nematostella, Lost in Medusozoa? Nv=HoxDa Anthox8b Nv=HoxDb PG9 Nd Nd Nd See the PG9/Cdx-related cnidarian families PG9/Cdx-related CnoxB Anthox1-Nv (HoxF) Cnox4 Pc, HoxB Hm Scox3 Cx CnoxC Cnox3 Hv=Cnox1 Hm, HoxC2 Hm, Nd Hydrozoan-specific? HoxC3 Hm, Cnox1 Ed, CnoxC Ch CnoxD Cnox3-Ed CnoxD Hm Hydrozoan-specific? CnoxE Anthox1a Nv (HoxE) Nd Scox1 Cx, Scox4 Cx
The novel genes reported here are written bold. a There is currently no cubozoan Hox/ParaHox sequence available. derived sequences that blur the phylogenetic picture, ii) to compare the Expression analyses respective evolutionary rates of Hox and ParaHox gene families and iii) to reconsider the metagrouping events, i.e. the clustering between the In situ hybridization were performed on Clytia embryos generously different H/P gene families. provided by Evelyn Houliston as described in (Chevalier et al., 2006) with the following modifications: samples were incubated ON at 4 °C in Materials and methods anti-DIG-AP antibody 1/2000 (Roche). Detection was performed in NBT/BCIP solution containing 100 μg/ml NBT, 175 μg/ml BCIP, 100 mM
Origins and culture of hydrozoan species Tris–HCl pH 8.5, 50 mM MgCl2, 100 mM NaCl. After extensive washes, samples were mounted in Mowiol and observed with an Axioplan Turritopsis dohrnii (Td) polyp colonies were collected from the microscope (Zeiss). For semi-quantitative RT-PCR, Turritopsis and Clytia Ionian sea (Schmich et al., 2007) and hosted as in (Piraino et al., cDNAs were produced with the SuperScript First Strand Synthesis 1996). Clytia hemisphaerica (Ch) colonies were collected from the System (Invitrogen) from mRNAs isolated from various stages with the Mediterranean sea and cultured according to (Carre and Carre, 2000; QuickPrep micro mRNA Purification kit (GE Healthcare). One microliter Stierwald et al., 2004) respectively. Animals were starved at least from each cDNA was PCR amplified first for 15 cycles (94 °C, 30 s; 65 °C, 3 days before mRNA extraction. 30 s; 72 °C, 30 s; −1 °C touch-down at each cycle), then for either 9, 14, 21 or 25 cycles (94 °C, 30 s; 50 °C, 30 s; 72 °C, 30 s) with the Pdx–TdF/– Cloning of the Pdx/Xlox, CnoxA and CnoxC genes TdR (324 bp), CnoxA–TdF/–TdR (550 bp), CnoxC–ChF/–ChR (384 bp), Actin-F01/-R01 primers (sequences on request). For the Pdx/Xlox Td, PdxXlox Ch and CnoxA Td genes, Turritopsis mRNA (900 ng) was extracted from polyps (n=50), medusa buds Phylogenetic analyses (n=50) and newly liberated medusae (n=100) using a Quickprep mRNA extraction kit (GE Healthcare) and Clytia mRNA was extracted The novel HD sequences were tested against the Swissprot, trEMBL from planulae with Dynabeads Direct kit (Dynal). Homology PCR was and Genbank protein databases through the BLASTp program (www. performed with degenerated primers (Murtha et al., 1991) under expasy.org/tools/blast/). The sequences providing the best scores standard conditions with low annealing temperature (37–40 °C). were selected, added to the currently available Hox-related cnidarian Those PCR fragments were then extended by RACE amplification and sequences and H/P sequences from slow-evolving species representing subcloned into the pCRII-TOPO vector (Invitrogen). For CnoxC Ch most bilaterian phyla (Table S1) and aligned with T-Coffee (Notredame cloning, Clytia cDNA was produced with the Sensiscript Reverse et al., 2000). The newly cloned cnidarian HD sequences were assigned Transcriptase (Qiagen) from 2 days-old larvae RNA (400 ng, RNeasy to the different H/P families using the maximum likelihood (PhyML) Qiagen). The PCR product (657 bp) was obtained by chance after 48 and Bayesian interference algorithms. The best model of protein cycles (profile: 96 °C 45 s, 50 °C 3 min, 72 °C 3 min plus 6 s extension evolution available within PhyML was determined using the ProtTest per cycle) with the degenerated primer (ckIckrttytgraaccadatytt) 1.3 program (Abascal et al., 2005). In both cases the JTT model of amino corresponding to the KIWFQNRR motif, which annealed on both sides. acid substitution was used. Bayesian phylogenetic analysis was It was subsequently inserted into the pGEM-T Easy vector (Promega). performed using MrBayes version 3.1.2 under a mixed rate model of The CnoxA Cr sequence was identified among Cladonema ESTs amino acid substitution (Ronquist and Huelsenbeck, 2003), assuming (National Institute of Genetics Mishima) kindly made available by the presence of invariant sites and using a gamma distribution Takashi Gojobori and Walter Gehring. approximated by four different rate categories to model rate variation 176 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 across sites. Starting from random trees two independent runs of four that they are expressed and thus likely functional in those hydrozoan incrementally heated Metropolis-coupled Markov chain Monte Carlo species. To detect whether these genes were regulated during chains were simultaneously performed for 1,000,000 generations with development, gastrulating embryos and growing planulae from trees being sampled every 100 generations. The likelihoods of the Clytia were hybridized to the Gsx Ch, Pdx Ch and CnoxC Ch riboprobes generations were scrutinized to estimate the beginning of the (Fig. 2). The FoxQ2a Ch riboprobe used in the same experiment stationary phase. Those trees were used to create a consensus tree (Fig. 2A) provided the anterior/aboral pattern at the gastrula and either after the first 100,000 generations (burn-in=1000) for the planula stages as previously described (Chevalier et al., 2006). The Gsx bayesian trees generated from the datasetA (21 distinct alignments Ch expressing cells were exclusively detected in endodermal cells of containing 34 to 50 HD sequences), or after the first 200,000 to 600,000 embryos and planulae, as large spots in the posterior/oral region at generations (burn-in=2000–6000) for the Bayesian tree generated the late gastrula stage then in the anterior/aboral half, extending from the datasetB (16 alignments containing 85 to 189 HD sequences). subsequently towards the oral pole until it covers the whole Tree topologies were also reconstructed on the same datasets by endodermal region (Fig. 2B). This pattern is consistent with that Maximum Likelihood using the PhyML v2.4.4 program (Guindon and described in Podocoryne (Yanze et al., 2001), although the initial Gascuel, 2003). The JTT+I+Γ (JTT plus Gamma distributed rates plus posterior wave of expression had not been reported in Podocoryne invariant residues) model of amino-acid substitutions giving the where the detection level was weaker than in Clytia. highest likelihood value and a gamma shape distribution with eight Interestingly Pdx Ch expression was turned on approximately 1 day discrete categories were used. Supports at nodes were assessed with later than Gsx Ch one: in fact Pdx Ch expressing cells were hardly the bootstrap method using 100 replicates. The gamma shape detectable at the gastrula stage, but formed in 1 day old planula a parameter and the proportion of invariant sites were estimated during spotty pattern that extended over both layers, leaving the anterior/ the ML search. aboral pole free of transcripts. In 3 days old planulae Pdx Ch expression was restricted to the endodermal region and rather diffuse (Fig. 2C). Results Similarly the CnoxC Ch expressing cells were also first detected in 1 day old planula, where they were clearly restricted to the tip of the Cloning of two hydrozoan Pdx/Xlox homologs anterior/aboral pole in the ectodermal layer. They subsequently extended in the endodermal region towards the posterior/oral pole Two partial cDNAs, respectively 464 bp and 310 bp long, encoding until they formed an ubiquitous pattern that left free the posterior/ 96% identical HDs were identified from two hydrozoan jellyfish, oral pole (Fig. 2D). Hence the Gsx Ch, Pdx Ch and CnoxC Ch genes Turritopsis and Clytia. These HDs showed highest identity rate with exhibit highly dynamic expression patterns during the early develop- bilaterian Pdx/Xlox sequences, either from deuterostomes (69–71%) ment of Clytia, suggesting that they play some role in the ongoing including vertebrates (Slack, 1995), amphioxus (Brooke et al., 1998), developmental processes. Moreover these three genes are also tunicates (Ferrier and Holland, 2002), hemichordates (Peterson, expressed at the medusa and polyp stages (Fig. S3). 2004), echinoderms (Hwang et al., 2003), or from protostomes as sipunculids (Ferrier and Holland, 2001b), molluscs (Canapa et al., Comparative phylogenetic analyses with three sampling strategies 2005; Barucca et al., 2006), and annelids (Park et al., 2006; Frobius and Seaver, 2006). In contrast identity was lower with the Nematos- To face the problem of the sampling influence on the clustering of tella XloxCdx HD (66.7%, Fig. S1A). Moreover, both HDs possessed the the sequences and the solidity of the nodes (Wallberg et al., 2004), H44 Pdx/Xlox signature residue (Hwang et al., 2003)(Fig. S1B), three distinct sampling strategies were applied to test the affiliation of indicating that these two genes likely belonged to the Pdx/Xlox the novel hydrozoan HD sequences (Fig. S4), two were based on the ParaHox group previously unmapped in cnidarians. alignment of HD sequences, the first one restricted to ParaHox families (datasetA), the second inclusive for all H/P families (datasetB), and The hydrozoan CnoxA and CnoxC families the third one was based on the alignment of full-length homeoprotein sequences (datasetC). The sequences from Turritopsis (Pdx Td, CnoxA We also isolated from Turritopsis a full-length (838 bp) homeobox Td), Clytia (Pdx Ch, CnoxC Ch) and Cladonema (CnoxA Cr) were aligned gene that we named CnoxA Td. The HD, located between positions 109 with the closest sequences identified in BLAST similarity search, the and 168, showed the highest identity (85%) with the orphan gene full set of cnidarian H/P sequences currently available (n=43) and a cnox2 Pc from Podocoryne carnea (Masuda-Nakagawa et al., 2000) set of previously aligned H/P HD sequences (Gauchat et al., 2000). (Fig. S1C). Concomitantly an additional cnox2 Pc homologue, CnoxA DatasetA contained up to 50 sequences representing the ParaHox, Cr, was isolated from Cladonema. In Neighbor-Joining analyses those CnoxA and Mox families (Fig. 3B, Fig. S4, Table S2), whereas datasetB CnoxA HDs, which did not contain any signature typical for any H/P that represented all H/P as well as Evx and Mox families was much family (Fig. S2), formed an orphan cnidarian family with the cnox2 Pc larger, up to 189 sequences (Fig. 5A, Fig. S4, Table S3). Three non-Hox sequence (not shown). Finally we isolated from Clytia a homeogene ANTP-class HD sequences (Msh Cv, NK-2 Hv, Cnox3 Pc) were used as whose HD sequence was closely related to the hydrozoan Cnox3 outgroups in most analyses. Consensus trees representative of the family, which shares some signature residues with the posterior Hox topology obtained with datasets A and B are depicted in Fig. 3A and PGs (Fig. S2) but failed to show convincing relationships with any H/P Fig. 4 respectively. bilaterian families in previous phylogenetic analyses (Gauchat et al., To test the stability of the nodes that define H/P families and 2000). To prevent confusion with numbers defining Hox paralogs, we identify the sequences that alter their robustness, datasets A and B named this novel gene “CnoxC Ch”. Therefore these three homeogenes were submitted to systematic variations of their composition, (CnoxA Td, CnoxA Cr, CnoxC Ch) indeed are related to the H/P gene resulting in 21 distinct alignments for datasetA (Fig. 3B, Table S2) families but in the absence of any obvious affiliation, were considered and 16 for datasetB (Fig. 5A, Table S3). Those alignments were tested as orphan cnidarian H/P-related genes. in both PhyML and Bayesian analyses (74 trees) with replicates to provide bootstrap proportion (BP) and posterior probability (PP). Temporo-spatial regulation of Gsx, Pdx and CnoxC expression in Each H/P family (F) tested in a given alignment was scored according developing Clytia to the formula: SF =BPF +[100× PPF], where the possible maximal SF value is 200. This approach allowed us to produce and compare up to Transcripts of those novel genes were detected by RT-PCR at both 37 scores for each H/P gene family, depicted as graphs in Fig. 3 medusa and polyp stages in Clytia and Turritopsis (Fig. S3), indicating (datasetA, Table S2) and Fig. 5 (datasetB, Table S3). M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 177
Finally, in an attempt to recover more phylogenetic information to The phylogenetic analyses performed on datasetB confirmed the analyze the possible affiliation of cnidarian H/P-related sequences affiliation of the hydrozoan Pdx sequences to the bilaterian Pdx/Xlox that displayed orphan positions in datasetA and datasetB trees, T- family with a significant support when XloxCdx-Nv and/or Antp-Nv euPdx/Xlox coffee alignments of 45 to 50 full-length homeoproteins were were absent (115≤S ≤178). When present the scores were euPdx/Xlox performed and tested in PhyML and Treefinder analyses (datasetC, significantly lower and more variable (4≤S ≤106) (Figs. 5A,F, Fig. S4). This approach confirmed the clustering of H/P gene families G, Table S4). For example the alignments B13 (Fig. 4) and B9 (Fig. S6) previously observed with HD sequences (Fig. S7), indicating that the that differ only by the presence of Xlox/Cdx-Nv and Antp-Nv scored background level of phylogenetic noise of those alignments did not 166 and 68 respectively (Fig. 5A, yellow curve). As in datasetA, the SPdx/ affect the overall phylogenetic signal. Xlox was drastically reduced in trees showing a complete disintegration of the Pdx/Xlox family in Bayesian analyses (B7, B8). By contrast the The hydrozoan Pdx/Xlox sequences display a high level of conservation variations in the bilaterian Pdx/Xlox sampling did not affect the stability of the Pdx group. However orphan bilaterian sequences could In datasetA the two hydrozoan Pdx sequences grouped with the also exert a negative effect as in B7 where the addition of Lox1 Hm and bilaterian Pdx/Xlox/IPF-1 sequences forming a well supported group Hox5 Dl to B5 alignment dramatically reduced SeuPdx/Xlox from 93 Pdx/Xlox (126≤S ≤188) when the two Nematostella sequences XloxCdx- down to 4 (Fig. 5A,F, Table S3). Nv and Antp-Nv were not included (Fig. 3, Table S2). In the six The affiliation of XloxCdx was tested in 14 alignments, 8 from alignments that included both XloxCdx-Nv and Antp-Nv, the Pdx/Xlox datasetA, 6 from datasetB. A similar behavior was recorded in both Pdx/Xlox group was poorly supported (12≤S ≤122); for example the A21 datasets, i.e. grouping together with the Pdx/Xlox family in all PhyML and A13 alignments that are identical except for these two sequences, trees, but appearing in Bayesian analyses either as a Pdx/Xlox family display SPdx/Xlox values of 179 and 13 respectively (Figs. 3, S5). The SPdx/ member (24 trees), or as an orphan (4 trees) when the Pdx/Xlox family Xlox was drastically reduced in A13 because the Pdx/Xlox group was was actually disintegrated (Fig. 6). But XloxCdx-Nv, either alone or in disintegrated in Bayesian analyses. The same phenomenon was combination with the Antp-Nv sequence, also affected the stability of observed in the alignments A10 and A12 where the SPdx/Xlox dropped the eumetazoan Cdx family as in 8 trees of datasetA where Cdx from 188 to 12. Except in those Bayesian trees, the Pdx/Xlox family appeared paraphyletic, branching from the Pdx/Xlox family (see A9, always formed and the two new hydrozoan Pdx sequences always A15-NA17 in Bayesian analyses and A14 and A18 in both analyses, Table grouped in. Moreover XloxCdx-Nv and Antp-Nv also affected the Gsx S2). In this latter case, XloxCdx-Nv branched at the root of Cdx within score in A12, A13, A14 where SGsx b110 (Fig. 3B), whereas the Mox, the Pdx/Xlox family. Similarly in datasetC, the clustering of XloxCdx- CnoxA and bilaterian Cdx scores remained unaltered (Fig. 3C). Nv at the root of the Cdx family was frequently observed (13/16 trees,
Fig. 2. Expression of Gsx Ch, Pdx Ch and CnoxC Ch in developing Clytia. (A–D) In situ hybridization patterns observed in mid/late gastrula (a panels), 1 day old (b panels) and 3 days old (c panels) planulae. Scale bar: 100 μm. (E) Expression detected by semi-quantitative RT-PCR in early gastrulae (e.g.), one (d1) and 2 days old (d2) planulae, in young medusa (y.m.). 178 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187
cdx+Cnox4-Ed 81%), rarely with the Pdx family (2/16 trees, 12.5%). These results indi- (31≤S ≤41), as supported only with the PhyML method. cate that the presence of the two hydrozoan Pdx sequences enhanced In the corresponding Bayesian analyses Cnox4-Ed was also attracted the attraction of XloxCdx-Nv towards the Pdx family in analyses towards the CnoxA family (3/16 trees, 18.7%) or orphan (2/16 trees, restricted to HD sequences. Hence, the systematic analysis of trees 12.5%). A similar behavior was noted in datasetC where Cnox4-Ed built from either large or small datasets provided clearcut evidences grouped with Cdx (12/23 trees, 52%), CnoxA (7/23 trees, 30%), or took that Pdx Td, Pdx Ch and XloxCdx-Nv belong to the Pdx/Xlox ParaHox an orphan position (4/23 trees, 17%). Finally in datasetB, the grouping gene family, XloxCdx-Nv being a derived Pdx/Xlox representative. of the Cnox4-Ed and Cdx sequences was less frequent (8/32 trees, 25%) likely prevented by the presence of the derived planarian Pnox6 Cnox4-Ed as a distantly-related hydrozoan Cdx gene Pn sequence, which favored the grouping of Cnox4-Ed with CnoxA (13/32 trees, 40.6%, Fig. 6, Table S3). These data confirm the frequent Previous analyses had shown that Cnox4-Ed and Cdx HD grouping of Cnox4-Ed with Cdx sequences in small datasets. In large sequences share some signature residues (Fig. S2) and phylogenetic datasets this grouping was more labile, altered by the influence of affinities (Gauchat et al., 2000; Chourrout et al., 2006; Kamm et al., derived sequences. 2006). In datasetA Cnox4-Ed that was included in 8 alignments (Table S2) was indeed frequently grouping with Cdx sequences (11/16 trees, Characterization of the hydrozoan CnoxA family Cdx + Cnox4-Ed 68.8%), although with low scores (31 ≤ S ≤ 104) as supported by both methods in only three alignments (A8, A11, A21). The three hydrozoan CnoxA sequences formed a robust node with CnoxA This grouping was still observed in the presence of the Xlox/Cdx-Nv high and stable S values in datasetA (S ≥166, Table S2), datasetB CnoxA and Antp-Nv sequences, however with S values drastically lowered (S ≥160, Fig. 5E, Table S3) and datasetC (BPN80, Fig. S7). The
Fig. 3. Phylogenetic relationships between the cnidarian and bilaterian ParaHox HD sequences (datasetA). (A) Tree drawn using the PhyML program, corresponding to the alignment A21 (15 cnidarian and 27 bilaterian HD sequences, see in (B) also tested in Bayesian analysis. For each node, the BP (top) and PP (bottom) values obtained after 100 and 10,000 replicates respectively are indicated. Gene families significantly supported are indicated on the right, cnidarian sequences are boxed in grey. For species code and accession numbers see Table S1. (B) Composition of the 21 alignments of datasetA. Cnidarian gene names are written red. Bilaterian sequences of a given family were grouped, e.g. “Gsx n4 (3)” means that the 3 sequences forming the Gsx group number 4 were included (+) or not (−). See Table S2 for the detailed composition of each tree. N: total number of sequences; light grey shading: alignments where 50bSPdx/Xlox b150; dark grey shading: alignments where SPdx/Xlox b50; pink shading: presence of Antp-Nv and Xlox/Cdx-Nv sequences. (C) Graph showing the variations of S values for each gene family according to the alignment composition. The compiled S scores that represent the robustness of a given family, were deduced from BP and PP values of PhyML and bayesian analyses (see in Table S2). Alignment numbers are indicated at the top. Note that the Pdx and Gsx families were supported in both analyses in all alignments except when the Antp-Nv and Xlox/Cdx-Nv sequences were included together with two bilaterian orphans (A12, A13, light grey box, Fig. S5). M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 179 affiliation of CnoxA to one or the other H/P family was ambiguous as in scoring b102 but one (Fig. 5C, SeuPG1 =45±33). In contrast the score of datasetA CnoxA either appeared as an orphan H/P-related family (15/ the bilaterian PG1 family were very high (SbiPG1 =186±6) whereas the 42 trees, 36%) or grouped with the Mox family (16/42 trees, 38%, hydrozoan PG1 family that was supported by both methods in 12/15 Table S2), this latter grouping being supported by both methods in alignments, also scored significantly higher than SeuPG1 (ShyPG1 =102± CnoxA + Mox seven alignments (107≤S ≤131) and not affected by the 32) (Fig. 5C,G, Table S3). Concerning the eumetazoan PG2 family, we presence of Xlox/Cdx-Nv and Antp-Nv. More rarely the CnoxA and similarly noted a large gap between the bilaterian and the eumetazoan Cdx sequences grouped together (5/42 trees, 12%) with weak scores (SbiPG2 =137±42 versus SeuPG2 =66±42). These significative CnoxA + Mox support (21≤S ≤74) and in two alignments CnoxA+ differences between eumetazoan and bilaterian scores of the PG1 and Mox+Cdx grouped together. PG2 families can only be explained by a strong divergence of the In datasetB the CnoxA family was also frequently orphan (27/32 cnidarian sequences. trees, 84%) but displayed a position in the vicinity of the root in 13/16 In contrast, the eumetazoan ParaHox families displayed higher Phyml trees, i.e. close to the Evx and Mox families, frequently and more stable scores: the eumetazoan Gsx family scored between associated with Cnox4-Ed as stated above. When tested in datasetC, 100 and 150 in 14/16 alignments (SeuGsx =121±27, Fig. 5B,G, Table S3) the CnoxA sequences that are all possibly full-length (Fig. S1C) and as reported above, the eumetazoan Pdx family scored high and frequently grouped with the Gsx family (15/21 trees, 71.4%) with stable in the 9 alignments where the XloxCdx and Antp-Nv sequences some bootstrap support (Fig. 6). In some trees this metagroup were excluded (SeuPdx =148±24), still relatively well when taking (CnoxA+Gsx) was associated with the Cdx (4/21, 19%) or Mox (3/17, into account all alignments (SeuPdx =112±52, Fig. 5A,G, Tables S3 and 17.6%) families. CnoxA sequences also attracted Cnox4-Ed (4/22 trees, S4). In case of the Gsx family, where a large number of cnidarian 18.2%) or derived cnidarian sequences (Cnox3-Ed, XloxCdx-Nv, 2/22 sequences is available, the eumetazoan, bilaterian and cnidarian scores trees, 9%). This result suggested that CnoxA and Gsx proteins share were close to each other (SbiGsx =136±26 and ScnGsx =151±33). In some common residues outside the HD as the Pro-, His- or Cys-rich summary the eumetazoan scores obtained by the Gsx and Pdx families stretches upstream to the HD, or the K61 residue immediately were about twice higher than those recorded for the PG1 and PG2 euGsx euPdx euPG2 euPG1 downstream, also present in the Cnox4-Ed and Cdx sequences. It families (S (121)∼S (112/148)NS (66)NS (45), should be noted that CnoxA never grouped with the other “orphan” Fig. 5G). This quantitative analysis clearly measures higher amounts cnidarian families (CnoxB to CnoxE). of phylogenetic signal in the cnidarian Gsx or the Pdx/Xlox ParaHox sequences compared to the PG1 and PG2 Hox ones, indicating a Characterization of the cnidarian CnoxB, CnoxC, CnoxD higher level of conservation for the ParaHox compared to the Hox and CnoxE families families in cnidarians.
Six sequences, representing five distinct hydrozoan genes form the PG1 does not have any ParaHox counterpart cnidarian CnoxC family (Fig. S1, Fig. 4). In datasetB this family CnoxC displayed a high phylogenetic score (S ≥150), insensitive to the When the clustering of the H/P gene families, named “meta- variations introduced in the dataset (Fig. 5). Moreover, three grouping”, was investigated in the trees generated from datasetB additional cnidarian families, CnoxB, CnoxD and CnoxE, containing (Fig. 6 and not shown), we noticed that the “anterior” Hox families, i.e. medusozoan sequences, formed in datasetB and datasetC trees (Fig. 4, PG1, PG2 and PG3 actually never clustered together but rather formed Fig. S7, Table 1). In datasetB, Anthox1-Nv grouped together with two distinct branches, PG1 on one side, PG2 and PG3 together on CnoxB sequences in 12/18 trees (67%) whereas Anthox1a-Nv grouped another side as depicted in Fig. 4. Moreover the Gsx and Pdx/Xlox with CnoxE in 6/28 trees (21.4%) (see MG1 and MG3 in Fig. 6), ParaHox sequences never clustered with the PG1 ones but repeatedly suggesting that Anthox1-Nv and Anthox1a-Nv are the anthozoan with the PG2/PG3 metagroup as observed in 34.4% of the trees built orthologs of the medusozoan CnoxB and CnoxE families respectively. on datasetB (Fig. 6), with the best scores recorded in the B1, B4, and The metagrouping of CnoxC together with CnoxB and Anthox1-Nv B13 trees (57, 58, 63 respectively). This indicates firstly that the PG1 sequences (metagroup 2, MG2) was observed in 13/18 trees (72%) Hox family on one side and the PG2/PG3 on the other side, should be MG2 with significant support (77≤S ≤149, Fig. 6). In datasetB, the considered as two distinct groups with separate origins; secondly that clustering of these four families (CnoxB, CnoxC, CnoxD and CnoxE) the Gsx and Pdx/Xlox ParaHox gene families should no longer be was recorded in the vicinity of the PG9/Cdx sequences in 5/16 trees considered as PG1-related but rather as homologous to the PG2/PG3 (31%) although without any supporting bootstrap value (MG9, Fig. 6). Hox families. In datasetC, these “orphan” cnidarian families that frequently clustered together (as noted in 63% of the trees, not shown), also Discussion appeared to share a common origin with PG9 and Cdx families (Fig. S7B). Surprisingly in datasetB the metagrouping of the PG9 and Cdx A systematic quantitative strategy to score the phylogenetic signals bilaterian families was noted in only 5/28 trees (17%) with very low present in H/P sequences scores (≤10). Therefore, in the absence of any other significant metagrouping event, we consider these four cnidarian families as With the increasing number of Hox-related HD sequences putative “PG9/Cdx” orthologs. identified from cnidarian species (up to 43 by now), phylogenetic analyses repeatedly established that most cnidarian Hox-like genes The Gsx and Pdx/Xlox families are more conserved than the PG1 and PG2 display rather divergent sequences when compared to bilaterian Hox families in eumetzoans ones, a feature that is not common within the ANTP-class (Gauchat et al., 2000; Ryan et al., 2006; Larroux et al., 2007). In fact there is a To evaluate the divergence of the Hox and ParaHox families strong contrast between the pre-bilaterian non-Hox ANTP-class gene between bilaterians and cnidarians, the eumetazoan phylogenetic families (Msx, NK2, Emx, …) that are highly conserved, and the scores obtained in the alignments of datasetB were systematically cnidarian Hox-like gene families that display limited support with compared to the bilaterian and/or cnidarian ones (Figs. 5A–F). The any bilaterian Hox sequences. Therefore the phylogeny of cnidarian mean values and standard deviations were calculated for each gene H/P H/P sequences remained difficult to establish and numerous gene family identified in eumetazoans (SeuF), bilaterians (SbiF), scenarios were proposed to describe the early evolution of H/P cnidarians (ScnF) and/or hydrozoans (ShyF)(Fig. 5G, Table S4). genes. Phylogenetic analyses applied to either large or restricted Concerning the eumetazoan PG1 family, the scores were low, all trees (Gauchat et al., 2000; Finnerty et al., 2004; Kamm et al., 2006) 180 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 181 sequence datasets, provided conflicting results, e.g. the presence of Phylogenetic analyses of the cnidarian H/P genes are obscured by some “posterior” H/P genes in cnidarians. In this study, we developed highly derived sequences strategies where the phylogenetic signal of each H/P gene family was systematically quantified in three different samplings, a first one Concerning the ambiguous Xlox/Cdx Nematostella sequence focused on the ParaHox sequences to assess the family identity of the (Chourrout et al., 2006), the presence of the H44 Pdx/Xlox signature novel sequences, a second one inclusive for the cnidarian Hox, residue together with the absence of Cdx signature residues in its HD, ParaHox and Hox-related sequences to analyse the metagrouping of provide support for a Pdx/Xlox origin of this gene. Moreover the the H/P families and identify the phylogenetic relationships of five presence of the hydrozoan Pdx/Xlox sequences clearly enhanced the cnidarian “orphan” Hox-related gene families, and finally, a third grouping of Xlox/Cdx towards the Pdx/Xlox family. In addition to the dataset that contained selected full-length homeoprotein sequences XloxCdx and Antp-Nv sequences from Nematostella but also Hox5 Dl to confirm the obtained results and detect when possible additional or Lox1 Him from bilaterians were identified as derived sequences, phylogenetic information. As H/P gene families presumably arose which drastically altered the robustness of the Pdx/Xlox group, also from non-Hox ANTP-class genes (Gauchat et al., 2000; Garcia- affecting in some cases the robustness of other gene families. Fernandez, 2005b; Larroux et al., 2007), all trees were rooted with Therefore those highly derived sequences considerably obscure the the non-Hox Msx, NK-2 sequences. phylogenetic picture of the cnidarian H/P genes and need to be systematically searched prior to drawing any conclusion. Additional The discovery of hydrozoan Pdx/Xlox orthologs highlights the different derived sequences with negative effects were also identified in this constraints applied on Hox and ParaHox genes in their early evolution study: Pnox6 Pn on the grouping of Cnox4-Ed with the Cdx family, Anthox8 Nv on the eumetazoan PG2 score, the urochordate Gsx These analyses reported two novel hydrozoan sequences, whose sequences on the eumetazoan Gsx score. This work also shows that identification as cnidarian Pdx/Xlox orthologs was unambiguously hydrozoans and anthozoans display strongly heterogenous evolu- proven in three samplings, including in alignments containing the tionary patterns: whereas the Hox families (PG1, PG2) exhibit a higher largest numbers of sequences, as the increase in the number of rate of conservation in anthozoans than in medusozoans, this is not homologous sequences is supposed to strengthen the significant the case for the Pdx/Xlox and Cdx families, more conserved in nodes (Wallberg et al., 2004). Moreover, they also confirmed the hydrozoans (Pdx/Xlox, Cnox4-Ed) than in Nematostella (XloxCdx). presence of Gsx, PG2 and PG1 orthologs in cnidarians. Among the H/P Consequently, data from both anthozoans and medusozoans are families, the Gsx and Pdx/Xlox ParaHox families displayed the needed when considering cnidarians in evolution. highest scores, indicating that cnidarian and bilaterian members of those families are more similar to each other than are the PG1 and The “posterior” H/P gene families are highly derived in cnidarians PG2 Hox family members. Therefore we assume that the Gsx and Pdx/Xlox ParaHox sequences have less evolved from the ancestral As anticipated, we did not observe any cnidarian sequences in any ParaHox sequences, suggesting that differential evolutionary rates tree that would branch together with the central PG4 to PG8 groups, applied to the cnidarian ParaHox and Hox gene families, the former implying that those Hox paralogs arose after Cnidaria divergence. ones being more constrained. Does it mean that ParaHox genes Concerning the posterior genes, it was previously reported that some represent better the ancestral status of the ProtoHox genes? This Hydra (Cnox3 Hv) and Nematostella (Anthox1, Anthox1a) sequences question remains open and data from basal non-cnidarian species as share few signature residues with the posterior family but when the ctenophora and placozoa are needed to tell whether it is a cnidarian residues were given the same weight, clustering no longer appeared in specificity or not. phylogenetic analyses (Gauchat et al., 2000). Here, in datasets B and C, the cnidarian CnoxB, CnoxC, CnoxD and CnoxE HD sequences The Gsx-PG2-PG3-Pdx/Xlox metagroup does not include PG1 frequently grouped together with Cdx/PG9 (Fig. 4, Fig. 6), rarely with [PG2–Gsx/PG3–Pdx], never with PG1. This metagrouping analysis In addition this new phylogenetic picture did not show the suggested a Cdx/PG9 origin for those families, without predicting from expected affiliation between the H/P gene families; the PG2, PG3, Gsx the data presented here the origin of the CnoxB, CnoxC, CnoxD and and Pdx/Xlox grouped together but never exhibited in the 32 trees CnoxE ancestor(s); they could be derived from the PG9/Cdx ancestor obtained from datasetB the linkage usually displayed (Ferrier and of the ProtoHOX cluster, but also from Cdx of the primordial ParaHOX Holland, 2001a), i.e. Gsx related to PG1/PG2 and Pdx/Xlox related to cluster or PG9 of the primordial HOX cluster (Fig. 7). Hence “posterior” PG3. By contrast, the systematic testing of those three different gene families, either Hox- or ParaHox-related, are highly divergent in samplings allowed us to sort out the bilaterian H/P sequences in four cnidarians but can nevertheless be identified when numerous datasets metagroups: PG1,[Gsx–PG2–PG3–Pdx/Xlox], [PG4–PG8], [PG9–Cdx]. are compared and when metagroupings are taken into consideration. The metagrouping of the [Gsx–PG2–PG3–Pdx/Xlox] gene families is Concerning the “posterior” ParaHox gene, the putative Cdx ortholog, reported here for the first time, indicating that, in contrast to the the hydrozoan Cnox4-Ed, indeed more frequently grouped with Cdx in prevalent view, PG2 and PG3 are twin families actually not closely small datasets but also in large datasets that did not include the derived related to any of the other Hox families, specially not to PG1. The sequence Pnox-6. Nevertheless additional medusozoan Cnox4-Ed like eumetazoan PG1 family contains three distinct sub-families, bilaterian, sequences are required before concluding about the presence of anthozoan and hydrozoan, highlighting the early divergence between genuine Cdx orthologs in cnidarians. Finally, the CnoxA cnidarian family Anthozoa and Medusozoa. The Evx and Mox families, which both is related neither to the “posterior” cnidarian families nor to the PG1 contain cnidarian homologs, rarely grouped together (Fig. 6) but families, but exhibits some Mox and Gsx phylogenetic signal. Therefore, frequently took positions close to the root of the tree in PhyML we propose that CnoxA has possibly arisen through duplication from analyses, suggesting that they represent intermediates between the more ancestral sequences than the extant H/P families, and might thus non-Hox and the H/P sub-classes of the ANTP-class, as previously represent ProtoHox gene families as the Gsx/Pdx/PG2/PG3 ancestor, proposed (Gauchat et al., 2000; Garcia-Fernandez, 2005a). likely arisen from Evx/Mox duplication (Fig. 7, right).
Fig. 4. Tree showing the phylogenetic relationships between cnidarian and bilaterian Hox and ParaHox HD sequences. This tree corresponds to the alignment B13 that contains 47 cnidarian and 140 bilaterian H/P sequences (see composition in Fig. 5F and Table S3). The robustness of the phylogenetic inference was tested through replicates, 100 in PhyML and 10,000 in Bayesian programs with BP and PP values noted on the nodes. Each gene family sits on a colored background, dashed outlined when cnidarian only, full when cnidarian and bilaterian, no outline when bilaterian only. Note the five Hox-related cnidarian families, CnoxA to CnoxE, on orange backgrounds. Except Pnox6 Pn, only the names of the cnidarian sequences are noted. The Nk2, Msh and Cnox3 Pc sequences were used as outgroups. 182 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187
Fig. 5. Analysis of the robustness and the stability of the H/P family nodes. (A–E) Variations in the S values supporting (A) the ParaHox families in eumetazoans except Cdx restricted here to bilaterian sequences, (B) the Gsx family in eumetazoans, bilaterians, medusozoans and anthozoans, (C) the PG1 family in eumetazoans, bilaterians, hydrozoans and anthozoans, (D) the PG2 family in eumetazoans, bilaterians and anthozoans and the bilaterian PG3 family, (E) the “orphan” cnidarian Hox-related families. The Mox (A) and Evx (C) families were included as control families that exhibit high and stable scores. See all BP and PP values in Table S3. (F) Composition in ParaHox, Hox and Hox-related sequences forming the 16 distinct alignments of datasetB that were tested in PhyML and Bayesien analyses. Left: Names of the H/P gene families (underlined) and cnidarian sequences (red). Numbers on yellow background indicate the number of sequences representing each gene family in a given alignment. n: total number of sequences included in each alignment that is numbered at the top. Note that the alignments B4 to B9 contain the XloxCdx sequence. (G) Graph aligning the means of the scores obtained by the various H/P families from datasetB (depicted in panels A–E); the blue bars correspond to the scores obtained by the H/P families in eumetazoans; red bars in bilaterians; yellow bars in cnidarians; green bars in medusozoans (Gsx and PG1 only). Two mean values are provided for the eumetazoan Pdx family, one taking into account all alignments (N=16, left) and a second one that considered only the alignments lacking the XloxCdx and Antp-Nv sequences (N=9, right). See in Table S4 the numerical values and standard deviations. Note the low mean values for the Cdx, PG1 and PG2 families in eumetazoans. M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 183
Fig. 6. Analysis of the metagrouping between the ParaHox, Hox and Hox-related families. Respective frequencies of the metagrouping events (MG) observed among the 32 trees built on alignments from datasetB. The percentages correspond to the number of trees where the indicated metagrouping event was observed over the number of expected events. The names of the gene families that appeared clustered are indicated on the corresponding bars. The mean values of the phylogenetic scores supporting each MG event are given on the right.
The various evolutionary models for the composition of the ancestral independently both PG3 and central genes. The A-3-P three-gene ProtoHOX cluster model is based on evolutionary parsimony (Finnerty and Martindale, 1999; Ferrier and Holland, 2001a): The apparent absence of central The finding of Hox and ParaHox cognate genes in Cnidaria but not ParaHox genes in bilaterians and cnidarians, coupled with the lack of in Porifera (Larroux et al., 2007) supports the emergence of the central Hox genes in cnidarians, suggested a simpler, three-gene ProtoHOX cluster after the divergence of Porifera and its segmental composition of the original ProtoHOX cluster, including representa- tandem duplication that led to the formation of the HOX and ParaHOX tives of the anterior, PG3 and posterior genes. This model assumes that clusters in the Cnidaria–Bilateria common ancestor. An open question cnidarians, following the ProtoHOX cluster duplication, lost their Hox relates to the configuration of the ProtoHOX cluster: How many and PG3 and the corresponding ParaHox (i.e., Pdx/Xlox). The radiation of what genes were originally included in this cluster ? Four hypothesis Bilateria was paralleled by the origin of central paralogs by tandem of H/P evolution were proposed since the discovery of the ParaHOX duplication on the HOX cluster. The A-P two-gene model assumes that complex in amphioxus (Brooke et al., 1998; Garcia-Fernandez, 2005b). the ProtoHOX cluster contained only two Hox-related genes, one In the A-3-C-P four-gene model, the ancestral ProtoHOX cluster is anterior and one posterior. According to this model, the absence of composed by four genes, anterior, group3, central, posterior as intermediate Hox/ParaHox genes in cnidarians is a plesiomorphic founders of the main paralog groups (Brooke et al., 1998). This character that should be interpreted as an evidence of ancient hypothesis is supported by the joint occurrence of anterior, inter- divergence of cnidarians from bilaterians. After the ancestral Proto- mediate (PG3, central) and posterior Hox genes in most bilaterians HOX cluster duplication, the radiation of Bilateria was linked to the (Ferrier and Holland, 2001a); it assumes that the lack of the central origin of PG3 on both HOX and ParaHOX clusters and PG4 to PG8 on ParaHox group in extant Metazoa would be due to an early loss from the HOX cluster by independent tandem duplication from the anterior the primitive ParaHOX cluster and that cnidarians would have lost one (Baguna and Riutort, 2004; Garcia-Fernandez, 2005b). The recent 184 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187
A-3 two-gene model takes into account the lack of posterior orthologs (Chourrout et al., 2006). Therefore this model also proposes that only in cnidarians as well as the faster evolutionary rate of the posterior two genes were present in the ProtoHOX cluster, one anterior and one Hox paralogs compared to the anterior or central ones in bilaterians PG3. Following trans-duplication in HOX and ParaHOX clusters, the M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 185 non-anterior genes would have appeared by separate tandem subsequently needed, cis-duplication of the PG1 ancestor in the three- duplication events on each of the two clusters, independently in gene ProtoHOX model, deletion of the PG1-like ancestor from the cnidarians and bilaterians. Finally, Ryan et al. (2007) dispute the twin ParaHOX cluster in the four-gene ProtoHOX model (Fig. 7). cluster model, arguing that the HOX and ParaHOX clusters arose as the The subsequent evolution of these two primordial clusters would be split of a cluster formed via repeated tandem duplications of identical whatever the initial hypothesis: the PG3 got likely lost from individual genes rather than via the duplication of a ProtoHOX cluster. the HOX cluster in the early evolution of Cnidaria but was maintained in This hypothesis is based on the analysis of Nematostella, amphioxus the radiation of Bilateria, where submitted to an early wave of tandem and Drosophila sequences, from which they concluded that PG3 duplications, it produced the central genes (PG4 to PG8). In cnidarians representatives were lost in cnidarians and that Gsx formed as an the HOX and ParaHOX clusters underwent some parallel evolution: independent lineage, as they observed the absence of grouping their clustered organisation became highly disintegrated (Chourrout between PG1, PG2 and Gsx. However the identification of genomic et al., 2006; Kamm et al., 2006) and the “posterior” genes highly regions in Nematostella that are syntenic with the human H/P derived compared to the other paralogous groups, submitted to several sequences clearly favors the hypothesis of a duplication that gave duplication events, leading to the formation of the CnoxB, CnoxC, rise to the HOX and ParaHOX clusters prior to the divergence of CnoxD, CnoxE families. In contrast, the posterior genes were maintained Cnidaria (Hui et al., 2008). in Bilateria, the PG9 ancestor being duplicated independently in Lophotrochozoa (post1, post2) and Chordata (PG9–PG15). The pristine composition of the ancestral Hox-like cluster The firstly evolved H/P genes rather supported cell differentiation The results presented in this paper fully support a segmental novelties than axis specification tandem duplication of a ProtoHOX cluster but do not support any of the models proposed above as they provide two major changes in the Being likely absent from poriferans (Larroux et al., 2007), we evolutionary picture of the H/P genes: firstly the presence of Pdx/Xlox assume that H/P genes arose early in eumetazoan evolution to be cnidarian orthologs, second the identification of a major and highly recruited into regulatory networks that allowed the differentiation of conserved metagroup, the Gsx/Pdx/PG2/PG3, which is clearly distinct more complex anatomies. The expression patterns presented here from “anterior” and “posterior” H/P gene families, i.e. PG1 and Cdx/ (Fig. 2) are consistent with an early developmental function for Pdx, PG9 respectively. Moreover, this study proposes the CnoxB to CnoxE Gsx and CnoxC in Clytia: Gsx was detected first in gastrulae, present gene families as cnidarian representatives of the ancestral Cdx/PG9 in endodermal cells of the posterior half. One day after fertilization, gene families. These new data favor two possible hypotheses. both Gsx and Pdx displayed a transient punctuated pattern, which In the first one, the four-gene ProtoHOX cluster, depicted in Fig. 7 transformed into a diffuse staining of the endoderm, more intense in (left), would originate from a PG1–PG9/Cdx ancestral ProtoHox gene, case of Gsx. In Podocoryne, Gsx exhibits a very similar pattern except i.e. an A/P ancestor as previously postulated (Zhang and Nei, 1996). the earliest transient wave of expression at the posterior pole of Hence, after three subsequent rounds of gene duplication, this gastrulae that was not reported (Yanze et al., 2001). In contrast CnoxC protoHOX cluster would have contained, in addition to the Evx/Mox expression pattern was clearly different, with CnoxC transcripts first ancestor, one gene related to the “anterior” group providing a localized at the anterior pole in 1 day old planula before extending common PG1 ancestor to cnidarians and bilaterians (although with subsequently all along the axis but leaving free the posterior pole. no counterpart in the ParaHOX cluster), a second one related to group Further studies should tell us whether Pdx and Gsx are chromosomally 2 (PG2/Gsx), a third one related to the group 3 (PG3/Pdx/Xlox) and a clustered in Clytia, potentially sharing some regulatory region that fourth one related to the posterior group (PG9/Cdx). The tandem would be reminiscent of those controlling temporal colinearity in duplication of this ProtoHOX cluster in the common cnidarian– vertebrate Hox genes (Kmita and Duboule, 2003). bilaterian ancestor would have led to the formation of two highly The currently available data indicate that the H/P genes at the time similar clusters, the ParaHOX and HOX clusters. cnidarians diverged, were already involved in cell differentiation as However, we favor a second more parsimonious alternative myogenesis (Aerne et al., 1995; Yanze et al., 1999) and neurogenesis scenario, the three-gene ProtoHOX cluster (Fig. 7, right), where the (Miljkovic-Licina et al., 2007) and recruited for some developmental ancestral ProtoHox gene would have rather resembled the Gsx/Pdx/ processes as apical patterning, but not yet following the rules that lead PG2/PG3 gene families instead of A/P ancestors, the posterior ancestors to body axis specification in bilaterians (Gauchat et al., 2000; Kamm being generated by cis-duplication at the subsequent stage either from et al., 2006; Chourrout et al., 2006). Interestingly, the Gsx/cnox2/Ind the Evx/Mox ancestor, or from the Gsx/Pdx/PG2/PG3 ancestor. Similarly, gene family is likely involved in neurogenesis from cnidarians in the absence of any “anterior” representative in the ParaHOX cluster, (Hayward et al., 2001; Miljkovic-Licina et al., 2007) to bilaterians, i.e. it is tempting to speculate that the PG1 ancestor might have actually Drosophila and mouse (Weiss et al., 1998; Toresson and Campbell, been absent from the ProtoHOX cluster, arising later onto the 2001; Yun et al., 2003). Similarly we expect some key cellular function primordial HOX-cluster from the PG2 cis-duplication. As a consequence for the cnidarian Pdx/Xlox, possibly restricted to jellyfish anatomy. In the ancestral ProtoHOX cluster would have contained only three genes fact, the functional and expression analyses performed in developing in addition to the Evx/Mox ancestor, Gsx/PG2, Pdx/PG3 and Cdx/PG9. vertebrates and annelids highlighted a role for Pdx/Xlox/IPF and Cdx The three-gene ProtoHOX cluster hypothesis is more parsimonious as it genes in cell and tissue differentiation rather than axis specification requires only three steps from the Evx/Mox ancestor up to the (Wysocka-Diller et al., 1995; Offield et al., 1996; Milewski et al., 1998; segmental duplication, whereas the four-gene ProtoHOX cluster Melloul, 2004; Frobius and Seaver, 2006; Young and Deschamps, in hypothesis requires four steps. In both scenarios an additional step is press). Hence the stabilization of ParaHox genes in early animal
Fig. 7. A three-gene ProtoHOX cluster derived from a Gsx/PG2-Pdx/PG3 ProtoHox gene as the most parsimonious model to describe the early evolution of the H/P gene families. In the absence of H/P genes in poriferans, ProtoHox genes likely appeared after Porifera divergence as a result of a cis-duplication event of a non-Hox ANTP-class gene, possibly an Evx/Mox ancestor gene. According to the nature of this ancestral ProtoHox gene, we describe two possible scenarios. In the first case (left), the repeated cis-duplication from a PG1/Cdx-PG9 ProtoHox gene led to the formation of a four-gene ProtoHOX cluster containing PG1, Gsx/PG2, Pdx/PG3 and Cdx/PG9. In the second case (right), three paralogs, corresponding to Gsx/ PG2, Pdx/PG3 and Cdx/PG9, arose from a Gsx/PG2-Pdx/PG3 ProtoHox gene. Subsequently the segmental tandem duplication of this ProtoHOX cluster led to the formation of the primordial ParaHOX and HOX clusters. The absence of PG1-related sequence among ParaHox genes can be interpreted in two ways, either secondarily lost (four-gene ProtoHOX model) or never present in the primordial ParaHOX cluster (three-gene ProtoHOX model). This work supports CnoxB, CnoxC, CnoxD, CnoxE gene families as derived from Cdx/PG9, and Cnox4-Ed gene as a possible Cdx ortholog. In cnidarians, the HOX and ParaHOX clusters got disintegrated while remaining intact in numerous bilaterian species. The emergence of the PG4 to PG8 paralogs is likely a more recent event that occurred after Cnidaria divergence. 186 M. Quiquand et al. / Developmental Biology 328 (2009) 173–187 evolution would have coincided with the establishment of evolutio- Gauchat, D., Mazet, F., Berney, C., Schummer, M., Kreger, S., Pawlowski, J., Galliot, B., 2000. Evolution of Antp-class genes and differential expression of Hydra Hox/ narily-conserved regulatory networks driving cellular novelties such paraHox genes in anterior patterning. Proc. Natl. 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Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
More constraint on ParaHox than Hox gene families in early metazoan evolution
Manon QUIQUAND1, Nathalie YANZE2, Jürgen SCHMICH3, Volker SCHMID2, Brigitte GALLIOT1*° and Stefano PIRAINO3°
SUPPLEMENTARY INFORMATION
FIGURE S1: Alignments of the novel hydrozoan Pdx/Xlox, CnoxA and CnoxC homeoproteins.
FIGURE S2: The Hox /ParaHox (H/P) homeodomain signatures.
FIGURE S3: Expression of Pdx Td, CnoxA Td, Pdx Ch and CnoxC Ch at the polyp and medusa stages.
FIGURE S4: Scheme describing the composition and the phylogenetic analyses applied to the datasets A, B and C used in this study.
FIGURE S5: Phylogenetic relationships between the cnidarian and bilaterian ParaHox HD sequences (datasetA, alignment A13, 44 HD sequences).
FIGURE S6: Phylogenetic relationships between the cnidarian and bilaterian Hox and ParaHox HD sequences (datasetB, alignment B9, 189 HD sequences).
FIGURE S7: Phylogenetic relationships between the cnidarian “orphan” Hox-related families and some eumatozoan H/P families when the alignments of full-length protein sequences were tested (dataset C).
TABLE-S1 : List of the Hox, ParaHox and Hox-related sequences used in datasets A, B and C
TABLE-S2: Table showing the respective scores of the paraHox (Gsx, Pdx/Xlox, Cdx), cnidarian CnoxA and Mox gene families obtained in both PhyML and Bayesian analyses applied to alignments A1 to A21 derived from dataset A.
TABLE-S3: Table showing the respective scores of the paraHox (Gsx, Pdx/Xlox, Cdx), Hox (PG1, PG2, PG4-PG8, PG9), cnidarian Hox-related (CnoxA, CnoxB, CnoxC, CnoxD, CnoxE), Mox and Evx gene families obtained in PhyML and Bayesian analyses applied to alignments B1 to B16 derived from dataset B.
TABLE-S4: Mean values and standard deviations of the phylogenetic scores obtained by the various H/P families tested in dataset B.
*author for correspondence: [email protected] 1 Department of Zoology and Animal Biology, University of Geneva, Switzerland; 2 Institute of Zoology, Biocenter/Pharmacenter, Basel University, Switzerland; 3 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, 73100 Lecce, Italy
- 1 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
FIGURE S1
A) Pdx / Xlox CNIDARIA (3) 1------10 Pdx Turritopsis QRRVRGISEVIDTISSLTSKGKLKTTSTNEQNQTEKEKTKDDKPPKAWQSAKV KKRNRTTYTR Pdx Clytia ------RKRTAYTR Xlox/Cdx Nemato ------RSRARTAYTA Consensus cnid. ------k2R3RT2YT2 DEUTEROSTOMES (9) Xlox Archaster ------SVVAHVRDD....LDEFAVITQINYCNVRGFVGIMFLA.....GASFVDFDE NKRTRTAYTR Splox Strongylo PLYTMASEYPMSSAKMSKNVNVGQNLQFPWMKTTKSHAHMWKANWP...... GASFADFDE NKRTRTAYTR Lox1 Ptychodera Q-QQVTHAGNPMNNVDPQ...RNQNLPYPWMKTTKSHAHMWKAQWP...... GANFNDLDE NKRTRTAYTR Xlox Diplosoma ------CTPPQFPEVDD NKRTRTAYTR IPF-1 Ciona --AGKLPAVGHPGKKE--ALAGTSSVKFPWMKNTRSHHLEWKAQWQRATGGIPAQFPEVDE NKRTRTAYTR Xlox Branchios ------NKRTRTAYTR Xlox Tetraodon QPGP.PAPGYP.DPGEQ...SRYALPFPWMKTTKSHSHAWKGQWTG...... SYVMAEAEE NKRTRTAYTR IPF-1 Homo PPAGPFPEGAEPGV...LEEPNRVQLPFPWMKSTKAHA..WKGQWAG.....GAYAAEPEE NKRTRTAYTR Xlh box8 Xenopus PHHQMPFPDDNESGTL..EERNRTLLPFPWMKSTKSHT..WKGQWTD.....GSYIMEQ.E NKRTRTAYTR Consensus deut. ------NKRTRTAYTR LOPHOTROCHOZOA (5) Xlox Phascolion ------GHTFNIEDE NKRTRTAYTR Xlox Capitella VMHHVGMAPSHHSAPDKV..KEQGKVHFPWMKTTKSHAHQWKANWS...... GANFQTFSE NKRTRTAYTR Xlox Perionyx ------NKRTRTAYTR Lox3a Hirudo ------GADAQSLDD NKRTRTAYSR Lox3 Helobdella ------VHILDD NKRTRTAYSR Consensus lopho. ------NKRTRTAYtR Consensus bilat. ------NKRTRTAYtR
CNIDARIA (3) ------20 ------30 ------40 ---4----50 ------60 Pdx Turritopsis VQQLELEKEY RYSKYISRAR RIELAKNLTL TEKHIKIWYQ NRRMKEKRDE EDALRNENSLDGQLRFF Pdx Clytia AQQLELEKEY RYNRYISRAR RIELAKNLTL TEKHIKIWYQ NRRMKEKRDE EDIMRGTTVLDPRSSYY Xlox/Cdx Nemato SQQLELEKEF LYSRYITRTR RKELANTLDL SEKHIKIWFQ NRRMKKKKTD ------Consensus cnid. 3QQLELEKEy 2Y2rYIsR2R R2ELA22L2L tEKHIKIWyQ NRRMK2Kr2e ------
DEUTEROSTOMES (9) AtXlox Archaster GQLLELEKEF HFNKYISRPR RIELAAMLNL TERHIKIWFQ NRRMKWKKEE AKRRPSGGKTDKEDGE Splox Strongylo GQLLELEKEF HFNKYISRPR RIELAAMLNL TERHIKIWFQ NRRMKWKKEE AKRKPLKQDADGSDVS Lox1 Ptychodera SQLLELEKEF HFNKYISRPR RIELAAMLNL TERHIKIWFQ NRRMKFKKEE AKRKPRGSTDSDQNDE Xlox Diplosoma KQLLELEKEF HFNKYISRPR RIELAAGLNL TERHIKIWFQ NR IPF-1 Ciona WQLLELEKEF HFSRYISRPR RIELAAMLNL TERHIKIWFQ NRRMKWKKDQ AANSKTGKVRDITAEIR Xlox Branchios GQLLELEKEF HFNKYISRPR RIELAAMLNL TERHIKIWFQ NRRMKWKKEQ ------Xlox Tetraodon AQLLELEKEF LFNRYISRPR RVELALTLNL TERHIKIWFQ NRRMKWKKEE DRRRAS IPF-1 Homo AQLLELEKEF LFNKYISRPR RVELAVMLNL TERHIKIWFQ NRRMKWKKEE DKKRGGGTAVGGGGVA Xlh box8 Xenopus AQLLELEKEF LFNKYISRPR RVELAVMLNL TERHIKIWFQ NRRMKWKKEE DKKRGRGSDPEQDSVVS Consensus deut. 5QLLELEKEF 2FNkYISRPR RiELA33LNL TERHIKIWFQ NRRMKWKKE2 LOPHOTROCHOZOA (5) Xlox Phascolion AQLLELEKEF HFNKYISRPR RIELAAMLNL TERHIKIWFQ NRRMKWKKDE ------Xlox Capitella AQLLELEKEF HFNRYITRPR RVELAAHLNL TEQHIKIWFQ NRRMKWKKDV DKKRPQQS Xlox Perionyx AQLLELEKEF HFDKYISRPR RVELAGLLNL TERHIKIWFQ NRR Lox3a Hirudo AQLLELEKEF HYDKYISRPR RLELAASLNL TERHIKIWFQ NRRMKWKKLE SGKITSTTGSAAFYGAT Lox3 Helobdella SQLLELEKEF HFDKYISRPR RVELASSLNL TERHIKIWFQ NRRMKWKKME AGKITSTTANNFEGANV Consensus lopho. AQLLELEKEF Hf2kYIsRPR RvELA34LNL TERHIKIWFQ NRRMKWKK3E Consensus bilat. 5QLLELEKEF 2f3kYIsRPR RiELA56LNL TERHIKIWFQ NRRMKWKK43
B) Pdx / Xlox signature residues (6) : N1, T4, P29, N39, H44, E60
------10 ------20 ------30 ------40 ------50 ------60 Pdx bilat NKRTRTAYtR 5QLLELEKEF 2f3kYIsRPR RiELA56LNL TERHIKIWFQ NRRMKWKK43 (14) Pdx prot D.N--T-----R --L------H--k----P------S-N- --CH------W--EE (5) Pdx deut D.N--T-----R --L------Nk----P------3M-N- --RH------W--EE (9) Pdx Clytia RKRTAYTR AQQLELEKEY RYNRYISRAR RIELAKNLTL TEKHIKIWYQ NRRMKEKRDE (3/6 residues) Pdx Turrit. V.KKRNRTTYTR VQQLELEKEY RYSKYISRAR RIELAKNLTL TEKHIKIWYQ NRRMKEKRDE (3/6 residues) Xlox/Cdx Nv .RSRARTAYTA SQQLELEKEF LYSRYITRTR RKELANTLDL SEKHIKIWFQ NRRMKKKKTD (1/6 residues) Antp Nv NHAPRSAFTT VQQLEIEKEF LYDHYVSRVR RIEIVMALDL SEKQVRTWFQ NRRMKLKREA (1/6 residues) Pdx cnid k2R3RT2YT2 3QQLELEKEy 2Y2rYIsR2R R2ELA22L2L tEKHIKIWyQ NRRMK2Kr2e (2)
P29 is also present in the PG2 and PG3 families
- 2 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
C) CnoxA CnoxA Turritopsis ------MWRMNCSTSSCETCSSRWSHVIPAPYQNRYCNPRRTVDDKYGKVRPWIVARNSVDNK CnoxA Cladonema MIKTSCPVTTSACRCCCCKEEELKR-YYHAYTPRETSSRYLSLEDKTKVRMDTSPTFSRKFENTHN cnox-2 Podocoryne ------MPLKLLHLPAAVIHLVVKVNTGQLVTKRKGTRIILLPVRPTIGIDHICTEK
CnoxA Turritopsis HLVSRDYRPPCHASR....VSLCHCRSCHL...... AAETQYITYFARQFIPRECNCIDCDREG CnoxA Cladonema SRSVFREESIDYYNTYKSRIETERYY....LKPQLGRSCY.....NMQLYPPV..YHDFHYGFYQDD cnox-2 Podocoryne EYDRSYYRYYNYYPRYDKRIVTCTNTCCMGFSSAVRKSCAPHYPSHNYYHTPVEQYNDYFFDFDRDG
1------10 ------20 ------30 ------40 ------50 ------60 CnoxA Turritopsis MKRFRTSSNT SQLTELEKEF QQNKYLTRRR RVELAVGLKL SEKQVKVWFQ NRRMKWKKQT CnoxA Cladonema LKRFRTSFST TQLTELEREF KYNKYLTRRR RVDLAVNLSL SEKQVKVWFQ NRRMKWKKQG cnox2 Podocoryne LKRFRTSFNT SQLTELEKEF QYNKYLTRRR RVELSVSLNL TEKQVKVWFQ NRRMKWKKQN consensus 2KRFRTS22T sQLTELEkEF 22NKYLTRRR RVeL2V3L3L sEKQVKVWFQ NRRMKWKKQ3
CnoxA Turritopsis KFEEEEEEGRFT CnoxA Cladonema KDEKEEDIGSSDLNTTTS cnox-2 Podocoryne KGEDLDEKEETE------
D) CnoxC CnoxC Clytia ------SAILEPNFPQGLNNNNTPPPLSSPSYLRNDANRYGGTGTPTTDSLEQSAFSHI.. cnox1 Eleutheria MEISRLQQIDSTQNNDNDDRYHYAHNSRTPALISSHATNFRSAFLPTTLSSHQYQSSVIRNIDNSTD
CnoxC Clytia SSPDTTANSPPMTEPTYSTTAGSTFDTSVQ.SYLANTSLANPPSTSAMS.FYNTPTSLLSNQHLSTDYS cnox1 Eleutheria TTSFGSFSHGNESSTHNSLPYPPNSTHIELPSYLSNTGL..PPATSSLSAFYNPIHSSSQAHLAEYQQW
1------10 ------20- CnoxC Clytia QFGYASPS-NYFYSSGYPSIGSGYNTYPGMTNTGPLPAGPWICRDID TKRKRMTYSR KQLLELEKEFH cnox1 Eleutheria PYGNVSPSGNYIYGYGSPMNNGFNPYSSVDSNGFAGSSSWLY.RDID SKRKRMTYSR KQLLELEKEFH cnox3 H.vulgaris ------SQIQTKH SKRKRMTYSK FQLHELEKEFS cnox1 H.magnipap ------MNSVFCFSDILVMSQIQTKH SKRKRMSYSK FQLHELEKEFS hoxC2 H.magnipap ------D SKRKRMTYSR HQLLELEKEFH hoxC3 H.magnipap ------D SKRKRMTYSR HQLLELEKDFH consensus ------sKRKRMtYSr 3QL2ELEKeF2
------30 ------40 ------50 ------60 CnoxC Clytia FSQFLKKER RSDLAKQLSL TERQIKIWFQ N cnox1 Eleutheria LSHFLKKER RVDLAKQLNL SERQIKIWFQ NRRMKFKKEN KKTSSSESIMNQEAPKIEEHPDIISENV cnox3 H.vulgaris FNHFLRKER RTELAKLLKF SDRQIKIWFQ NRRMKFKKEI NKVKLRNLPEDTNMS------cnox1 H.magnipap FNHFLRKER RTELAKLLKF SDRQIKIWFQ NRRMKFKKEI NKVKLRNLPEDTNMS------hoxC2 H.magnipap FNHFLKKER RTELSKKLNL SERQIKIWFQ NRRMKFKKEM ------hoxC3 H.magnipap FNHFLKKER RAELAKQLNL SERQIKIWFQ NRRMKFKKEV ------consensus F2HFLkKER R4eLAK3L32 seRQIKIWFQ NRRMKFKKE4 cnox1 Eleutheria TEQQETNENIENEEKDKKMNIQQINLNDMKNFMMSHVARI
Figure S1: Alignments of the novel hydrozoan Pdx/Xlox, cnoxA and cnoxC homeoproteins. A-D) Alignment of the Pdx/Xlox (A, B), CnoxA (C) and CnoxC (D) homeoproteins with their closest relatives (see accession numbers in Table-S1). The homeodomain is boxed; the underlined RRMKWKK motif corresponds to a nuclear localization signal (Moede T, Leibiger B, Pour HG, Berggren P, and Leibiger IB, Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1. 1999. FEBS Lett. 461:229-234). B) Each cnidarian Pdx/Xlox homeodomain sequence was compared to the deuterostome and lophotrochozoan derived consensus; signature residues are written bold, those that are specific for the Pdx/Xlox family are written red. The number of Pdx/Xlox signature residues present in each cnidarian HD sequences is indicated on the right. D) Note that in the CnoxC family, the cnox-1 Hm and cnox-3 Hv sequences are identical.
- 3 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
FIGURE S2 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 .hh.hh.hh. .ih.hhh..h ....i..... h.hhh....h ...... hiih ihi.h.hh.. ANTP-CLASS *RK*RT*FT* *QL*ELEK*F ****YLS**E R*ELA**L*L TE*QVKVWFQ NRR*K*KR** KR YS V R R VT R IS S I I Y K RK Non-Hox ------*------S*-E -*--*------*T------*------Hox/paraHox ------L----E- HFN---TR-R -I------R------M------PRD-CLASS ------PDi-- -E------R-Q---3 ---A------
Mox cons 4RKERTAFsK 5Qi4eLE4EF 732NYLTRLR RYEiAV3L4L 4ERQvKVWFQ NRRMKWKR3k (8) Mox bilat 3-----A-tK 3-i3---2-- 22H------3-3- 2------2--2- (3) Mox cnid 2-----2-s2 3-l3---2-- 5RN------S-2- 3------W--2- (5)
Evx cons 7RR3RT3FTR eQl4rLEKEf 7rENYvSR2r R3ELA45L5L 3E4TIKvWFQ NRRMK3KRQR (13) Evx bilat 5--Y--3--- e--3------6--N----P- -C---33-4- P-s------D--Q- (10) Evx anth 2--2--A--- 2--2------3--2----T- -3---23-2- 2-2------2--R- (3) CnoxA hydroz 2KRFRTS22T sQLTELEkEF 22NKYLTRRR RVeL2V3L3L sEKQVKVWFQ NRRMKWKKQ3 (3) PG1 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 PG1 cons 6773R44F34 42l3ELEKEF 2f2kYLtR4R RiE2A85L8L 3E4QiKIWFQ NRRMK3Kk28 (16) PG1 bilat 235G-TN-t2 kQLT------H-N-----A- ---I-54-6- N-T------Q--R6 (8) PG1 cnid 45kK-33-32 42iv------2-2-----4- ---2-54-3- t-4------3--E3 (8) PG1 anth SNKKRFTFTQ RQLV------HFS-----T- ---IATs-KL TET------W-REF (3) PG1 hydr 33kKR22F32 32Ii------2YN-----2- -V-2A33-eL sE2------Q-kEQ (5)
PG2-Gsx 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 PG2 cons 3rR3RT3yTN 3QLLELEKEF HfNKYlC43R R4EiA44L3L TERQVKvWFQ NRRMk5Kr34 (8) PG2 bilat 2--L--A--- T------rP- -i---23-D------3--Q3 (6) PG2 anth s--2--S--- k------2s- -R---2A-2------2--DE (3)
Gsx cons 3kR2RTAftS 4QLLeLEkEF 55N3YLSRLR RI2IA37L5L sEKQVKIWFQ NRRVK5KK44 (17) Gsx bilat S--M------T------43-M------E--24-3------4--33 (8) Gsx cnid 3--I------2------22-R------Q--A3-D------W--DK (9) PG3-Pdx/Xlox 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 PG3 bilat 5KR3RTAYtS AQLVELEKEF HFNRYLCRPR RiEMA32L3L sERQIKIWFQ NRRMk3KK34 (9)
Pdx cons 3KR4RTAYtR 4Q2LELEKEf 3f3kYIsR3R RiELA76L3L tErHIKIWfQ NRRMK4Kk54 (17) Pdx bilat NK-T--A--- 5-L------2-3-----P------56-N------W--43 (14) Pdx cnid k2-3--2--- 3-Q------2-2-----2------22-2------2--2e (3) Antp Nv NHAPRSAFTT VQQLEIEKEF LYDHYVSRVR RIEIVMALDL SEKQVRTWFQ NRRMKLKREA PG9-Cdx 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 PG9 bilat 5RKKR2PYtk fQT22LE2EF L4N3Yltr3r R5Ei234l4L tERQVKiWFQ NRRMK4KK42 (7) cdx bilat KDKYRvVYtd 4QrlELEKEF 5434YITI4R K3ElA107l5L sERQvKIWFQ NRRAKeRK57 (13) cnox4-Ed AMRSRPCFSS HQTRELEKEF LVCQYVTRRR RIELAFSLNL SEKQIKTWFQ NRRVKERKQK (1) Pnox6 Pn KMRLRTSFHS HQLNILEKEF LVNMYLTRMR RIEMASALEL GEKQIKIWFQ NRRVRLKKIC (1) PG9-Cdx related 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 CnoxB 3rRKRTAYtr 2QL42LE3EF 3323FLtreR R322A23L4L sERQiKIWFQ NRRMK3KK24 (4) CnoxC sK---Mt-Sr 3-L2E--K-- 2F2H--k--- -4eLAK3-32 s-----I------M-F--E4 (6) CnoxD 2K---2S-22 2-I2E--N-- 22s2--s--- -i2LS22-NL T-----T------M-s--22 (2) CnoxE k2---M2-t2 3-3LE--K-- 2ysR--2--- -2e2232-3L t-----I------3-2--33 (3)
PG4-PG8 1~~~|~~~10 ~~~~|~~~20 ~~~~|~~~30 ~~~~|~~~40 ~~~~|~~~50 ~~~~|~~~60 PG4 bilat 3KR3RTAYTR 2QvLELEKEF HFNRYLTRRR RIEIAH3L4L sERQIKIWFQ NRRMKWKKdN (10) PG5 bilat 5KR5RT3YTR 3QTLELEKEF HFNRYLTRRR RIEIAH5L4L TERQIKIWFQ NRRMKWKKe3 (9) PG6 bilat 7kR4RQTYTR 3QTLELEKEF 23NRYLTRRR RIEIA35L4L tERQIKIWFQ NRRMKWKKe5 (14) PG7 bilat RKRGRQTYTR YQTLELEKEF HFNrYLTRRR RIEIAHALCL TERQIKIWFQ NRRMKWKKEN (5) PG8 bilat rkRGRQTYtR 3QTLELEKEF 4FN3YLTRrR RIEIAH6L3L tERQIKIWFQ NRRMK5KKE7 (8)
FIGURE S2: The Hox /ParaHox (H/P) homeodomain signatures. Alignment of the ParaHox, Hox and Hox-related consensus homeodomain sequences derived from bilaterian (bilat) and cnidarian species (cnid; hydroz : hydrozoan ; anthoz : anthozoan). The number of sequences used to derive the consensus are indicated on the right. The signature residues for a given H/P family are written bold-red when unique, plain-red when shared by two H/P families and bold-
- 4 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information) black when shared by more than two H/P families. Lowercase letters indicate conservative substitutions (E/D, F/Y, I/L/V, K/R, N/Q, S/T) ; dashes represent identical residues ; numbers in the consensus indicate the number of different residues observed at a given position. Some derived sequences tested in the phylogenetic analyses were included in the alignment (Cnox4-Ed, Pnox6 Pn, XloxCdx Nv). h : highly conserved residue; i : invariant residue among all homeodomains ; asterisk : variable position.
FIGURE S3
FIGURE S3: Expression of Pdx Td, Cnox-A Td and Cnox-C Ch at the polyp and medusa stages. Expression was detected by semi-quantitative RT-PCR after 24 (lanes 1, 5), 30 (lanes 2, 6), 36 (lanes 3, 7) or 40 (lanes 4, 8, 9) cycles on polyp (lanes 1-4) or medusa (lanes 5-8) cDNAs.
- 5 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
FIGURE S4
FIGURE S4: Scheme describing the composition and the analyses applied to the datasets A, B and C used in this study.
- 6 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
FIGURE S5: Phylogenetic relationships between the cnidarian and bilaterian ParaHox HD sequences (datasetA). Tree drawn using the PhyML program, corresponding to the alignment A13 (44 HD sequences) also tested in Bayesian analysis. For each node, the bootstrap proportion (BP) is indicated at the top and the corresponding posterior probability (PP) at the bottom. Gene families significantly supported are indicated on the right. The only difference with the tree A21 (42 HD sequences) presented in Fig.3 is the addition of the Xlox/Cdx-Nv and Antp-Nv. Note that the score of the Pdx/Xlox family (SeuPdx/Xlox) is drastically reduced from 173 in A21 to 13 in A13 as in the presence of the Xlox/Cdx-Nv and Antp-Nv sequences the Pdx/Xlox family is disintegrated in Bayesien analysis.
- 7 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
FIGURE S6: Phylogenetic relationships between the cnidarian and bilaterian Hox and ParaHox HD sequences (datasetB). Tree drawn using the MrBayes program, corresponding to the alignment B9 (189 homeodomain sequences) also tested in PhyML analysis. For each node, the PP and BP are indicated (PP/BP). Gene families significantly supported are indicated on the right. The only difference with the tree B13 (187 HD sequences) presented in Fig.4 is the addition of the Xlox/Cdx-Nv and Antp-Nv sequences. Note that the score of the Pdx/Xlox family (SeuPdx/Xlox) is drastically reduced from 166 to 68. CnoxC*: Cnox1 Hm, Cnox3 Hv, HoxC2 Hm, HoxC3 Hm, Cnox1 Ed, CnoxC Ch.
- 8 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
FIGURE S7: Phylogenetic relationships between the cnidarian “orphan” Hox-related families and some eumatozoan H/P families when the alignments include the full-length protein sequences (dataset C). Bootstrap values obtained after 100 replicates with the PhyML program are indicated. Partial cnidarian sequences restricted to the HD were not included (HoxC2 Hm, HoxC3 Hm, Cnox-D Hm). In both alignments shown here, Cnox-A grouped together with Gsx. Note that Cnox4-Ed no longer grouped with the Cdx family (A) when the XloxCdx sequence was added, as shown in (B) where XloxCdx was at the root of the Cdx family.
- 9 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
TABLE-S1 : List of the Hox, ParaHox and Hox-related sequences used in datasets A, B and C
GENE FAMILIES PHYLA SPECIES NAMES GENE NAMES ACCESSION N. Pdx / Xlox (18 genes) Cnidaria Cnidaria: hydrozoan Clytia hemisphaerica Pdx Ch FM207043, FM207044 Turritopsis dohrnii Pdx Td FM207048 Cnidaria: anthozoan Nematostella vectensis XloxCdx, NVHD065 DQ500749,DQ301955 Lophotrochozoa Sipuncula Phascolion strombi Xlox Ps AF363233 Annelida: polychaeta Capitella sp. Xlox Casp DQ102390 Annelida: clitellata, oligochaeta Perionyx excavatus Xlox Pe AY769113 Annelida: clitellata, hirudinida Hirudo medicinalis Lox3a Him S79684 Helobdella triserialis Lox3 Ht Y09622 Deuterostomes Echinodermata: asterozoa Archaster typicus Xlox At AF439973 Echinodermata: echinozoa Strongylocentrotus purpuratus Splox/Xlox Sp AF541970 Hemichordata Ptychodera flava Lox2 Pf AY436763 Urochordata: ascidiacea Ciona intestinalis IPF1 Ci AJ296167 Urochordata: Diplosoma listerianum Xlox Dl AF375978 Cephalochordata Branchiostoma floridae Xlox Bf AF052464 Vertebrata Xenopus laevis XlHBOX8 Xl X16849 Tetraodon nigroviridis Xlox Tn Q4S567 Mus musculus IPF1 Mm X74342 Homo sapiens IPF1 Hsa AF035260 Gsx (21 genes) Cnidaria Cnidaria: hydrozoan Hydra viridissima Cnox2 Cv AJ871179 Hydra vulgaris Cnox2 Hv AJ277388 Eleutheria dichotoma Cnox2 Ed Kuhn et al., 1996 Podocoryne carnea Gsx Pc AF268446 Hydractinia symbiolongicarpus Cnox2 Hs = Gsx Hs AF031953 DQ298519 Sarsia sp. Cnox 2 Ssp AF285145 Cnidaria: anthozoan Nematostella vectensis Anthox2 Nv AF085283 Acropora millipora Cnox2 Am AF245689 Cnidaria: scyphozoan Cassiopea xamachana Scox2 Cx AF124592 Placozoa Trichoplax adhaerens Trox2 Ta AY319762 Lophotrochozoa Annelida: polychaeta Capitella sp. Gsx Casp DQ132894 Annelida: clitellata, oligochaeta Perionyx excavatus Gsx Pe AY769112 Mollusca Eupryma scolopes Gsx Es AY675202 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Ind Dm AF095926 Deuterostomes Hemichordata Ptychodera flava Gsx Pf AY436761 Urochordata: ascidiacea Ciona intestinalis Gsx Ci AF305500 Diplosoma listerianum Gsx-Dl AF375980 Urochordata: appendicularia Oikopleura dioica Gsx-Od AY705721 Cephalochordata Branchiostoma floridae Gsx Bf AF052463 Vertebrata Oryzias latipes Gsh1 Ol AF035573 Mus musculus Gsh1 Mm U21224 Cdx / cad (14 genes) Cnidaria Cnidaria: hydrozoan Eleutheria dichotoma Cnox4 Ed U41841 Lophotrochozoa Platyhelminthes Symsagittifera roscoffensis Cad Sr AY117550 Nemertea Lineus sanguineus Cdx Ls P81193 Annelida: polychaeta Capitella sp. Cad Casp DQ102389 Platynereis dumerilii Cad Pd DQ188196 Annelida: clitellata, oligochaeta Perionyx excavatus Cdx Pe AY769114 Mollusca Patella vulgata Cdx Pv AJ518062 Ecdysozoa Arthropoda: crustacea Artemia franciscana Cad As AJ567452 Arthropoda: hexapoda, insecta Bombyx mori Cad Bm D16683 Drosophila melanogaster Cad Dm M21070 Deuterostomes Urochordata: ascidiacea Halocynthia roretzi Cad Hr AB031032 Cephalochordata Branchiostoma floridae Cdx Bf AF052465 Vertebrata: teleost Oncorhynchus mykiss Cdx1 Om AY434713 Vertebrata: mammals Mus musculus Cdx1 Mm M37163 PG1 / lab (18 genes) Cnidaria Cnidaria: hydrozoan Chlorohydra viridissima Cnox1 Cv X64625 Hydra vulgaris Cnox1 Hv Q9NFW5 Hydra vulgaris Cnox3b Hv L22787 Hydra magnipapillata Cnox4 Hm S39067 Podocoryne carnea Cnox1 Pc X81455 Eleutheria dichotoma Cnox5 Ed U41842 Cnidaria: anthozoan Nematostella vectensis Anthox6 Nv, HoxA DQ206301 Nematostella vectensis Anthox6a Nv, NVHD060, HoxB DQ206321 Metridium senile Anthox6 Ms AY096246 Acropora formosa AntpC Af S36771 Lophotrochozoa Platyhelminthes Polycelis nigra Pnox3 Pn L41848 Nemertea Lineus sanguineus Hox1 Ls Y16570 Annelida: polychaeta Nereis virens Lab Nvi AF151663 Ecdysozoa Arthropoda: chelicerata, arachnida Cupiennius salei Lab Cs AJ007431 Arthropoda: hexapoda, insecta Drosophila melanogaster Lab Dm X13103 Deuterostomes Urochordata: appendicularia Oikopleura dioica Hox1 Od AY449462 Cephalochordata Branchiostoma floridae Hox1 Bf Z35142 Vertebrata Mus musculus Hoxb1 Mm X53063 PG2 / Pb (9 genes) Cnidaria Cnidaria: anthozoan Nematostella vectensis Anthox7 Nv, HoxC AF020962 Anthox8a Nv, HoxDa AF020963 Lophotrochozoa Annelida: polychaeta Chaetopterus variopedatus Hox2 Cva AF163857 Ecdysozoa Arthropoda: hexapoda, insecta Bombyx mori Hox2 Bm AB120762 Drosophila melanogaster Pb Dm X63729 Deuterostomes Urochordata: ascidiacea Ciona intestinalis Hox2 Ci AJ535672 Cephalochordata Branchiostoma floridae Hox2 Bf AB028207 Vertebrata Lampetra japonica Hox2 Lp AY497314 Mus musculus HoxA2 Mm M95599
- 10 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
PG3 / Zen (12 genes) Lophotrochozoa Nemertea Lineus sanguineus Hox3 Ls Y16571 Brachiopoda Lingula unguis Hox3 Lu AF144673 Annelida: polychaeta Nereis virens Hox3 Nvi AF151665 Annelida: clitellata, oligochaeta Perionyx excavatus Hox3a Pe AY439319 Mollusca Euprymna scolopes Hox3 Es AY330185 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Zen Dm X78058 Tribolium castaneum Zen Tc X97819 Tribolium castaneum Ftz Tc AF321227 Deuterostomes Urochordata: ascidiacea Ciona intestinalis Hox3 Ci AB210495 Cephalochordata Branchiostoma floridae Hox3 Bf X68045 Vertebrata Lampetra japonica Hox3d Lj AB125270 Mus musculus Hoxb3 Mm X66177 PG4 / Dfd (19 genes) Lophotrochozoa Platyhelminthes Schistosoma mansoni Dfd Sm AY348575 Dugesia japonica Plox3 Dj AB024407 Nemertea Lineus sanguineus HoxA4 Ls P02833 Brachiopoda Lingula unguis Lox4 Lu AF144678 Bryozoa Bugula turrita Dfdb Bt AY497424 Annelida Nereis virens Dfd Nvi AF151666 Echiura Urechis unicinctus Lox4-Uu AY770072 Mollusca Euprymna scolopes Lox4 Es AY052759 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Dfd Dm X05136 Bombyx mori Dfd Bm D83534 Arthropoda: crustacea Artemia sanfranciscana Dfd As DQ371220 Deuterostomes Hemichoradata Ptychodera flava Hox4 Pf AY436754 Urochordata: ascidiacea Ciona intestinalis Hox4 Ci AJ535673 Cephalochordata Branchiostoma floridae AmphiHox4 Bf AB028208 Vertebrata Latimeria menadoensis HoxB4 Lm AY183736 Latimeria menadoensis HoxC4 Lm AY183742 Oreochromis niloticus Hox On AY757325 Mus musculus Hoxa4 Mm S70444 Mus musculus HoxC4 Mm D11328 PG5 / Scr (15 genes) Lophotrochozoa Brachiopoda Lingula unguis Scr Lu AF144674 Bryozoa Bugula turrita Lox5-Bt AY497425 Annelida Nereis virens Scr Nvi AF151667 Mollusca Eupryma scolopes Scr Es AY052756 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Scr Dm X14475 Bombyx mori Scr Bm D83533 Deuterostomes Echinodermata Heliocidaris erythrogramma Hehbox9 He U31563 Hemichordata Ptychodera flava Hox5 Pf AY436755 Urochordata: ascidiacea Ciona intestinalis Hox5 Ci AJ002028 Cephalochordata: Branchiostoma floridae Hox5 Bf Z35145 Vertebrata Lampetra japonica Hox5w Lj AB125277 Brachydanio rerio HoxC5 Br X68324 Triturus viridescens HoxC5 Tv M84001 Latimeria menadoensis HoxC5 Lm AY183743 Mus musculus HoxC5 Mm U28071 PG6 / Antp (11 genes) Lophotrochozoa Platyhelminthes Dugesia tigrina Dutarh4 Dt Z34089 Nemertea Lineus sanguineus Hox6 Ls Y16572 Annelida: polychaeta Nereis virens Lox5 Nvi AF151671 Annelida: clitellata, hirudinida Helobdella robusta Lox5 Hr AF004387 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Antp Dm X03790 Deuterostomes Echinodermata Tripneustes gratilla HB3 Tg X13146 Hemichordata Ptychodera flava Hox6 Pf AY436756 Urochordata: ascidiacea Ciona intestinalis Hox6/7 Ci AJ535674 Cephalochordata Branchiostoma floridae Hox6 Bf Z35146 Vertebrata: Brachydanio rerio Hoxc6B Br AF071266 Mus musculus Hoxb6 mm M18166 PG7 / Ubx (4 genes) Lophotrochozoa Nemertea Lineus sanguineus Hox7 Ls Y16573 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Ubx Dm X05723 Deuterostomes Cephalochordata Branchiostoma floridae Hox7 Bf Z35147 Vertebrata Mus musculus Hoxb7 Mm M18167 PG8 / abdA (7 genes) Lophotrochozoa Platyhelminthes Polycelis nigra Pnox1a Pn L41845 Annelida: clitellata, hirudinida Hirudo medicinalis Lox2 Him X17566 Annelida: polychaeta Nereis virens Lox4 Nvi AF151669 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster abdA Dm X54453 Deuterostomes Echinodermata Tripneustes gratilla HB1 Tg X14508 Cephalochordata Branchiostoma floridae Hox8 Bf Z35148 Vertebrata Lampetra japonica Hox8p Lj AB125273 PG9 / abdB (8 genes) Lophotrochozoa Nemertea Lineus sanguineus Hox9 Ls Y16574 Annelida: polychaeta Nereis virens Post2 Nvi AF151673 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster AbdB Dm U31961 Deuterostomes Echinodermata Tripneustes gratilla HB4 Tg X13147 Hemichordata Ptychodera flava Hox9/10 Pf AY436757 Urochordata: ascidiacea Styela clava Hox2 Sc S73920 Cephalochordata Branchiostoma floridae Hox9 Bf Z35149 Vertebrata Mus musculus Hoxb9 Mm S66855 Cnox A (3 genes) Cnidaria Cnidaria: hydrozoan Turritopsis dohrnii CnoxA Td FM207047 Cladonema radiatum CnoxA Cr FM207046 Podocoryne carnea Cnox2 Pc AB014684 Cnox B (4 genes) Cnidaria Cnidaria: hydrozoan Hydra magnipapillata HoxB Hm Chourrout et al. 2006 Podocoryne carnea Cnox4 Pc AY036893 Cnidaria: scyphozoan Cassiopea xamachana Scox3 Cx AF124593 Cnidaria: anthozoan Nematostella vectensis Anthox1 Nv (HoxF) AF020953
- 11 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
Cnox C (6 genes) Cnidaria Cnidaria: hydrozoan Clytia hemisphaerica CnoxC Ch FM207045 Eleutheria dichotoma Cnox1 Ed DQ451870 Hydra vulgaris Cnox3 Hv AJ252182, Q9NFW4 Hydra magnipapillata Cnox1 Hm Z22638 Hydra magnipapillata HoxC2 Hm Chourrout et al. 2006 Hydra magnipapillata HoxC3 Hm Chourrout et al. 2006 Cnox D (2 genes) Cnidaria Cnidaria: hydrozoan Hydra magnipapillata HoxD Hm Chourrout et al. 2006 Eleutheria dichotoma Cnox3 Ed U41840 Cnox E (3 genes) Cnidaria Cnidaria: scyphozoan Cassiopea xamachana Scox1 Cx AF124591 Cnidaria: scyphozoan Cassiopea xamachana Scox4 Cx AF124594 Cnidaria: anthozoan Nematostella vectensis Anthox1a Nv (HoxE) U42728 Mox (9 genes) Cnidaria Cnidaria: hydrozoan Hydra magnipapillata Hox5 Hm, Mox Hm Z22640, Nematostella vectensis MoxA Nv DQ500752 Nematostella vectensis MoxB Nv DQ500753 Nematostella vectensis MoxC Nv DQ500754 Nematostella vectensis MoxD Nv DQ500755 Lophotrochozoa Platyhelminthes Dugesia tigrina Dutarh2 Dt Z32525 Mollusca Haliotis rufescens Hrox1 Hr X75217 Deuterostomes Urochordata: ascidiacea Ciona intestinalis Mox Ci AB210559 Vertebrata Mus musculus Mox1 Mm Z15103 Evx / eve (12 genes) Cnidaria Cnidaria: hydrozoan Sarsia sp. Evx Ss Q9BJW6 Cnidaria: anthozoan Acropora formosa Eve Af S36770 Nematostella vectensis Evx Nv DQ206338 Lophotrochozoa Annelida: clitellata, hirudinida Helobdella robusta Eve Hr AF409098 Annelida: polychaeta Platynereis dumerilii Eve Pd DQ188195 Ecdysozoa Arthropoda: hexapoda, insecta Drosophila melanogaster Eve Dm P06602 Bombyx Mori Eve Bm D38486 Arthropoda: crustacea Artemia sanfranciscana Eve As AJ567453 Deuterostomes Urochordata Ciona intestinalis EvxA Ci AB210415 Cephalochordata Branchiostoma floridae EvxA Bf AF374191 Vertebrata Oryzias latipes Evx Ol AB232920 Mus musculus Evx1 Mm X54239 Orphans (4 genes) Cnidaria Cnidaria: anthozoan Nematostella vectensis Antp-Nv, NVHD117 DQ500750 Lophotrochozoa Platyhelminthes Polycelis nigra Pnox6 Pn L41852 Annelida: clitellata, hirudinida Hirudo medicinalis Lox1 Him Q25097 Deuterostomes Urochordata: ascidiacea Diplosoma listerianum Hox5 Dl Q86DH0 Outgroups (3 genes) Cnidaria Cnidaria: hydrozoan Podocoryne carnea Cnox3 Pc AB014685 Hydra Magnipapillata Nk2 Hv AF012538 Chlorohydra viridissima Msh Cv X64629
- 12 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
TABLE-S2
TABLE-S2: Table showing the respective scores of the paraHox (Gsx, Pdx/Xlox, Cdx), cnidarian CnoxA and Mox gene families obtained in both PhyML and Bayesian analyses applied to alignments A1 to A21 derived from dataset A. The composition of each alignment is depicted as a column and the bootstrap prevalence (BP) and posterior probablility (PP) values supporting the Gsx and Pdx/Xlox families are indicated on left and rigtht semi-columns respectively. The score value (S) that corresponds to [BP + (100xPP)] is shown on the line corresponding the gene family name. S maximal value is 200; the 4 highest S values obtained by each gene family are underlined whereas the scores lower then 120 are written red. The BP and PP values are only given when S values vary according to the input sequences. nPP: no posterior probability, N = total number of sequences included in a given alignment. The names of cnidarian sequences are written red; hatched squares indicate orphan positions in either PhyML and/or Bayesian tree. The A21 tree is drawn in Figure 3A and a comparative analysis of the different scores is shown in Figure 3C. The Xlox/Cdx-Nv, Antp-Nv, Cnox4-Ed sequences exhibit a versatile clustering and are boxed with colored backgrounds that indicate to which family they belong in a given tree. The alignments are available upon request.
- 13 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
TABLE-S3
- 14 - Quiquand et al. Early evolution of Hox/ParaHox genes (supplementary information)
TABLE-S4: Table showing the respective scores of the paraHox (Gsx, Pdx/Xlox, Cdx), Hox (PG1, PG2, PG4-PG8, PG9), cnidarian Hox-related (CnoxA, CnoxB, CnoxC, CnoxD, CnoxE) and, Mox and Evx gene families obtained in PhyML and Bayesian analyses applied to alignments B1 to B16 derived from dataset B. The composition of each alignment is depicted as a column and the bootstrap prevalence (BP) and posterior probablility (PP) values supporting each gene family are indicated on left and rigtht semi-columns respectively. The score value (S) that corresponds to [BP + (100xPP)] is shown above on the line corresponding to gene family name. S maximal value is 200 and the 4 highest S values obtained by each gene family are underlined. nPP: no posterior probability, N = total number of sequences included in a given alignment. The names of cnidarian sequences are written red; hatched squares indicate orphan positions in either PhyML and/or Basesian tree. Alignments B15 and B16 were only tested in Bayesian analysis. Within the Pdx/Xlox family the IPF1 Hsa, Lox3 Ht and Xlox Tn were tested as an independent subset of the other Pdx/Xlox bilaterian Pdx HD sequences. The number of sequences clustering in either PhyML or Bayesian analyses can vary as some ambiguous HD sequences exhibit a versatile clustering (Antp Nv, HoxB Nv, Cnox4 Ed, Cnox3 Pc in cnidarians, Zen-Dm, Pnox6-Pn in bilaterians). The color of the background indicates to which family those sequences are related within a given tree. Three B16 is depicted in Figure 4, a comparative analysis of the respective scores of the ParaHox, PG1, PG2, PG3 and cnidarian Hox-related families is shown in Figure 5A-F. The analysis of the metagrouping displayed by those gene families in the B1 to B16 trees are displayed in Figure 6A. The alignments are available upon request.
H/P Number of Eumetzoan Bilaterian Cnidarian Medusozoan families S values scores scores scores scores (Sme) eu bi cn (N) (S ) (S ) (S ) Gsx N = 16 121 ± 26.5 136 ± 26.2 151 ± 32.8 158 ± 29.2 Pdx N = 16 112 ± 52.1 N = 9 * 148 ± 24.3 Cdx N = 8 27 ± 13.8 198 ± 2.7
PG1 N = 16 45 ± 32.8 186 ± 6.0 102 ± 32.3 PG2 N = 16 66 ± 41.7 137 ± 42.3
PG3 N = 16 77 ± 57.7 PG9 N = 15 125 ± 35.8
Mox N = 16 174 ± 15.1
Evx N = 13 191 ± 11.7
CnoxA N = 16 182 ± 8.4 CnoxB N = 14 157 ± 12.4 CnoxC N = 15 177 ± 11.1 CnoxD N = 6 169 ± 4.9 CnoxE N = 15 141 ± 34.2
Table-S4: Mean values and standard deviations of the phylogenetic scores obtained by the various H/P families tested in dataset B. The corresponding graph is depicted in Figure 5G. N: number of S values used to calculate the mean value. Each S value corresponds to a given alignment. *: alignments that do not contain the XloxCdx and Anpt-Nv sequences were selected. Anthozoan scores are not given the low number of species wheresequences represent each family.
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II.2. CHAPITRE 2 : Analyse d’un régulateur précoce de la neurogénèse chez Nematostella vectentsis, Anthox2
Nous avons confirmé que les cnidaires possèdent un répertoire non négligeable de gènes Hox-like (antérieurs et postérieurs) plutôt divergents, et montré que les trois familles ParaHox (Gsx, Pdx, Cdx) sont présentes et exprimées. À ce jour, les seuls groupes paralogues non identifiés chez les cnidaires sont les groupes PG3 à PG8 (groupes paralogues « centraux »). Si les cnidaires disposent de la quasi-totalité des familles Hox et ParaHox dans leur génome, alors comment les utilisent-ils ?
Bien que les gènes Hox de cnidaires soient exprimés de manière différentielle le long des axes de la larve et du polype, une corrélation entre les axes de polarité des bilatériens et ceux des cnidaires n’est pas clairement établie. La colinéarité spatiale semble donc plus vraisemblablement être une innovation bilatérienne (Gauchat et al., 2000; Schierwater and Desalle, 2001; Chourrout et al., 2006; Kamm et al., 2006; Lee et al., 2006). Les gènes Hox de cnidaires pourraient accomplir des fonctions d’avantage liées à des processus cellulaires qu’à la mise en place du plan général d’organisation du corps (Chiori et al., 2009).
Les gènes ParaHox ont des fonctions conservées chez les bilatériens. Les gènes Pdx et Cdx sont tous deux impliqués dans la mise en place de l’intestin et s’expriment principalement dans les dérivés endodermiques (Offield et al., 1996; Guo et al., 2004). La fonction du gène Gsx semble d’avantage conservée dans la mise en place du système nerveux (Hsieh-Li et al., 1995; Li et al., 1996; Weiss et al., 1998).
Notre laboratoire a démontré l’implication du gène Cnox2 (orthologue de Gsx) de l’hydre dans le maintien du système nerveux apicale dans les conditions homéostatiques ainsi que pour son développement lors de la régénération (Miljkovic- Licina et al., 2007).
Nous avons voulu savoir si l’orthologue de Nematostella (Anthox2) jouait un rôle dans le développement nerveux comme c’est le cas chez les bilatériens. Une approche par perte de fonction a été utilisée par le biais de l’injection de morpholinos. Par ailleurs, la majeure partie de ce travail a consisté à mettre au point la technique d’injection de gènes rapporteurs, utilisée pour la première fois chez Nematostella, afin d’étudier le promoteur de ce gène ParaHox.
89 L’impact de ce travail est à deux volets : d’une part nous apportons une contribution à la compréhension de la fonction ancestrale du gène Gsx; d’autre part nous utilisons une nouvelle méthode permettant l’analyse fonctionnelle des gènes régulateurs au cours du développement de Nematostella.
90 Preliminary version
The ParaHox gene Gsx/Anthox2 regulates neurogenesis in developing Nematostella vectensis
(Preliminary version)
Manon QUIQUAND and Brigitte GALLIOT
INTRODUCTION ...... 2 MATERIALS & METHODS ...... 3 Animal culture and gametogenesis induction...... 3 Phylogenetic and genomic analyses ...... 3 RT-PCR expression analyses...... 4 In situ hybridization (ISH)...... 4 Immunohistochemistry (IHC)...... 4 Morpholino (MO) injection...... 4 Cloning of Anthox2 upstream sequences...... 4 Reporter gene constructs...... 4 RESULTS...... 5 The three ParaHox families, Gsx, Pdx/Xlox and Cdx, are highly conserved in cnidarians...... 5 Extended conservation of the Gsx / Anthox2 / cnox2 structure...... 6 Onset of Anthox2 expression precedes neurogenesis in developing Nematostella...... 6 Staging of neurogenesis in developing Nematostella ...... 6 Anthox2 loss of function assays in developing Nematostella...... 10 Anthox2 inhibition decreases the survival rate...... 10 Anthox2 inhibition disrupts the !-tubulin pattern in the ectoderm...... 12 Anthox2 silencing delays formation of the RFamide nerve net ...... 12 Analysis of Anthox2 regulatory sequences during Nematostella development ...... 12 Different efficiency of the Ax2_DsRed2 constructs...... 12 Anthox2 upstream sequence drive neuronal expression...... 13 SoxB2, a putative regulator of the Anthox2 gene...... 14 Conservation of putative regulatory elements in Gsx homologs ...... 14 DISCUSSION...... 14 The ParaHox gene families appear eumetazoan specific...... 14 Conservation of two functional domains in the Gsx protein from cnidarian to bilaterians...... 16 Conservation of the Gsx genomic structure from cnidarians to bilaterians...... 16 Conserved expression of Gsx in the neuronal cell lineage among cnidarians...... 16 The formation of the neuronal network involves Anthox2 ...... 17 Anthox2 promotor contains a repressor element and drives expression in apical nerve cells...... 17 Evolutionary conservation of Gsx neurogenic function...... 17
Acknowledgements...... 18 REFERENCES ...... 18
ABSTRACT Background: Neurogenesis in bilaterians rely on a shared genetic circuitry that involves different classes of transcription factors including homeobox genes. Among those, the Gsx/Ind gene family is involved in dorso-ventral patterning of the neural tube, brain formation and neuronal identity. In cnidarians, the first phylum that acquired a nervous system, the Gsx homologs (cnox2, Anthox2) are highly conserved and expressed in the nervous system as in Hydra, Clytia, Acropora. Here we investigated the neurogenic function of Anthox2 during Nematostella development. Results: The phylogenetic analysis confirmed the higher conservation of the cnidarian ParaHox families than the Hox- like ones. Moreover the structural conservation in Gsx orthologs possibly reflects the conservation of an ancestral function. We found that Anthox2 expression precedes onset of neurogenesis early during embryogenesis. Subsequently Anthox2 is expressed in putative neuronal precursors and differentiated neurons, which are exclusively detected in tentacles after metamorphosis. Anthox2 morpholino inhibition altered the formation of the nerve net and the survival of the planulae. We also performed reporter assays and found that 3 kb of upstream Anthox2 sequences suffice to drive expression in apical neurons. These sequences contain putative regulatory elements also present in the Hydra cnox2 upstream sequences, suggesting a conserved genetic regulation for Gsx between anthozoans and hydrozoans. Significance: These data suggest an essential role for Anthox2 in Nematostella neurogenesis, likely shared among Gsx cnidarian homologs Hence the high degree of conservation of the ParaHox genes from cnidarians to bilaterians could reflect the constraints driven by their essential function in cell type innovation as neurogenesis, an innovation that was maintained and complexified among eumetazoans.
- 1 - Preliminary version
Cnidarians and ctenophores are the first phyla where the INTRODUCTION neuromuscular system allows active behaviour such as The cnidarian phylum (sea anemones, hydra, medusae) feeding, which requires coordinated tentacle that diverged about 700 millions years ago (Chen et al., mouvements (Westfall and Kinnamon, 1984; Westfall, 2002) is supposed to be, together with the Ctenophora 1996). The organization of the cnidarian nervous system (combjellies), the closest outgroup to bilaterians (Philippe is traditionally considered as diffuse nerve net. et al., 2009). Hence, comparative studies of the signaling Nevertheless, a specific structure highlighted with the pathways and regulatory genes at work in developing RFamide antibody is recognized as a nerve ring, bilaterians and cnidarians, are essential to trace back the reminiscent to a primitive centralized nervous system core developmental mechanisms already at work in the (Koizumi, 2007) placed at the base of the hypostome cnidarian-bilaterian ancestor as well as to infer the (Koizumi et al., 1992) and the bell margin (Mackie, 2004) molecular changes that led to the formation of more in polyps and jellyfishes respectively. Cnidarian nervous complex organisms. Classically, the cnidarian anatomy is systems are composed of two main cell types: the defined as diploblastic i.e with two cell layers organized neurons and the mechanoreceptor cells named around a radial symmetry and a unique oral-aboral axis. nematocytes. In hydrozoans and anthozoans neurons More recently cnidarians started to be regarded as are of two types: Sensory cells have their cell bodies organisms displaying bilaterian and triploblastic features located in the ectoderm and extend their processes as the presence of a directive axis in Nematostella, towards the surface; ganglion neurons are bipolar or perpendicular to the oral-aboral axis (Finnerty et al., multipolar and spread in both cell layers along the 2004) and some evidences for mesodermal derivatives in mesoglea (Grimmelikhuijzen and Westfall, 1995). Thus numerous species (Seipel and Schmid, 2005, 2006). The cnidarians provide a unique standpoint to study the early Anthozoa class that contains species living exclusively as evolution of neurogenesis. polyps (sea anemone, corals…), is the sister group to the In cnidarians, neurogenesis takes place in different other major cnidarian class, the Medusozoa that contexts: in developing hydrozoans and anthozoans, alternate during their life cycle between a pelagic neurogenesis process starts in the endoderm as soon as (medusa) and a benthic (polyp) form (Odorico and Miller, the two cell layers are defined. Nematoblasts and 1997) (Fig. 1). According to molecular criteria, the neuroblasts then migrate towards the ectodermal layer anthozoans likely represent the cnidarian ancestral (Groger and Schmid, 2001). In hydrozoans, anthozoans condition. Cnidarians are particularly informative to trace and scyphozoans, the neuronal network develops in the back the early steps of neurogenesis as together with the planula in a regionalized manner starting from the Ctenophora they were the first phylum along animal anterior pole, corresponding to the future aboral side of evolution to establish the conditions for an efficient the metamorphosed polyp, where anthozoans bear a neuro-muscular transmission. sensory organ, the apical tuft (Groger and Schmid, 2001;
FIGURE 1: Cnidarian tree of life. Anthozoans are the only cnidarians without a medusa stage. Gsx ortholog genes were characterized in 15 different cnidarian species (3 different hydra species).
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Hayward et al., 2001; Nakanishi et al., 2008; Rentzsch et nematoblasts in the body column and is necessary to al., 2008). After metamorphosis, the nervous system maintain the apical nerve net in the adult polyps and for undergoes a complex reorganization with degeneration promoting de novo apical neurogenesis during head at the aboral pole and densification at the oral one regeneration (Miljkovic-Licina et al., 2007) suggesting an (Kroiher et al., 1990; Martin, 2000; Nakanishi et al., ancestral function in neuronal cell development. 2008). In the hydrozoan adult polyp, nematocytes and neuronal cells derive from the pluripotent interstitial stem To investigate whether the neurogenic function of the cells and appear to share a common bipotent progenitor Gsx/Cnox2 ortholog can also be traced in anthozoans, belonging to the interstitial stem cells (Holstein and we investigated its regulation and function during David, 1990; Miljkovic-Licina et al., 2007). Produced in neurogenesis in the developing sea anemone the body column they migrate to their definitive location Nematostella vectensis. We show that Anthox2 is indeed (Campbell and Marcum, 1980). In non-hydrozoan polyps, expressed very early during embryogenesis, subsequently in putative neuronal precursors and where the interstitial cell lineage was not characterized, neurons are proposed to derive directly from the differentiated neurons. Loss-of function assays showed epithelial cells (Nakanishi et al., 2008). In adult medusae, that Anthox2 is necessary for the proper formation of the neurons and nematocytes are intensively produced in the nerve net, moreover the expression of reporter gene manubrium and the tentacle bulbs (Grimmelikhuijzen and constructs revealed that 3 kb of the Anthox2 regulatory Spencer, 1984). Moreover medusae display sensory sequences could mimic the endogenous Anthox2 organs as ocellus, camera eye and rhopalia that are not expression in tentacle neurons. Interestingly, the found in polyps. upstream sequences of Nematostella Anthox2 and Hydra Cnox2 genes, share response elements for transcription The recent genomic analyses have shown that the factors involved in neurogenesis in higher eumetazoans. regulatory pathways and transcription factors involved in developmental processes in bilaterians were already present and diversified in cnidarians (Miller et al., 2005; Technau et al., 2005; Putnam et al., 2007; Ryan et al., MATERIALS & METHODS 2007; Larroux et al., 2008). Actually all the major gene families encoding regulatory genes used in bilaterians Animal culture and gametogenesis induction during neurogenesis or to pattern the central nervous system are also active in cnidarians (for review see Nematostella culture and gametogenesis induction were (Galliot et al., 2009) as for instance the BMP/DPP factors carried out as described in (Hand and Uhlinger, 1992; and their antagonists (Hayward et al., 2002; Finnerty et Fritzenwanker and Technau, 2002). Adults were fed at al., 2004; Rentzsch et al., 2006), or the homeobox neural least five times a week with freshly hatched Artemia identity gene ind/Gsh in anthozoans (Finnerty and nauplii. Adults and embryos were raised at 18 °C. Martindale, 1999; Hayward et al., 2001), hydrozoans (Schummer et al., 1992; Kuhn et al., 1996; Gauchat et Phylogenetic and genomic analyses al., 2000; Yanze et al., 2001) and scyphozoans (Kuhn et The cnidarian HD sequences related to the Hox and al., 1999). ParaHox bilaterian sequences in Blastp analyses, Ind/Gsh/Gsx homolog genes belong to the ParaHox sub- namely those related to PG1, PG2, Gsx, Pdx and Cdx class; the homeodomain (HD) sequences encoded by were selected and 65 bilaterian and cnidarian HD the ParaHox genes (Gsx, Pdx, Cdx) are highly related to sequences were aligned using ClustaW2 (Accession the Hox genes and these three genes are often numbers are available in Table S1). The Nematostella organized in cluster as in the amphioxus (Brooke et al., Xlox/Cdx sequence was not considered as it is highly 1998), fishes (Mulley et al., 2006) and tetrapods (Coulier derived (Quiquand et al., 2009). The best model of et al., 2000; Illes et al., 2009). Hence the ParaHOX protein evolution was choosen using the Protest 1.3 cluster is considered as the evolutionary sister of the program. Phylogenetic reconstruction was performed HOX clusters, both evolved from the same ProtoHOX using phyML (v2.4.4) (Guindon and Gascuel, 2003) and cluster (Brooke et al., 1998) that had already duplicated MrBayes (v3.1.2) (Ronquist and Huelsenbeck, 2003) in the last common cnidarian-bilaterian ancestor program. The maximum likelihood analysis used the JTT (Finnerty and Martindale, 1999; Garcia-Fernandez, 2005; model of amino acid substitutions and estimation of a Chourrout et al., 2006; Chiori et al., 2009; Quiquand et gamma shape distribution with eight substitution rate al., 2009). categories as well as invariant residues (JTT+I+!). Supports at nodes were evaluated with the bootstrap The Ind/Gsh/Gsx gene, along with the other neural method using 100 replicates. The Bayesian analysis was identity genes, is a conserved regulator of the dorso- performed under a mixed rate model of amino acid ventral patterning of the nervous system in bilaterians substitutions. The presence of invariant sites were (Weiss et al., 1998; Cornell and Ohlen, 2000; De assumed and a gamma distribution was approximated by Robertis, 2008; Mieko Mizutani and Bier, 2008). It is also four different rate categories to model rate variation implicated in brain formation (Hsieh-Li et al., 1995; across sites. From random trees, two independent runs Szucsik et al., 1997; Toresson and Campbell, 2001) as of four incrementally heated Metropolis-coupled Markov well as neuronal identity (McDonald et al., 1998; Weiss et chain Monte Carlo chains were simultaneously al., 1998; Kriks et al., 2005; Mizuguchi et al., 2006). performed for 1,000,000 generations. Trees were Interestingly, the cnidarian orthologs sampled every 100 generations. A consensus tree was (Gsx/cnox2/Anthox2) (Fig.1) are expressed in restricted created after the first 100,000 generations (burn in manner along the oral-aboral axis in putative neuronal =1000). precursors and differentiated sensory neurons during development and in homeostasis conditions (Hayward et For structural analysis, the N-termini and the HD al., 2001; Miljkovic-Licina et al., 2007; Chiori et al., 2009; sequences of 21 Gsx proteins were aligned and Quiquand et al., 2009). In Hydra, cnox2 is also consensus sequences were deduced using the expressed in dividing interstitial cells and clusters of MacVector program version 10.0. Genomic sequences
- 3 - Preliminary version were searched on the Metazome database Morpholino (MO) injection (http://www.metazome.net/) and intron positions were deduced after alignment of the mRNA with the genomic After 4% cysteine treatment, one cell stage fertilized DNA for 24 Gsx genes (Table S2). embryos were injected using the Eppendorf FentoJet Transjector 5246 and needles from Clark electromedical instrument GC100-10 (1,0 mm OD x 0.58 mm I.D) RT-PCR expression analyses (injection volumes estimated at 3% of egg) with the Nematostella mRNA was extracted from embryos taken Anthox2 MO (5’-ATATCAGGAGATGTGTGCCGTCTGG- at 1, 4, 7, 11, 17, 21 hours post fertilization (hpf, n=30) 3’, matching the 5’ UTR of the Anthox2 sequence, Fig. using the QuickPrep micro mRNA extraction kit S2) and the control (5’- (Amersham, Biosciences). 200 ng mRNA per condition CCTCTTACCTCAGTTACAATTTATA-3’, no target in were used to produce 20 !l cDNA with the Superscript Nematostella coding sequences) MOs, both modified on First-Strand Synthesis for RT-PCR kit (Invitrogen) with a the 3’ end with carboxyfluorescein, diluted in water at mixture of oligo (dT)12-18 and random hexamers (0.5 µg various concentrations (from 0.25 mM to 1 mM). and 50 ng per reaction respectively). The Anthox2, Embryos were then scored each day for survival and SoxB2, Sox2 and Actin genes were PCR amplified (see fluorescence under a GFP filter. Fluorescein negative primers in Table S3) (profile: 94°C 30s, 55°C 30s, 72°C animals were considered as uninjected but were 1min) from 1 µl cDNA and migrated after 35 cycles on nevertheless manipulated along with fluorescein positive 1% agarose gel. animals to form an additional cohort of control animals. Cellular phenotypes were observed after !-tubulin IHC In situ hybridization (ISH) performed at 4 days post-injection (dpi) or RFamide IHC performed at 3 and 5 dpi. DIG-labelled riboprobes were synthesized from the Ax2- 954, Ax2-585, Ax2-354 (Fig. S1), and SoxB2-819 pGEM- T easy plasmids with the T7 polymerase after PCR Cloning of Anthox2 upstream sequences amplification of the Ax2-954 plasmid with the M13 Genomic DNA was extracted from 200 juvenile primers and linearization of the Ax2-585 and SoxB2-819 Nematostella polyps dissociated for 3 hours at 55°C in plasmids with the Nde1 enzyme and the Ax2-354 with 500 µl PK buffer (4.4% 20x SSC, 1% Tris 1 M pH7.5, the Pst1 enzyme. ISH was performed according to 0.02% EDTA 0.5 M, 10% SDS 10%) containing 20 !g (Finnerty et al., 2003). For DIG detection, we used fast proteinase K (Roche). Genomic DNA was then extracted red (DAKO corporation) or NBT/BCIP stainings, which with phenol chloroform, precipitated with isopropanol (1 were monitored under the steromicroscope. volume) at RT, picked out directly and dissolved in H2O. The Anthox2 upstream sequences were isolated by three Immunohistochemistry (IHC) rounds of inverse PCR (iPCR) using Nhe1, EcoRV and Embryos were dejellied in 4% cysteine (pH 7.4) and again Nhe1 restriction enzymes and three different pairs polyp stages were relaxed in 7% MgCl2 for 15 min prior of primers namely Ax2F2/R3, Ax2F6/R5 and Ax2F7/R6 to fixation. All incubations were done for 15 min unless (Fig. S2) respectively. The 1774, 532 and 1477 bp PCR stated differently. Animals were fixed ON at 4°C in 4% products were cloned independently in pGEM-T easy PFA/PBS, washed 5x in PBS, TritonX-100 1% (PBST), vector (Promega) providing the Ax2-1774-Nhe1, Ax2- then 3x in methanol 100% and stored at -20 °C. Samples 532-EcoRV and Ax2-1477-Nhe1 clones that contain were stepwise rehydrated in 75%, 50%, 25% methanol in 1653, 458 and 1018bp of Anthox2 upstream sequences. PBST, washed 4x in PBST, blocked for 30 min at RT in After sequencing of these iPCR clones, 3122 bp of filtrated blocking solution (BS: BSA 2%, PBS) and Anthox2 upstream sequences were amplified with the incubated ON at 4°C either in the rabbit polyclonal anti- Ax2F8 and Ax2R7 primers with the Expand Long RFamide (kind gift of C. Grimmelikhuijzen, 1/1000), or in Template PCR system (Roche) and inserted into pGEM- the mouse monoclonal anti-Tyrosine-tubulin (anti-tyr-tub) T easy (Ax2-3122) (Fig. S1-S2). (Sigma, clone TUB-1A2, 1/800) or in the rabbit polyclonal anti-DsRed2 (Abcam, Ab34771, 1/400) antibodies diluted Reporter gene constructs in BS. Samples were then washed 4x in PBST, incubated in the appropriate secondary antibodies (Molecular The CMV promoter was excised from the CMV-DsRed2 probes, 1/400) for 3 hours at RT, washed 4x in PBST, vector (provided by Pr. Ivan Rodriguez laboratory) by Hoechst stained (Molecular probes, 33342, 1!g/ml), digestion with the Mlu1 and Nhe1 restriction enzymes. briefly washed 3x in PBS and mounted in Mowiol. 3122 bp of Anthox2 upstream sequences were isolated from Ax2-3122 by Not1 and Xmn1 digestion then For !-tubulin immunostaining, animals were fixed for 20 inserted upstream of the DsRed2 reporter gene to obtain hours in Helly fixative (Ott, 2008), washed extensively (at Ax2-3000_DsRed2. 1999 bp and 996 bp were amplified least 10x, ON) in PBST, 3x in methanol and stored at - from Ax2-3122 with the Nru1-Ax2pr-2000F / Afl2- 20°C. Samples were then stepwise rehydratated in 90%, Ax2ATGR and Nru1-Ax2pr-1000F / Nhe1-Ax2ATGR 70%, 50%, 30% methanol, Tris 0,1M (pH 7,4), washed pairs of primers respectively, digested at the restriction 4x in Tris 0,1M (pH7,4), blocked as above, incubated in sites present at the 5’ end of each primer and inserted the mouse monoclonal anti-!-tubulin antibody (Sigma, upstream of the DsRed2 reporter gene to obtain Ax2- clone 2-28-33, 1/1000) for 3 days at 4°C, washed 2000_DsRed2 and Ax2-1000_DsRed2 (Fig. S1-S2). For extensively in PBST, incubated in the secondary these two constructs, the CMV promoter was removed antibody (Molecular probes, 1/400) for 2 days at 4°C, from CMV-DsRed2 by digestion with Nru1 and Afl2 in the extensively washed in PBST, Hoechst stained and first case, with Nru1 and Nhe1 in the second. Prior to mounted in Mowiol. Imaging was done either on a Zeiss injection, each reporter construct was excised with Axioplan 2 motorised microscope or on a Leica SP2 appropriate double digest, purified with Gelase confocal. (Epicentre Biotechnologies), and resuspended in H2O at 100 ng/!l. After injection at the one cell stage, the
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expression rate was calculated every day over the ParaHox gene families showing that the ParaHox genes number of living animals under rhodamin filter. are more conserved than Hox genes (Quiquand et al., 2009). However this study did not include the new Cdx RESULTS sequence identified in Clytia (Chiori et al., 2009), therefore we used this more complete set of sequences, along with the cnidarian ParaHox sequences and Hox The three ParaHox families, Gsx, Pdx/Xlox sequences widely recognized namely PG1 and PG2 and Cdx, are highly conserved in families (Ryan et al., 2006; Chiori et al., 2009; Quiquand cnidarians et al., 2009). A dataset of 65 HD sequences containing 24 cnidarian and 41 bilaterian HDs was submitted to In a previous work using the HD sequences we phyML and MrBayes analysis (Fig. 2). extensively reconstructed the phylogeny of Hox and
FIGURE 2: Phylogenetic relationships between the PG1, PG2, Gsx, Pdx and Cdx Hox/ParaHox families in eumetaozoans. The topology of the tree was obtained in PhyML analysis of homeodomain (HD) sequences aligned with ClustalW. For each node, the BP and PP values obtained in PhyML and Bayesian analyses respectively are indicated (left / right). Families significantly supported are indicated on the right. Cnidarian sequences are boxed in grey. The Vnd1 Am and Nk2 Hv sequences were used as outgroups. Note the higher support for the ParaHox families than for the Hox ones (black dots). Species code: Am: Acropora millipora, Bf: Branchiostoma floridae, Ce: Caenorhabditis elegans, Ch: Clytia hemisphaerica, Ci: Ciona intestinalis, Csp: Capitella sp., Cv: Chlorohydra viridissima, Cx: Cassiopea xamachana, Dm: Drosophila melanogaster, Dr: Danio rerio, Ed: Eleutheria dichotoma, Hm: Hydra magnipapillata, Hs: Hydractinia symbiolongicarpus, Hv: Hydra vulgaris, Lg: Lottia gigentea, Mm: Mus musculus, Nv: Nematostella vectensis, Pc: Podocoryne carnea, Pf: Ptychodera flava, Sk: Saccoglossus kowalevski, Sp: Strongylocentrotus purpuratus, Ssp: Sarsia sp., Ta: Trichoplax adhaerens, Td: Turritopsis dohrnii. See accession numbers in Table S1.
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constrained domains at its N and C termini from The obtained trees confirm that two hydrozoans indeed cnidarians to mammals. express genes that belong to the Pdx and to the Cdx families which are not well represented in anthozoans so far except the Xlox/Cdx sequence that diverge. The Onset of Anthox2 expression precedes obtained bootstraps and posterior probabilities strongly neurogenesis in developing Nematostella supported the three eumetazoan ParaHox families (Fig. 2). The eumetazoan PG2 node was only supported with To characterize the embryonic regulation of Anthox2 phyML and the eumetazoan PG1 grouping did not expression we first performed RT-PCR analysis: Anthox2 appear in any analysis. Nevertheless the affiliation of transcripts were first detected at 4 hpf and then cnidarian sequences to the PG1 and PG2 families was significantly increased between 17 to 21 hpf (Fig. 4A). already described several times and is well recognized Later, at 6 days post-fertilization (dpf), Anthox2 positive even if supports are low (Gauchat et al., 2000; Chourrout cells were detected by ISH in the vicinity of the future oral et al., 2006; Kamm et al., 2006; Quiquand et al., 2009). pole, forming symmetrical territories surrounding the Here we showed that the order of eumetazoan future oral opening (asterisk) and corresponding to the Hox/ParaHox families conservation is as followed: Pdx ~ future tentacle buds (Fig. 4B, E). At the tentacle bud Cdx> Gsx >> PG2 >> PG1. The topology of the tree stage (7 dpf), Anthox2 expression was restricted to the presented in Fig. 2 confirmed that PG1 family appears buds (Fig. 4F oral view). After metamorphosis, Anthox2 orphan as it does not cluster with any of the other was exclusively detected in the tentacles with decreasing families. expression levels as the polyp ages (Fig. 4C, D, G, H). The fluorescent detection allowed us to analyze more Extended conservation of the Gsx / precisely the Anthox2 expressing cell types. At 6 dpf, Anthox2 / cnox2 structure Anthox2 transcripts were noted in cell bodies and processes of neuronal shape cells (Fig. 4I, K, Phylogenetic analysis perform on the HD sequences arrowheads). The same cell type was identified in showed that the eumetazoan Gsx family is significantly tentacles of the juvenile polyp (20 dpf) (Fig. 4L-N, P), less conserved than the two other ParaHox families. isolated (Fig. 4L, M, arrowheads) or found as pairs Interestingly, this family displays a unique feature: in resembling to two newly differentiated neurons (Fig. 4N, addition to the HD, the first 11 amino acids of the P, arrows). In addition at 6 and 20 dpf, Anthox2 was also Nterminus are also highly conserved from cnidarians to detected in pairs of non-neuronal cells, suggesting bilaterians (Fig. 3A), except in few sporadic species dividing progenitors (Fig. 4J, O asterisks). (Sarsia, Ciona). This consensus amino-acid sequence shares 4/8 residues with the so-called “HEP peptide” Taken together this data show that Anthox2 gene starts (present in H2.O/engrailed/paired homeobox genes) to be expressed as early as 4 hpf. Subsequently, described in (Allen et al., 1991; Williams and Holland, Anthox2 positive cells can be detected before the 2000). One of the differences is a conservative metamorphosis (6 dpf) in the posterior ecdoderm into substitution. The most conserved sites are an invariant symmetrical domains corresponding to the future site of phenylalanine at position 5 (found in 15/18 sequences the tentacle buds of the polyp. At the pre-metamorphosis analyzed) and an almost invariant isoleucine at position 7 stage and after the metamorphosis, Anthox2 expression is restricted to the oral part of the polyp. At the cellular (Williams and Holland, 2000) (found in 9/18 sequences analyzed, 8/9 cnidarian sequences) of the Nterminal level, Anthox2 positive cells belong to two categories: domain. The Nterminal domain of the Gsx family could dividing cells likely corresponding to neuronal precursors, therefore corresponds to a derived amino terminal HEP- and cells with extended projections suggesting motif (Ferrier and Holland, 2001; Finnerty et al., 2003). differentiated neuronal cells, isolated or as pairs. Anthox2 transcripts were not detected in any other tissues. To analyze the conservation of the Gsx gene structure we compared the HD position, the exonic/intronic Staging of neurogenesis in developing organisation and amino-acid sequences surrounding the splice sites among 24 Gsx genes (Fig. 3B, Table S2). As Nematostella previously reported (Schierwater et al., 2002), Gsx genes To investigate the formation of the nervous system in are composed of two exons and one intron, the splice developing Nematostella, we used a combination of three site being located upstream to the homeobox, relatively different antibodies, anti-Tyr-tubulin together with anti- close to it (from 26 to 101 bp) (except in Ciona Gsx). No RFamide and anti-!-tubulin; these three antibodies are consensus sequence was characterized at this position markers of the nervous system in bilaterians (Nassel et neither across eumetazoans nor across bilaterians. The al., 1988; Siddiqui et al., 1989; Farina et al., 2001; total gene size does not vary significantly from cnidarians Hessling and Westheide, 2002) but also in cnidarians to mammals (from 1.1 kb to 1.3 kb) and the second exon (Grimmelikhuijzen, 1985; Groger and Schmid, 2001; is smaller than the first one, usually less than 400 bp up Satterlie, 2002; Miljkovic-Licina et al., 2007; Rentzsch et to the stop codon (Table S2). However some arthropods, al., 2008; Marlow et al., 2009). echinoderms and urochordates display some atypical Gsx genomic structure as shown in Fig. 3B. For genomic The first RFamide mature sensory neurons were structure description and atypical feature see detected at 47 hpf after gastrulation process (Fig. 5B supplementary file Table S2 and Addendum 1. arrow). Subsequently, RFamide immunostaining detected the larval nerve ring that forms in between the The high degree of conservation in the size of the second ectoderm and the endoderm of the planula, from 57 hpf exon could be linked to the presence of the HD; although (not shown) to 130 hpf (Fig. 5B arrowheads). Later on, more variable, the size of the first exon is relatively well projections developed perpendicularly to the mesoglae, conserved in length. It carries sequences coding for the throughout the ectoderm. Those projections were more Nterminus, which is highly conserved too. Its function is concentrated in the anterior region of the apical tuft. currently unknown but this domain is probably important (Fig.5A-B), which is not surprising as this structure was to give the proper function to the protein that shows
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FIGURE 3: Gsx homologs across the animal kingdom. A) Schematic structure of the Gsx/Anthox2 protein. Two domains are conserved across species, the 11 first residues at the N-terminus and the HD, represented with red rectangles. The consensus sequence of the HEP peptide is in red. The 21 Gsx sequences used to build the tree were aligned. A derived consensus sequence is shown at the top: grey indicates identical residues or conservative substitutions (D/E, A/G/I/L/V, N/Q, F/W/Y, R/H/K, S/T, C/M). B) Genomic organization of representative Gsx orthologs. This panel of 24 Gsx sequences corresponds to 14 sequences used in Fig. 2 where genomic sequences were available plus those from Apis mellifera (Ame), Bos taurus (Bt), Homo sapiens (Hsa), Oikopleura dioica (Od), Pan troglodytes (Pt), Rattus norvegicus (Rn), Tertraodon nigroviridis (Tn). Red rectangles correspond to exons, black lines to introns, blue lines to the homeobox. The last 5 residues encoded by the first exon (the last one is numbered) and the first 5 residues encoded by the second exon are indicated. See the accession numbers in Table S1 and values Table S2. Abbreviations: Verteb: Vertebrata, Cephal: Cephalochordata, Urocho: Urochordata, Echino: Echinodermata, Arthro: Arthropoda, Mollus: Mollusca, Cnidar: Cnidaria, Placoz: Placozoa.
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FIGURE 4: Anthox2 expression in developing Nematostella. A) RT-PCR analysis of Anthox2 expression at indicated hours post-fertilization (hpf). The two Anthox2 PCR products correspond to cDNA (954 bp) and contaminating gDNA in some samples (1131 bp). B-P) Anthox2 expression detected by in situ hybridization after NBT-BCIP (B-D, O) or Fast Red (E-N, P) staining at 6 dpf (B, E, I-K), 7 dpf (F), 20 dpf (C, G-H, L-P) and 35 dpf (D); dpf: days post-fertilization. In B-H, the oral pole is orientated to the left except in F where the view is oral. Asterisk marks the mouth in B and F. F) tentacle bud stage, H) Enlarged view from G. I-P) Magnified views of Anthox2 expressing cells. At 6 dpf Anthox2 is expressed in pairs of cells corresponding to putative neuronal precursors (I,J asterisks) but also in cells with extended projections suggesting mature neurons (I,K, arrowheads). At 20 dpf, Anthox2 is expressed in putative neuronal precursors (O, asterisk) in neurons (L-M arrowheads) sometimes detected as pairs (N, P arrows). Scale bars ("m): 50 (E-H), 20 (I, L left, T), 10 (M, N), 5 (K, L right). described as the prominent neural structure of the Fig. S3D), particularly evident at the planula stage, here planula stage (Marlow et al., 2009). At 104 hpf the nerve shown at 37 hpf (Fig. S3F). Finally, the anti-Tyr-tubulin ring reached 2/3 of the larvae circumference (Fig. 5B) revealed the apical sensory organ named apical tuft as excluding the posterior side (future oral pole of the soon as it forms at the aboral side, here detected at 37 polyp). After metamorphosis, the diffuse nerve net hpf (Fig. 5A arrowheads, Fig. S3F arrows). This sensory spreads throughout the polyp body but the RFamide structure of the larvae facing the swimming direction is positive neurons were denser in the oral region and involved in the settlement. This cross-reactivity was especially in the tentacles (Fig. 5A-B). We defined four particularly useful as provided clues to orientate the distinct stages for the formation of the RFamide nerve larvae (Rentzsch et al., 2008). net: stage 0 no positive RFamide cells; stage 1 RFamide The !-tubulin positive neurons were elongated whatever cells with limited processes; stage 2 differentiated RFamide neurons with extended processes; stage 3 the stage with cell bodies located at the surface of the advanced RFamide nerve net with neuron ectodermal ectodermal layer, perpendiculary to the mesoglea (Fig. projections perpendicularly to the mesoglae and 5C-D). From 80 hpf, those cells elongated and their connected to an extended larval nerve ring (Fig. 5B, Fig. processes crossed the ectodermal layer to reach the 7D). These results show that the anti-RFamide antibody mesoglea (Fig. 5D). After metamorphosis, the !-tubulin is a valid marker to monitor neurogenesis in developing positive cells were preferentially detected in the Nematostella. ectodermal layer of the tentacles although also present in the endodermal layer of the body column (Fig. 5D Immediately after fertilization, the anti-Tyr-tubulin arrows). The anti-!-tubulin antibody identified a distinct antibody cross-reacted with mitotic spindles (Fig. S3A-E) subset of neuronal cells strictly restricted to the detecting mitotic cells at the metaphase and anaphase ectodermal layer up to the metamorphosis. The stages up to the gastrulation stage. As previously expression pattern detected with the !-tubulin antibody reported (Fritzenwanker et al., 2007) cells synchronously was not as dynamic as the Tyr-tubulin or the !-tubulin divide up to 11 hpf, before the gastrulation starts (Fig. ones. Nevertheless it highlighted a characteristic S3C). From 7h30pf, the anti-Tyr-tubulin antibody also population of ectodermal neuronal cells throughout the labeled cell membranes up to 8 dpf (Fig. S3B-D, G) and larval development of Nematostella. ciliary epithelia from the gastrulation stage (i.e 22 hpf, - 8 - Preliminary version
FIGURE 5: Formation of the nerve net in developing Nematostella. The formation of the nerve net was detected from 2 to 11 dpf (47 to 241 hpf) with three antibodies detecting Tyr-tubulin (A), RFamide (B) and !-tubulin (C, D). A, B) Tyr-tubulin and anti-RFamide immunodetections were performed on the same specimens. Here, from 3 dpf, the Tyr-tubulin antibody labels the apical tuft located in the aboral region (oriented upwards, e,i,m,q arrowheads). The RFamide neuropeptide is expressed in sensory mature neurons, first detected here at 47 hpf (b, arrow and inlet), subsequently forming a nerve ring between endoderm and ectoderm (indicated by two arrowheads in f,j,n,r). Staging was defined as follows: stage 0: no RFamide neurons; stage 1: few RFamide+ neurons with limited processes and no or limited larval nerve ring, stage 2: differentiated RFamide+ neurons with extended processes with or without larval nerve ring; stage 3: differentiated RFamide+ neurons connected to an extended larval nerve ring. C, D) Detection of !-tubulin in ectodermal neuronal cells, first at 47 hpf in cells that display an asymmetrical staining, stronger at the surface of the ectoderm (c-d), later in cells that elongate perpendicularly to the surface and cover the entire ectoderm (g-h, k-l, o-p, s-t). After metamorphosis, !- tubulin positive cells are numerous in the ectodermal layer of the tentacle but also in neurons located in the endoderm of the body column (w-x, arrows). The oral pole is marked with an asterisk (u-x). (Scale bars (µm): A-B: 50, C: 75, D: 20).
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To address the network progression with the time in a ones as populations are composed most of the time of 3 given population of animals spawned and fixed at the different stages and they always kept representatives of same point, we monitored the formation of the RFamide the stage 1. Globally, water injected populations are nerve net according to the stages defined above, in slightly delay compared to uninjected populations. different populations of non-injected embryos fixed at different time points. (Fig. 6 non-injected embryos (-), To investigate the function of the Anthox2 gene we used Table S4). Except at 1 and 6 dpf (31 and 142 hpf), a MO approach to perform loss of function assays. We animals fixed at the same time point were quite compared several criteria: the survival rate and the heterogenous as different stages of RFamide nerve net formation of the nerve net monitored with the !-tubulin were represented in the same population. Usually two or and the RFamide immunostaining. three distinct stages were represented in a given population. Heterogeneity can be due to the fact that Anthox2 inhibition decreases the survival rate fertilization was not properly synchronized. Nevertheless, For each MO concentration (0.25 to 1 mM) the survival we assume that even in the case of synchronized rate of fluorescein positive animals was monitored from 1 fertilization Nematostella larvae populations do not to 6 dpi (Fig. 7A-B, methods, Addendum 2). The graph develop strictly their nerve net at the same speed. The showed in Fig. 7B clearly indicates that the survival rate RFamide stage 1 appeared at 47 hpf, the stage 2 at 72 in Anthox2 MO-injected animals was decreased hpf and the stage 3 at 104 hpf. At 142 and 152 hpf, compared to control MO-injected animals (Table S5). We majority of the uninjected animals were in the advanced could detect some correlation between the MO stage 3. Stages 0 disappeared at 4 dpf (95 hpf). The concentration and the mortality from 3 to 5 dpi where the RFamide nerve net development seems to be relatively survival rate of control and Anthox2 MO-injected animals irregular. Indeed, the population of a given time point can decreased when the MO concentration decreases. be delayed compared to the population of the previous Indeed, the best survival rate corresponded to sample time point. RFamide antibody is therefore a good marker injected with MOs at 0.25 mM and the lowest survival of the mature sensory neurons that can be use to study rate was observed at 0.75 mM. The difference between the early stages of neurogenesis in Nematostella. the survival rates of control and Anthox2 MO-injected animals increased when the MO concentration Anthox2 loss of function assays in increased. However surprisingly embryos injected at 1 developing Nematostella mM survived better than those injected with 0.75 mM. From 6 dpi usually the ratio approaches 0 meaning that To control the effect of the water injection on the almost all Anthox2 injected animals were dead. For neuronal development we compared not injected or further studies, we decided to use the intermediate water injected populations (Fig. 6 water injected embryos concentration giving the intermediate mortality: 0.5 mM. (+), Table S4). Water injected animals have a more heterogenous nerve net formation than the uninjected
FIGURE 6: Staging of early neurogenesis in developing Nematostella. The progressive formation of the nerve net was assessed by RFamide immunostaining as defined in Fig. 5. The stacked histograms show the distribution of the different stages at different time points of development. Neurogenesis in non-injected (-) and water injected (+) embryos was compared: injected animals normally achieved neurogenesis but were slightly delayed. 6 to 13 animals were staged for each condition. See values in Table S4.
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FIGURE 7: Anthox2 phenotypes observed in morpholino (MO)-injected animals. A) The fluorescence emitted by the carboxyfluorescein attached to the 3’ end of the MO proves the injection efficiency. B) Survival rate of MO-injected animals exposed to increasing concentrations (0.25, 0.5, 0.75, 1 mM). Only fluorescein+ animals at 1 day post-injection (dpi) were taken into consideration. Each curve corresponds to the ratio of the survival rates in Anthox2 MO over control MO injected animals recorded from 1 to 6 dpi. See values Table S5. C) Neurogenic phenotypes observed at 4 dpi in MO-injected planula (0.5 mM) detected by !-tubulin immunostaining. Class I corresponds to the dense ectodermal !-tubulin pattern as noted in uninjected animals (Fig. 5K) and in most Control MO animals (left panels); class II to a reduced ectodermal !-tubulin pattern as observed in Anthox2 MO animals (middle panels); class III to no ectodermal !-tubulin pattern as observed in Anthox2 MO animals (right panels). Scale bars (!m): 50 (upper), 20 (lower). D) Alterations in the formation of the RFamide nerve net in Anthox2 MO animals at 3 dpi (upper, 0.5 mM) and 5 dpi (lower, 0.25 mM). E) Stacked histograms showing the distribution of the different !-tubulin patterns detected at 4 dpi in animals injected with water (H2O) or MOs (0.5 mM). Non fluorescent MO-injected animals at 1 dpi (fluo-) were scored separately. Note that 45% of the Anthox2 MO animals belong to class III versus 15% in control MO animals. See values in Table S6A. F) Stacked histograms showing the distribution of the different RFamide nerve net stages detected at 3 and 5 dpi in non-injected animals (uninj) or animals injected with water (H2O) or MOs (0.5 and 0.25 mM). Note the delayed formation of the nerve net in Anthox2 MO animals. See values in Table S6B.
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Anthox2 inhibition disrupts the !-tubulin the formation of the RFamide nerve net was delay when pattern in the ectoderm Anthox2 expression was inhibited. To test whether Anthox2 inhibition affect neurogenesis The results obtained at 5 dpi confirmed those observed (Addendum 3), we first looked at the !-tubulin ectodermal at 3 dpi even though the control MO-injected planulae neuronal cells at 4 dpi (100 hpf) (Fig. 7C). We noticed were delayed compared to the H2O, uninjected and fluo that in Anthox2 MO-injected animals the ectodermal layer (-) controls. However the concentration (0.25 or 0.5 mM) did not seem to significantly affect this delay. was abnormal. The elongated !-tubulin neuronal cells were either significantly reduced (Fig. 7C class II) either In contrast, the Anthox2 MO-injected populations behave completely absent (Fig. 7C class III, note that the three dramatically different with a significant delay in nerve net lower panels are shown at the same scale). formation that was increased from 0.25 mM to 0.5 mM. When injected at 0.25 mM, most of the animals (85%) This alteration of the ectodermal nerve cell pattern was were in stage 2 whereas none of those injected at 0.5 quantified by sorting the animals in each given class at 4 mM had reached that stage (20% were still in stage 0 dpi (Fig.7E, Table S6A). The fluorescein negative and 80% in stage 1). This result indicates that beside animals that likely correspond to animals not properly initiation of the nerve net formation, the progression injected at the one cell stage displayed a normal through neurogenesis is actually dramatically altered in ectodermal !-tubulin pattern (Fig. 7C class I, Fig. 7E) Anthox2 MO-injected animals. similar to the WT pattern (Fig. 5 C-D k-l). Most of the animals injected with water also displayed a normal !- In addition, the control MO-injected population was more tubulin ectodermal neuronal pattern (60%, class I), heterogenous, suggesting that the Anthox2 MO however 40% of these animals showed some alterations population was stacked in their neuronal development at (class II). The animals injected with the control MO a point depending on the level of Anthox2 inhibition. At showed similar distribution with 55% forming a normal 0.25 mM the neurogenesis was stacked predominantly in neuronal pattern and 32% abnormal one. However, few stage 2 whereas at 0.5 mM it appeared stacked in stage animals (13%) belonged to the extreme class III 1. Thus, highest was the concentration earliest the phenotype when injected with the control MO. Finally, the population was stacked. percentage of normal animals (class I) was lower in Anthox2 MO-injected animals (30%) than in control MO Those quantitative data suggest that Anthox2 gene is or water injected sample. In addition, the percentage of involved in the proper formation of the RFamide nerve animals showing the extreme phenotyp was much higher net. When inhibited, the progression of the network is compared to the same controls (class III, .45%). delayed and probably stacked in precocious stages. The fact that even after Anthox2 MO injection, the RFamide These results showed that Anthox2 inhibition significantly nerve net was still able to developed suggest that either affected the formation of the ectodermal neuronal cells an other process replaced the Anthox2 gene or that a as either the pattern was disrupted, or it never formed. small quantity of Anthox2 gene persisted, allowing to As Nematostella larvae were likely submitted to the develop an incomplete Rfamide network. Anthox2 silencing from the one cell stage we rather favor the second hypothesis, suggesting an essential role for Analysis of Anthox2 regulatory sequences Anthox2 in the formation of the ectodermal nerve net. during Nematostella development Anthox2 silencing delays formation of the RFamide nerve net Different efficiency of the Ax2_DsRed2 constructs To confirm the effect of Anthox2 on the nerve net formation, we monitored the RFamide nerve net We set up a DsRed2 reporter construct assay to study progression at 3 and 5 dpi in animals that was exposed the regulation of the Anthox2 gene. Five different to Anthox2 MO at two different concentrations (0.25 and DsRed2 reporter constructs were injected at the one cell 0.5 mM) (Fig. 7F, Table S6B). 4 conditions were used as stage of fertilized embryos. Three of them were under the controls: uninjected, H2O injected, injected but negative control of the Anthox2 upstream sequence: respectively for fluorescein and control MO-injected. As previously 1, 2 and 3 kb long. Two additional constructs were noted, the formation of the nerve net was slightly injected as controls: the CMV_DsRed2 construct under modified in H2O injected compared to uninjected the control of the Cytomegalovirus promoter, and the populations and the RFamide nerve net formation was empty_DsRed2 construct that do not contain any delay in H2O injected animals. Indeed, at 3 dpf 43% of promoter (Fig. 8A). the H2O injected animals did not yet started to form the The expression rate among the survivors was recorded RFamide network (stage 0) versus 13% in uninjected each day for each condition and plotted on a graph (Fig. population. The negative effect of H2O injection was 8B, Table S7, Addendum 4). The empty_DsRed2 confirmed in the fluorescein negative population, which construct gave a non-significant expression of the was even more advanced than the uninjected population DsRed2 reporter gene in less than 10% of animals. The as all animals had already started to form a nerve net. In expression rates of the Ax2-3000_DsRed2 and MOs injected animals, we observed that the formation of CMV_DsRed2 constructs were almost similar. The day the RFamide nerve net in control-injected animals was after the injection experiment, expression rate was intermediate between uninjected and H2O injected around 10% of positive animals and the highest populations (control (MO) 0.5) meaning that there was no expression rate was recorded between 2 and 4 dpi, significant difference between these 3 populations. In reaching 40 %. In contrast, the expression rate of the contrast, the proportion of animals exposed to Anthox2 deleted reporter constructs, (Ax2-2000_DsRed2 and MO with no nerve net at 3 dpi was higher (55%) than in Ax2-1000_DsRed2), presented a higher expression rate any control condition. In fact, the Anthox2 MO-injected as early as 1 dpi up to 9 dpi, compared to the three other population was the only one at this time point to miss the constructs. At 3 and 4 dpi, the same expression rate was stage 2. These results showed that the initial phase of - 12 - Preliminary version
FIGURE 8: Anthox2 promoter activity in Nematostella embryos. A) Schematic representation of the DsRed2 reporter gene constructs injected in one-cell stage fertilized embryos. DsRed2 expression was directed either by the Anthox2 upstream sequences of various length (3, 2, 1 kb) or by the CMV promoter. B) Graph showing the mean expression rate of each construct calculated among survivors from 1 to 9 dpi. Note that the highest expression rate was obtained whith 2 kb and 1 kb of Anthox2 upstream sequences drive DsRed2 expression. C) DsRed2 positive cells detected by immunohistochemistry with an anti-DsRed2 antibody 7 and 13 dpi after injection of the Ax2-3000_DsRed2 construct (a-f) and CMV_DsRed2 construct (g-k). The ratios represent the number of animals observed with the neuronal expression pattern over the total number of observed animals. Yellow staining shows the colocalisation between the DsRed2 protein and the secondary antibody coupled to Alexa- 488. Note the similarity with the expression pattern of the Anthox2 gene detected by in situ hybridization 20 dpf (fig. 4 K, P). (Scale bars (µm): a, c, g: 50, b, d, f, h: 5, e: 30, j: 75, k: 20). recorded for both constructs, between 80 and 85%. From day 5, the Ax2-2000_DsRed2 samples presented 15 to Anthox2 upstream sequence drive neuronal 20% more positive animals than in sample injected with expression the Ax2-1000_DsRed2 construct. The highest expression rate was actually recorded at day 5 in Ax2-2000_DsRed2 The expression pattern of the Ax2-3000_DsRed2 and injected animals and reached 95%. CMV-DsRed2 constructs was examined at the cellular level. Animals that were screened positive for DsRed2 The percentage of animals expressing the DsRed2 expression were fixed after metamorphosis and analyzed reporter gene was higher in both Ax2-2000_DsRed2 and after anti-DsRed2 IHC. DsRed2 expression, driven by the Ax2-1000_DsRed2 than in Ax2-3000_DsRed2 and 3 kb Anthox2 upstream sequences, was detected in the CMV_DsRed2 constructs. Therefore, the 1 kb of the tentacle buds at 7 dpi and in the tentacles at 13 dpi (Fig. Anthox2 promoter placed in the 5’ end of the Ax2- 8Ca-d arrows). Confocal view of those regions revealed 3000_DsRed2 could carry a repressor element. When absent in the shorter constructs the expression rate could be increased.
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sequences from Hydra viridissima and Hydra vulgaris that the DsRed2 protein, detected in both red (Fig. 9C). We focused on the transcription factors known (fluorescence) and green (antibody) chanels, was to be involved in neurogenesis in bilaterians. The position expressed in the ectoderm of the tentacles in elongated of two putative binding sites for Sox5 and some for cells that projected processes towards the endodermal GATA1/3 were conserved in all three upstream part. This expression pattern was very similar to that sequences. Putative binding sites for HNF3/3B, Ftz and observed for Anthox2 mRNA (Fig. 4 G, L). The same Oct transcription factors were conserved between type of positive elongated cells in the same location was Nematostella and Hydra viridissima showing a similar observed in five metamorphosed animals out of seven positions. Upstream sequences of Hydra magnipapillata treated in the same conditions. In contrast, positive also carry putative Oct and HNF 3/3B binding sites in elongated cells in the tentacles of metamorphosed different regions. Other putative binging sites like Croc, animals were observed only once out of four in Zen-1, GATA1/1A/2 are shared between Nematostella CMV_DsRed2 injected animals (Fig. 8Cg-h arrow). and Hydra viridissima but placed at different positions Moreover this latter construct also led to DsRed2 (Fig. 9C). These factors are known to regulate the expression in isolated round positive cells of the developing nervous system in bilaterians (Latchman, endoderm of the body column (Fig. 8Ck asterisk). In 1999; Jeffrey et al., 2000; Hsia and McGinnis, 2003; animals injected with Ax2-3000_DsRed2 and LaVoie, 2003; Anderson et al., 2006; Leone et al., 2008). CMV_DsRed2 constructs, the anti-DsRed2 antibody Finally, we were also able to detect by manual search a reacted with scattered cells in the ectodermal layer of the consensus DNA-binding domain for the Gsh1 protein as body column (Fig. 8C arrowheads). Because those cells described by (Valerius et al., 1995) were only positives in the green channel (reflecting the (GCT/CA/CATTAG/A) located in the most upstream anti-DsRed2 antibody), we considered these signals as region at position -2665 of the Nematostella upstream non-specific cross-reactivity of the anti-DsRed2 antibody. sequence (Fig. 9C, S2). Therefore this putative auto- These experiment indicate that 3 kb of the Anthox2 regulatory element was present only in the Ax2- promoter region are sufficient to mimic the endogenous 3000_DsRed2 construct. expression of Anthox2 in differentiated neurons of the oral region. DISCUSSION SoxB2, a putative regulator of the Anthox2 gene The ParaHox gene families appear The expression pattern of different members of the Sox family was reported in Nematostella (Magie et al., 2005). eumetazoan specific Among those SoxB2 and Sox2 exhibit very early up- We readressed the phylogeny of the eumetazoan regulations during development. Therefore we decided to ParaHox families relative to the Hox ones using new data monitor the relative temporal regulation of SoxB2, Sox2 available. We showed that cnidarian ParaHox genes are and Anthox2 (Fig. 9A) as they are express in common more closely related to their bilaterian counterparts than territories with Anthox2. SoxB2 is expressed in broader cnidarian Hox are. The node supporting the eumetazoan domains than Anthox2 from the gastrula to the polyp Gsx and Pdx families were not significantly different from stage, but similarly to Anthox2 its expression pattern those obtained in our previous study (Quiquand et al., suggests a role in the specification of neural cell fates 2009). In contrast, the support at the eumetazoan Cdx even if the SoxB2 expressing cell types were not clearly node was remarkably higher than those reported before identified. Sox2 is also expressed in the entire ectoderm where only the cnidarian Cdx-like HD (Cnox4-Ed) was of the gastrula and at subsequent stages, specially from aligned in the dataset (Gauchat et al., 2000; Kamm et al., the tentacle bud stage its expression in the oral region 2006; Chiori et al., 2009; Quiquand et al., 2009). In our resembles that of Anthox2. new analysis, probably that the two cnidarian Cdx The RT-PCR analysis (Fig. 9A) showed that the initiation sequences had an attractive effect on each others to the of the expression of the three genes after fertilization is Cdx bilaterian family as we already shown in (Quiquand clearly different. SoxB2, likely maternal, is expressed et al., 2009) where we described the same effect on the from 1 hpf, whereas Anthox2 expression started to be Xlox/Cdx-Nv sequence to the Pdx family thanks to the detected at 4 dpf and that of Sox2 from 7 hpf. Pdx-Ch and Pdx-Td sequences. In addition, the presence of two cnidarian sequences related to the Cdx family As SoxB2 expression precedes that of Anthox2 during probably enhances the support for the eumetazoan Cdx embryonic development, we looked at SoxB2 expression node. at the cellular level. Interestingly at 6 dpf, the shape of the SoxB2 positive cells in the tentacle buds was This new analysis reinforced our previous one, showing relatively similar to the shape of the Anthox2 positive that the eumatazoan ParaHox sequences are more cells in the same region (Fig. 9B) suggesting that those conserved than the Hox ones. Those results support the two genes could be co-expressed in nerve cells. idea that the conservation of the ParaHox genes in early Therefore we searched for Sox-binding sites inside the animal reflects conserved regulatory network driving Anthox2 upstream regulatory sequences (Fig. 9C). cellular innovations such as neurogenesis (Quiquand et Interestingly, the Anthox2 promoter present putative Sox al., 2009). This assumption would not be surprising as binding regions. the cellular mechanisms controlling the cnidarian homeostasis as neurogenesis (Galliot et al., 2009) are more similar to bilaterian than their axes where no clear Conservation of putative regulatory elements in homology is yet reported. Interstingly, Gsx represents the Gsx homologs Hox-extended family showing the clearest conservation To map the putative regulatory elements conserved in in the cell lineage in which it is detected from cnidarians the promoters of cnidarian Gsx homologs, we conducted to bilaterians. This reinforce the idea that ParaHox genes a comparative analysis between the Nematostella are conserved because of the cellular events that they Anthox2 upstream sequences and the Cnox2 upstream drive.
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FIGURE 9: Putative regulators of Anthox2 expression. A) Temporal regulation of the expression of SoxB2, Anthox2 and Sox2 detected by RT-PCR in Nematostella embryos. SoxB2 expression, likely maternal, precedes that of Anthox2. B) Detection by in situ hybridization of SoxB2 and Anthox2 transcripts at 5 and 6 dpf respectively. Anthox2 and SoxB2 are both expressed in ectodermal neuronal cells in the tentacle area. Lower panels: enlarged views from the upper ones. (Scale bars (µm): a-b: 50, c: 5, d: 10). C) Common putative regulatory binding sites identified in the upstream sequences of Anthox2 from Nematostella vectensis and cnox2 from Hydra viridissima and Hydra magnipapillata with the Transcription Element Search System program (http://www.cbil.upenn.edu/cgi-bin/tess/tess33?RQ=WELCOME) and the Consite program (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite). Colored rectangles indicate regulatory elements whose position is conserved between those cnidarian genes. All three carry putative Sox binding sites. Grey boxes correspond to exons.
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The combination of the new data published (Chiori et al., vertebrates and amphioxus (Garcia-Fernandez and 2009; Quiquand et al., 2009) highlighted for the first time Holland, 1994; Wada et al., 1999) except for the a cnidarian species carrying all tree ParaHox genes in its sequence coding for the hexapeptide motif that we could genome (namely the hydrozoan Clytia) expressed during not retrieve in Gsx sequences. Our genomic structure the larval development. The fact that cnidarians analysis reveals that at least in the case of the Gsx expressed the tree ParaHox genes favors the hypothesis ParaHox family, this organization extents to the cnidarian of a three gene ProtoHOX cluster. Nevertheless, no tools phylum meaning that likely Gsx gene sequences are yet available to look at the organization of those conserved an ancient intron position already existing in genes in Clytia genome. The expression patterns the Gsx common ancestor sequence and even probably published do not reveal a collinear expression as in the common sequence to Hox and ParaHox genes reported in Branchiostoma (Brooke et al., 1998), (Williams and Holland, 2000). Strongylocentrotus (Arnone et al., 2006) and Nereis According to the amino-acid sequence encoded by the (Kulakova et al., 2008). Gsx genes, the intron lies between the DNA sequences coding for the two conserved domains of the protein Conservation of two functional domains in the which probably performed two distinct functions: the HD Gsx protein from cnidarian to bilaterians involved in sequence-specific binding to DNA, and the Comparison of the support obtained for the ParaHox HEP motif mediating a putative role in the transcriptional families showed that the Gsx one is the least supported. activity. Interestingly, this protein presents a peculiar feature The Gsx gene probably existed in one single copy in the already reported (Finnerty and Martindale, 1999; Finnerty cnidarian-bilaterian ancestor and underwent one gene et al., 2003), consisting of a highly conserved peptide duplication event in the vertebrate lineage (Hsieh-Li et domain covering the 11 first amino-acids of the al., 1995; Valerius et al., 1995; Deschet et al., 1998). The Nterminus, possibly corresponding to a highly derived same kind of duplication events are reported for many “HEP peptide” (Ferrier and Holland, 2001; Finnerty et al., classes of transcription factors in early vertebrate 2003). evolution including homeobox genes as Hox, Otx, Msx, Given the high level of identity in this part of the protein Cdx, En, Emx, Pax as well as bHLH genes, duplication from cnidarians to deuterostomes, one could expect a probably affected a large proportion of the genome in functional constraint specific to this domain in addition to early vertebrate evolution (Holland et al., 1994; Holland, the one applied on the HD region. The global role of Gsx 1999). Because Gsx genes underwent few duplication homolog proteins could be driven by those two since the base of the eumetazoans, one could expect a conserved regions. This protein organization reflects the functional conservation across evolution (Finnerty et al., feature usually found in transcriptional regulators that 2003). Indeed, in vertebrate, those two copies of Gsx function according to two distinct activities. Through their gene seem to provide partially redundant functions as in DNA-binding domains they interact with specific sets of Gsx2 mutant mice, Gsx1 can partially compensate the target genes and on the other hand, through activation or Gsx2 loss of function (Toresson and Campbell, 2001; repression domains they affect the expression of those Yun et al., 2003; Wang et al., 2009) meaning that even in target genes. Those two functional roles are usually organism that possess two Gsx genes their function did physically separated in two functional domains (Smith not diversify completely. and Jaynes, 1996; Carlsson and Mahlapuu, 2002). Conserved expression of Gsx in the In addition to the HD proteins where it was characterized (H2.O, engrailed, paired), many other classes of HD neuronal cell lineage among cnidarians transcription factors (Msx, Nk-1, Nk-2, gsc, Not, Prd, Among the Hox/ParaHox genes, the Gsx homologs are HoxA7, HoxD9) as well as the forkhead domain, and zinc the most studied in cnidarians. It was characterized in 15 finger protein contain regions similat to the HEP motif different cnidarian species (Murtha et al., 1991; (Smith and Jaynes, 1996; Stein et al., 1996; Galliot et al., Schierwater et al., 1991; Schummer et al., 1992; Naito et 1999; Finnerty et al., 2003). The motif is shared by Hox al., 1993; Shenk et al., 1993; Finnerty and Martindale, and ParaHox proteins suggesting an ancient origin of the 1997, 1999; Kuhn et al., 1999; Hayward et al., 2001; motif that may have been already present in the common Yanze et al., 2001; Hill et al., 2003; Chiori et al., 2009; ancestor of those genes and subsequently lost in several Quiquand et al., 2009) coming from hydrozoan, of the descendants (Finnerty et al., 2003). The sequence anthozoan and scyphozoan classes (Fig. 1). For a long related to the HEP motif in the engrailed gene (eh1 motif) time cnidarian Gsx homologs were studied in order to of Drosophila was shown to mediate transcriptional find a conserved expression pattern to infer an ancestral repression when attached to a DNA-binding domain role of this gene in axial patterning. It revealed that (Smith and Jaynes, 1996) whereas in protein of the cnidarian Gsx homologs exhibit a high degree of forkhead domain transcription factors it mediates variability in their expression domains as Gsx was found transcriptional activation (Pani et al., 1992). In the Gsx expressed either orally or aborally depending of the family, its precise function should be investigated even if cnidarian species (Shenk et al., 1993; Hayward et al., we could expect that it acts as a transcriptional 2001; Yanze et al., 2001; Finnerty et al., 2003; Cartwright modulator. et al., 2006; Jakob and Schierwater, 2007; Miljkovic- Licina et al., 2007; Quiquand et al., 2009). Nevertheless, Conservation of the Gsx genomic structure cellular types expressing Gsx homologs were from cnidarians to bilaterians characterized in several cnidarians suggesting a putative conserved function in neuronal differentiation, rather than The genomic structure of cnidarian Gsx homologs is very in axis specification (Hayward et al., 2001; Finnerty et al., similar to that of vertebrate genes, with two conserved 2003; Miljkovic-Licina et al., 2007; Chiori et al., 2009). exons and one intron placed just 5’ to the homeobox. This structure is characteristic of the stereotyped Anthox2 expression pattern has been studied elsewhere genomic organization of the Hox and ParaHox genes in (Finnerty et al., 2003; Ryan et al., 2007) and was first
- 16 - Preliminary version detected after the gastrulation in the endoderm at the formation (Miljkovic-Licina et al., 2007). In Nematostella, posterior side of the planula. Before the tentacle bud Anthox2 showed an effect on the sensory neuronal stage, we found Anthox2 positive cells in the oral region. population differantiation meaning that it probably acts In contrast to the pattern described by (Finnerty et al., also at the beginning of the neuronal cell lineage 2003; Ryan et al., 2007) we could not detect transcripts probably in concert with other proteins. The putative role in the pharyngial zone as just before the tentacle bud of Anthox2 on the formation of sensory structures could stage we detected Anthox2 transcripts exclusively in the now be addressed more largely as on the apical tuft of tentacles until the metamorphosis include. At the cellular the larvae for exemple as its formation is regulated by level, we were able to recognize, according to their FGF signals (Rentzsch et al., 2008). morphology, putative dividing precursors as well as differentiated neurons isolated or by pairs. This cellular Anthox2 promotor contains a repressor analysis confirmed the suggestions done in (Hayward et element and drives expression in apical al., 2001; Finnerty et al., 2003; Ryan et al., 2007), that the Anthox2/Cnox2 positive cells in anthozoan nerve cells correspond to putative neuronal precursors and We were able to set a reporter gene construct approach differentiated bi, tri or multipolar neuronal cell populations in developing Nematostella. By this way, we showed that at the oral pole. Those observations are also in 3 kb of Anthox2 upstream sequence were sufficient to agreements with the hydrozoan situation where drive reporter gene expression in the tentacles of the Cnox2/Gsx positive cells in Hydra were identified as metamorphosed polyp. Even though mosaic, this neuronal precursors, dividing nematoblasts and neurons expression was reminiscent of the Anthox2 pattern (Miljkovic-Licina et al., 2007). In Clytia medusae, Gsx detected by ISH. This suggest that these sequences positive cells were suggested to be neuronal precursors probably carry elements necessary for Anthox2 neuronal or differentiated neurons located in the ectoderm of the expression. To further investigate these sequences we medusae tentacle bulbs in a neuron and sensory cell-rich performed different deletions in the promoter of the region (Denker et al., 2008; Chiori et al., 2009). These reporter constructs. We show in this work that removal of data suggest that Gsx homologs perform a common the most 5’ sequences (1 kb) led to a significant increase function in cnidarians, in the determination of the of the expression rate of the reporter gene. We propose neuronal stem cells committed to give neurons and that a repressor element was removed in the constructs nematoblasts. Ax2-2000_DsRed2 and Ax2-1000_DsRed2. Interestingly, exactly in this region we were able to detect a Gsh The formation of the neuronal network binding site, suggesting a negative autoregulatory involves Anthox2 function of Anthox2. Depending on the developmental phases of the Interestingly both the mouse Gsh and the Drosophila ind developing Nematostella (swimming larvae or benthic genes carry autoregulatory binding sites in their promoter polyp), the neuronal concentration was located in two (Li et al., 1996; Von Ohlen et al., 2007). In addition, the opposed territories; the apical sensory organ of the function of ind on its proper gene was characterized as planula, corresponding to the anterior region of the larvae activator or as derepressor of a repression on the and to the aboral side of the futur polyp or the oral pole of transcriptional activity (Von Ohlen et al., 2007). In the polyp corresponding to the posterior region of the Anthox2, a similar mechanism could apply explaining larvae. At each life stage, the neuronal concentration why when the autoregulatory site is removed, the takes place where the active behavior is localized repression of the transcription is no longer active and the (sensing or feeding). gene is more expressed. To characterize the nerve net formation in Nematostella The reporter construct assay that we were able to set up we used three different antibodies. The Tyr-tubulin in developing Nematostella provides a very useful tool, staining was particularly interesting in our study to label still missing up to now, to study in vivo gene expression the apical sensory organ (apical tuft) of the larvae, which and to dissect gene regulatory sequences. Nematostella develops from 37 hpf at the anterior pole, corresponding expressing reporter genes is a first step to transgenic to the future aboral side of the mature polyp. The anti- anemones. Given the high efficiently of the method, it RFamide antibody labeled sensory neurons surrounding provides a complement to transgenesis, providing the endoderm as well as in the ectodermal layer both at mosaic expression faster than raising transgenic lines. the larvae and polyp stage. We showed that the RFamide nerve net is formed progressively from 47 hpf Evolutionary conservation of Gsx to give an diffuse nerve net located perpendicularly in the neurogenic function ectodermal layer that connect to neurons surrounding the endodermal layer organized as a larval nerve ring in a Comparaison of the function of one given developmental defined temporal manner. In addition, sensory nerve cells gene between cnidarians and bilaterians may help to developed at a higher density where the apical tuft elucidate its evolutive history. Gsx homologs are develops. The !-tubulin antibody highlighted elongated expressed during brain development of all bilaterian cells in the ectodermal layer that probably correspond to studied yet. In mice, the two Gsx duplicated genes (Gsh1 a third neuronal population. and Gsh2) are both expressed in the developing central nervous system (Hsieh-Li et al., 1995; Valerius et al., After Anthox2 inhibition, the RFamide and the !-tubulin 1995; Toresson and Campbell, 2001). In fishes, Medaka network were affected. The !-tubulin positive cells failed and Danio, the Gsh1 gene is found similarly as in mice in to appear and the formation of the RFamide nerve net the three parts of the developing brain as well as the was delayed. This showed that Anthox2 is necessary to hypothalamus and the spinal cord (Valerius et al., 1995; the proper formation of the neuronal diffuse nerve net in Deschet et al., 1998; Cheesman and Eisen, 2004). In Nematostella. Those results are in line with the function Ciona and Branchiostoma, Ci-Gsx and AmphiGsx are of Cnox2 in hydra that is implicated in the apical network detected in the posterior sensory vesicle and the cerebral
- 17 - Preliminary version vesicle respectively, both considered as homolog to the forebrain / midbrain of vertebrates (Brooke et al., 1998; is involved in the dorso-ventral patterning of the Hudson and Lemaire, 2001). In Polychaetes and in neuroectoderm (Weiss et al., 1998). At last, in Drosophila Gsx/Ind homologs are found in the Polychaetes (Denes et al., 2007; Kulakova et al., 2008), Orizia (Nguyen et al., 1999) and Xenopus (Illes et al., neuroectoderm (Weiss et al., 1998; Frobius and Seaver, 2006; Denes et al., 2007; Kulakova et al., 2008). 2009), Gsx positive cells were associated to cell proliferation location Gsh1/Gsh2 are involved in the dorso-ventral patterning of the embryonic mouse telencephalon and spinal cord Among bilaterians, the Gsx expression patterns suggest (Corbin et al., 2000; Toresson et al., 2000; Toresson and a conserved function in brain development both at the Campbell, 2001; Yun et al., 2001; Yun et al., 2003; Kriks proliferation and at the differentiation level, depending on et al., 2005). At the cellular level, Gsh2 positive cells of the context. The fact that Gsx genes are expressed in the the mouse telecenphalon were recognized as neuronal cell lineage from Cnidaria to Bilateria suggests an ancient role of Gsx in the nervous system patterning telencephalic progenitors and Gsx is required to their proper proliferation and identity specification (Toresson before the cnidarian divergence (Hayward et al., 2001; et al., 2000; Toresson and Campbell, 2001; Yun et al., Finnerty et al., 2003; Ryan et al., 2007; Chiori et al., 2001; Wang et al., 2009). Both Gsh1/2 are expressed in 2009). the sensory interneuron progenitors of the mouse spinal Our finding that the Anthox2 gene is a regulator of the cord where they control their differentiation during nerve net formation in Nematostella adds supplementary development (choice between excitatory and inhibitory proofs at a functional level, for the involvement of this fate) (Kriks et al., 2005; Mizuguchi et al., 2006). Gsh1 is gene in the neuronal specification and confirm data involved in the regulation of cell proliferation (Li et al., proposed by (Miljkovic-Licina et al., 2007) on the cnox2 1999). In addition, Gsh2 would act upstream of and/or in function in Hydra. In any case, double labeling with concert with the neural identity determinants of the known markers will be necessary to address more proneural bHLH protein family (Mash1 and Ngn1 and 2) precisely cell type expressing Anthox2 as well as their in some context to specify neuronal progenitor state in the cell cycle (proliferation vs differentiation). differentiation in the mouse dorsal spinal cord (Kriks et al., 2005; Mizuguchi et al., 2006) as well as in the At last, more global approach using microarray or large telencephalon (Toresson et al., 2000; Wang et al., 2009). scale real time PCR would be extremely useful to Therefore Gsh2 would be an early patterning gene, decipher the gene regulatory network to which Gsx gene regulator of the neuronal fate even maybe an initial belong in different phylum. A module of regulation could transcriptional determinant of the dorso-ventral patterning have been conserved along the evolution or at contrary that function to restrict the expression domains of maybe the Gsx gene kept its function but its upstream neuronal determination genes (Kriks et al., 2005). Gsh and downstream genes derived according to the system. genes are probably key components of the neural Acknowledgements transcriptional regulatory network in vertebrates. We are grateful to Simona Chera for excellent advices for IHC In Drosophila the Gsx homologue, ind, is required to give experiments. The work in our laboratory is supported by the the proper identity to the neuroblasts located in the Swiss National Fonds, the Canton of Geneva, the Claraz intermediate region of the neuroectoderm and therefore Donation and the Geneva Academic Society.
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Preliminary version
The ParaHox gene Gsx/Anthox2 regulates neurogenesis in developing Nematostella vectensis
Manon QUIQUAND, Ueli TECHNAU and Brigitte GALLIOT
Supplemental data
Table S1: Hox (PG1, PG2) and ParaHox (Gsx, Pdx, Cdx) genes used in this study (Fig. 2 and 3).
Table S2 : Table listing some criteria of the Gsx genomic organization in various phyla.
Addendum 1: Gsx genomic structure.
Table S3 : Primer sequences.
Figure S1: Map of the Nematostella Anthox2 gene (genomic and cDNA).
Figure S2: The Anthox2 genomic sequence.
Figure S3: The Tyr-tubulin pattern in the developing Nematostella from 64 cells stage (6 hours post-fertilization) to post-metamorphosis (11 days post-fertilization).
Table S4: Staging of nerve net formation in developing Nematostella.
Table S5: Survival rates in animals injected with Anthox2 and control morpholinos (MOs) from 1 to 6 days post-injection (dpi).
Addendum 2: Survival rate calculation.
Table S6: Table showing the distribution of the MO induced phenotypes.
Addendum 3: Calculation of MO injected animal phenotypes.
Table S7: DsRed2 expression rate recorded over from 1 to 9 dpi among the survivors.
Addendum 4: Expression rate calculation.
- 1 - Preliminary version
GENE FAMILIES PHYLA SPECIES NAMES GENE NAMES ACCESSION NUMBERS Pdx / Xlox (9 genes) Cnidaria Cnidaria: hydrozoan Clytia hemisphaerica Pdx Ch FM207043 Turritopsis dohrnii Pdx Td FM207048 Lophotrochozoa Annelida: polychaeta Capitella sp. Xlox Csp DQ102390 Mollusca Lottia gigantea Pdx Lg Contig4058/, LgGsHFWreduced.7013 Deuterostomes Echinodermata: echinozoa Strongylocentrotus purpuratus Lox Sp AF541970 Hemichordata Ptychodera flava Lox1 Pf AY436762 Urochordata: ascidiacea Ciona intestinalis IPF1 Ci AJ296167 Vertebrata: teleost Danio rerio Pdx1 Dr AF036325 Vertebrata: mammals Mus musculus IPF1 Mm X74342 Gsx (34 genes) Placozoa Trichoplax adhaerens Trox2 Ta AY319762 Cnidaria Cnidaria: hydrozoan Chlorohydra viridissima Cnox2 Cv AJ871179 Hydra vulgaris Cnox2 Hv AJ277388 Hydra magnipapillata Cnox2 Hm XM_002155415 Eleutheria dichotoma Cnox2 Ed Kuhn et al., 1996 Podocoryne carnea Gsx Pc AF268446 Hydractinia symbiolongicarpus Cnox2 Hs = Gsx Hs AF031953 DQ298519 Sarsia sp. Cnox 2 Ssp AF285145 Clytia hemisphaerica Gsx Ch FJ392846 Cnidaria: anthozoan Nematostella vectensis Anthox2 Nv AF085283
Acropora millipora Cnox2 Am AF245689 Cnidaria: scyphozoan Cassiopea xamachana Scox2 Cx AF124592 Lophotrochozoa Annelida: polychaeta Capitella sp. Gsx Csp DQ132894 Mollusca Lottia gigantea Gsx Lg Contig4058/ LgGsHFWreduced.7014 Ecdysozoa Arthropoda: hexapoda, Anopheles gambiae Gsx-Ag AJ439353 insecta Apis mellifera Gsh1-Ame XM_001120954 Bombix mori Gsx-Bm Bmb029189
Drosophila melanogaster Ind Dm AF095926 Tribolium castaneum Gsx-Tc AY695257 Deuterostomes Echinodermata: echinozoa Strongylocentrotus purpuratus Gsx-Sp XM_779393.2 Hemichordata Ptychodera flava Gsx Pf AY436761 Urochordata: ascidiacea Ciona intestinalis Gsx Ci AF305500 Urochordata: appendicularia Oikopleura dioica Gsx-Od AY705721 Cephalochordata Branchiostoma floridae Gsx Bf AF052463 Vertebrata: teleost Danio rerio Gsh1 Dr, Gsh2 Dr AY486348, CAN88050.1 Tetraodon nigroviridis Gsh1-Tn CAG00567.1 Vertebrata: mammals Mus musculus Gsh1 Mm, Gsh2 Mm U21224, S79041 Rattus norvegicus Gsh1 Rn, Gsh2 Rn XM_221885, EU202675 Bos taurus Gsx2 Bt XM_585770 Pan troglodytes Gsx1 Pt XM_522643 Homo sapiens Gsx1 Hsa, Gsx2 Hsa AB044157, AB028838 Cdx / cad (11 genes) Cnidaria Cnidaria: hydrozoan Eleutheria dichotoma Cnox4 Ed U41841 Clytia hemisphaerica Cdx Ch FJ392842 Lophotrochozoa Annelida: polychaeta Capitella sp. Cdx Csp DQ102389 Mollusca Lottia gigantea Cdx Lg Contig4118/LgGsHFWreduced.268gDNA Ecdysozoa Arthropoda: hexapoda, Drosophila melanogaster Cad Dm M21070 Nematodainsecta Caenorhabditis elegans Cdx Ce C38D4.6a.2 Deuterostomes Echinodermata: echinozoa Strongylocentrotus purpuratus Cad Sp XM_784065.2 Hemichordata Saccoglossus kowalevskii Cad Sk EU908781 Urochordata: ascidiacea Ciona intestinalis Cdx Ci AB210341 Vertebrata: teleost Danio rerio Cdx4 Dr X66958 Vertebrata: mammals Mus musculus Cdx1 Mm M37163 PG1 / lab (14 genes) Cnidaria Cnidaria: hydrozoan Hydra vulgaris Cnox1 Hv Q9NFW5 Hydra vulgaris Cnox3 Hv L22787 Podocoryne carnea Cnox1 Pc X81455 Eleutheria dichotoma Cnox5 Ed U41842 Cnidaria: anthozoan Nematostella vectensis Anthox6 Nv, HoxA DS469567 Lophotrochozoa Platyhelminthes Dugesia japonica Plox3 Dj AB024407 Annelida: polychaeta Capitella sp. Lab Csp EU196537 Mollusca Lottia gigantea Lab Lg Contig4114/ LgGsHFWreduced.206 Ecdysozoa Arthropoda: hexapoda Drosophila melanogaster Lab Dm X13103 Deuterostomes Echinodermata Strongylocentrotus purpuratus Hox1 Sp XM_776873.2 Hemichordata Ptychodera flava Hox1 Pf AY436753 Urochordata: ascidiacea Ciona intestinalis Hox1 Ci AB210490 Cephalochordata Branchiostoma floridae Hox1 Bf AB028206 Vertebrata: teleost Danio rerio Hoxb1a Dr BC162942 Vertebrata: mammals Mus musculus Hoxb1 Mm X53063 PG2 / Pb (8 genes) Cnidaria Cnidaria: anthozoan Nematostella vectensis Anthox7 Nv (HoxC) DS469567 Anthox8a Nv (HoxDa) DS469567 Lophotrochozoa Annelida: polychaeta Capitella sp. Hox2 Csp EU196538 Ecdysozoa Arthropoda: hexapoda, Drosophila melanogaster Pb Dm X63729 Deuterostomes insectaHemichordata Saccoglossus kowalevskii Hox2 Sk DQ985445 Cephalochordata Branchiostoma floridae Hox2 Bf AB028207 Vertebrata: teleost Danio rerio HoxA2b Dr AF307010 Vertebrata: mammals Mus musculus HoxA2 Mm M95599 Outgroups (2 genes) Cnidaria Cnidaria: hydrozoan Hydra Magnipapillata Nk2 Hv AF012538 Acropora millipora Vnd1 Am EF044214
TABLE S1: Hox (PG1, PG2) and ParaHox (Gsx, Pdx, Cdx) genes used this study (Fig. 2-3). Color code is as follows : yellow : Gsx sequences used in phylogenetic and gene structure analyses ; blue : Gsx sequences used in phylogenetic analyses ; no color : Gsx sequences used in gene structure analysis only ; grey : Accession numbers corresponding to sequences not annotated yet.
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A amino acids HD Gsx genomic sequence total encoded Intron HD position Exon 1 Intron Exon 2 position / composition (bp) lenght by the position / gDNA cDNA acceptor site Gsx Deuterostomes Gsx1 Vertebrates Gsx1-Hsa 412 463 383 1258 D138 413-875 902-1081 439-618 Gsx1-Pt 412 464 383 1259 D138 413-876 903-1082 439-618 Gsh1-Rn 661 510 377 1548 D221 662-1171 1198-1377 688-867 Gsh1-Mm 409 479 377 1265 D137 410-888 915-1985 436-606 Gsh1-Dr 379 89 353 821 E127 380-468 495-674 406-585 Gsh1-Tn 379 124 374 877 E127 380-503 530-709 406-585 Gsx2 Vertebrates Gsx2-Hsa 574 663 341 1578 G192 575-1237 1267-1446 604-783 Gsx2-Bt 577 683 341 1601 G193 578-1260 1290-1469 607-786 Gsh2-Rn 577 676 341 1594 G193 578-1253 1283-1462 607-786 Gsh2-Mm 577 628 341 1546 G193 578-1205 1235-1414 607-786 Gsh2-Dr 388 301 341 1030 G130 389-689 719-898 418-597 Gsx-Bf Cephalochordate 379 2966 374 3719 G127 380-3345 3375-3554 409-588 Gsx-Ci Urochordates 177 184 873 1234 T59/S60 178-361 1022-1201 838-1117 Gsx-Od 406 45 355 806 E124 407-451 553-773 472-651 Gsx Protostomes Gsx-Lg Mollusc 196 472 407 1075 E66 197-668 701-880 229-408 Ind-Dm Arthoropods 963 NO NO 963 NO NO 679-858 Gsh1-Ame 265 76 761 1102 D89 266-341 425-604 349-528 Gsx-Bm 885 NO NO 885 NO NO ND ND Gsx-Tc 675 NO NO 675 NO NO ND ND Gsx-Ag 1017 NO NO 1017 NO NO ND ND Gsx Cnidarians Cnox2-Cv 466 397 305 1168 V156 467-863 938-1120 541-723 Cnox2-Hv/Hm 463 340 305 1108 V155 464-803 881-1060 541-720 Cnox2-Ssp 274 323 311 908 V92 275-597 681-860 358-537 Gsx-Ch 496 469 356 1321 V166 497-965 1061-1240 591-770 Anthox2-Nv 319 177 311 807 A112 320-496 544-723 367-546
Trox2-Ta Placozoan 334 491 314 1139 D112 335-825 864-1043 373-552 B
Gsx genomic sequence Total composition: averages and Exon 1 Intron Exon 2 lenght standard deviations Average Gsx size 1 416 384 393 1193 SD Gsx size 131 204 145 268 Average Gsx size 2 424 399 374 1197 SD Gsx size 129 197 121 274 Average Gsx1 Vertebrates 442 355 375 1171 SD Gsx1 Vertabrates 108 193 11 274 Average Gsx1 Mammals 474 479 380 1333 SD Gsx1 Mammals 125 22 3 144 Average Gsx2 Vertebrates 539 590 341 1470 SD Gsx2 Vertabrates 84 163 0 247 Average Gsx2 Mammals 576 663 341 1580 SD Gsx2 Mammals 2 24 0 24 Average Gsx Cnidarians 404 341 318 1062 SD Gsx Cnidarians 100 108 22 206
TABLE S2 : Table listing some criteria of the Gsx genomic organization in various phyla. A) Precise length of the exons. Intron and homeodomain (HD) positions in the genomic Gsx sequences. B) Averages and standard deviations of the total length of Gsx genes, of exons and intron. Average Gsx size 1 corresponds to all Gsx sequences except Bf, Sp, Dm, Bm, Tc, Ag. Average Gsx size 2 corresponds to all Gsx sequences except Bf, Sp, all insects. Data shown in Figure 3B.
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ADDENDUM 1: Gsx genomic structure. Gsx genomic structures of vertebrates are relatively well conserved and particularly in mammals. Vertebrates gsx genes are divided into two sub-families following a duplication event: Gsx1 and Gsx2. As expected, the genomic structure is more conserved in between sequences of different vertebrate species belonging to the same gene sub-family than in between the two gsx genes of the same species. On average, Gsx2 genes are longer than Gsx1 ones. In both families, the second exon size is more conserved and smaller than the first one. The size of the second is a bit longer in the Gsx1 family. All sequences analyzed in the Gsx2 family have exactly the same second exon size. The size of the first exon as well as the intron is more conserved and both are larger in the Gsx2 than in the Gsx1 genes. In both families, the intron position is highly conserved relative to the 5’ end of the homeobox; between 26-29 nucleotides. The consensus sequences of the five amino acid encoded by the upstream region of the acceptor site are CISV/I/LX and CLT/SXG respectively for Gsx1 and Gsx2 family. The consensus sequences encoded by the downstream region of the donor site are G/ASDXS for Gsx2. No consensus sequence could be retrieved for Gsx1. Nevertheless, we took a Tetraodon sequence, known to be a species highly derived, to extract consensus of the Gsx1 family. By removing it, consensus amino acid sequence encoded by the upstream sequence of the acceptor site and by the downstream sequence of the donor site are CISV/IDE and XSSXQ respectively. In both cases, fishes gsx genomic coding sequences are shorter than mammalian counterparts due to a reduction of the intron size. The five last and first amino acids encoded by the acceptor and the donor splicing site respectively are less conserved compared to mammalian’s one. In both gsh1 fishes genomic sequences the first exon is the same size and the sequence upstream of the acceptor site encodes for a consensus sequence: CISXE. The genomic sequence downstream of the donor site encodes for a sequence less well conserved. In mammals, the structure of the genes in both families is well conserved and especially for the Gsx2 family. As when considering vertebrates, the size of both exons is more conserved in the Gsx2 family. Nevertheless, the intron size is less variable in the Gsx1 family but the difference is probable not significant. The intron of the mammals Gsx2 family is placed in the 192 or 193th codon. In mammalian, the five last amino-acids encoded by the first exon as well as the five first encoded by the second exon are well conserved in both family: CISVD and SSSNQ, CLT/SMG and GSDXS in the Gsx1 and Gsx2 families respectively. The position of the intron is more variable in the Gsx2 family and the amino acids encoded by the upstream and downstream sequences of both splicing site are less conserved than in the Gsx1 family. Among cnidarians, the structure of the gene is less conserved than among mammals. The genomic sequence size is comprised between 807 and 1321 bp. The size of the second exon is more conserved as its length is comprised between 305 and 356 bp whereas the length of the first one is comprised between 274 and 493 bp. The Trox2-Ta gene structure resembles more to cnidarian one than to any other. Compare to vertebrates, the intron position is shifted to a more 5’ position relative to the Nterminal end of the HD. Some species display atypical Gsx genomic structure: Branchiostoma and Strongylicentrotus displayed longer Gsx gene than the other counterparts: 3719 bp and 9628 bp respectively. This difference is due to very long intron, especially in the Strongylocentrotus sequence but the size of their exons is not so different from their counterparts. Strongylocentrotus gsx genomic sequence is the only one to be composed of three exons and two introns and to carry the splice site of the second intron within the homeobox. In adition, this splice site is placed in between two codons as for the intron of Ciona. In addition, Ciona has a homeobox distantly located from the splice site (660 nucleotides). In all insects Gsx genomic sequences analyzed (Bombyx, Tribolium, Anopheles – not shown - Dorsophila, Apis) we didn’t detect any intron except in Apis, which also presents a very long second exon. In Lottia and Sarsia the first exon is smaller than the second one. The Gsx genomic structure is relatively well conserved across the evolution. Branchiostoma and Strongylocentrotus were probably submitted to insertion events in their Gsx gene. The genomic structure of Gsx genes in fishes is more different to mammalian counterparts than cnidarians structures are.
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Gene names Templates Clones Primer names Primer sequences References GCTTTGTTTAAACGATTTTGCT Anthox2 gDNA Ax2-4300 Ax2F8 TG This paper Ax1R1 TGACAAGCTTTTATTCACTTCC GCTTTGTTTAAACGATTTTGCT Ax2-3122 Ax2F8 TG This paper Ax2R7 GATATCAGGAGATGTGTGCCG (Finnerty, Anthox2 cDNA Ax2-954 Ax2F1 AAGTCGGTACCAACGAGTAC 2004) Ax2R1 TGACAAGCTTTTATTCACTTCC TAGCTATCGCGATGAATGAAG Anthox2 Ax2-3122 Ax2-1000_DsRed2 Nru1-Ax2pr-1000F TCTTGTCGCCGG This paper TAGCTAGCTAGCGATATCAGG Nhe1-Ax2ATGR AGATGTGTGCCG TAGCTATCGCGAGAATAAATG Ax2-2000_DsRed2 Nru1-Ax2pr-2000F TTCTTGTATTCAATCTAGTTC This paper TAGCTACTTAAGGATATCAGG Afl2-Ax2ATGR AGATGTGTGCCG ATGGGCAAGCAAGAAGACGG SoxB2 cDNA SoxB2-819 SoxB2-F1 G (Magie, 2005) CTAGAGCAATACCGGTACGTA SoxB2-R1 AGC ATGACCAAACCAGGAGATCAT Sox2 cDNA Sox2-705 Sox2-F1 ATT (Magie, 2005) Sox2-R1 TCAGTAACCCTCCGGCCG ACCACCTACAACTCCATCATG Actin cDNA Actin-225 Ch, Nvactin-F1 AA This paper TTTGAAGATCCACATCTGTTG Aauactin-R G
TABLE S3 : Primer sequences. Sequences of the primers used for the RT-PCR assay and the cloning of Anthox2 cDNAs, gDNAs from Nematostella and Anthox2 reporter contructs.
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FIGURE S1: Map of the Nematostella Anthox2 gene (genomic and cDNA). Rectangles represent exons, red line the homeobox. Ax2-4300 is the genomic clone full length presented in Fig. S2. Ax2-3122 is the clone containing 3122 bp of the upstream region of Anthox2 used to build all the Anthox2 reporter constructs. Ax2-954 corresponds to the cDNA clone used to synthesize riboprobes and to product Ax2-585 and Ax2-354, two other plasmids also used as template for riboprobes synthesis. Ax2-1999 and Ax2-996 represents region amplified to build Ax2-1999_DsRed2 and Ax2-996_DsRed2 constructs respectively.To simplify the 3 reporter constructs are named Ax2-3000_DsRed2, Ax2- 1999_DsRed2 and Ax2-1000_DsRed2.
- 6 - Preliminary version
Nhe1 Ax2F8-> gctagcgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtattttgattttctggtgagtcgttttgttttcggtcgcc 100 acgtctatgtcccgcagttctttttgtactaccgtcacaaggccctcaaaaatacctgtgaacataaaagccaaacacaacagcctcaaagtgagcaaaa 200 aacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaactgcaggatcacaaaaagtgtttcatgtttccttttctct 300 cgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggcttgcgacgggtttgcggaggggcgaatacgctcctggtta 400 Gsh ttgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaatttcaggccaattaaattaattttccatatgtcagagaccttg 500 ccatcttactaggacttgatcaatcgttttttttttttctttttggtcaattgtgtttacgctgttaactcgatcatgactttaattaagtcgcgttcgc 600 aagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctatttgcgcgcgattcaaggctcattggtaatcttttgagctttgacttgcaagac 700 taaatgagctccaactaggtactaaagctttagagatgtttcccgtggaattaaagaggcataagtttgctttagtgttaccggggtaacttctaagaag 800 aattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgcttgaattttttgtactgttgttattttttccatactaaaatcgtacatc 900 GATA1/1A ttctgttttacgccttgtttttagtactttgaaaacctttttcacaaataagtcagattgtttaagagaaatacattccagaagtttaaagataatgtat 1000 EcoRV <-Ax2R6 Ax2F7-> attgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttgattttcgatcgtttgtgaggatgatttgtcttttcgttcataaccttcgttcg 1100 Nru1-Ax2pr-2000F-> Croc tttttacggttaatgttcttgacatttttgaataaatgttcttgtattcaatctagttctcaaaacaccaggcttttatatttattgaagaggctgttcc 1200 GATA-3 tatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaactccgtgataaaattcagaattagatcatttgct 1300 Sox5 Oct2.1/HNF-3B atcggcatataaggaactaaatttaccaggcaatcttttgataaatattacccgatgagaattgtttattttgtctgcgaaagcaaatcaaagagctgac 1400 Nhe1 ttctgtatgaagcctatttaccttcaacattcatttcatttccttagtcaagtatttgagaggaagaaacttcggctagcgtattttgttcgagttcgtt 1500 <-Ax2R5 Ax2F6 EcoRV Ftz/GATA-1 aaaattatgtttccggaaagattagagaaacaattattatagtaaatacaggccgttacaatttgatatctttaattgttatctagccgttgtagtcctt 1600 ttcatttagattagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattcggaacgaaaaatacttgagaatccttttttatatatattctt 1700 tgagatactaagagattagatcaatcagaagagttaatgaactggtaggttaagaagtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaa 1800 tagttgattcttgtttttgttaatgaacgattcattcgtatctttgacgggttaaagttccccactgggcggcacctaatcttgtgtaactaacaagata 1900 gtgaacctacgtggtgtcatgatttacctgctacgcacaaactaagcgcatgtaatgtgttcaccaagacgagaggcattcaatattttatcgctcagtt 2000 gacgtacaatcgcttcaattgccccccagcgttcactaactctactgttttcttcacgcaaaacgcaaacacgatcttttaatattttcctagaaaataa 2100 Nru1-Ax2p-1000F -> Zen1/2 ttgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaata 2200 Croc cctattctccttcggtctagtcgccaggctatttatttttcttgttatatgattaaaacatatttcacattttggaattgggaaacaattacccatgacg 2300 cgtgcagaacggccattctaacgtcctggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaac 2400 HNF-3/3B aaataagttccgaaagaaaacttgcaggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaaga 2500 attatggtctacatctggaagtttggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgag 2600 aagacgtcgattgcttacacaggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgac 2700 Sox5 aattacggacgaaaaaaggccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttat 2800 actcaagggggaaaataattctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaa 2900 tctccttaaaacatgtcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttg 3000 tgttgcgaacaataggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcgcagc 3100 Ax2R7 / Afl2-Ax2ATGR / NheI-Ax2ATGR Ax2R3 TCCCAGACGGCACACATCTCCTGATATCATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGTCGGTACCAACGAGTACTTCAGAGCCG 3200 M S S S F Y I D S L I S K A K S V P T S T S E P Ax2F2 -> Nhe1 CGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTA 3300 R H T Y E S P V P C S C C W T P T Q P D P S S L C Q L C I P T S A S
GCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACA 3400 V H P Y M H H V R G A S I P S G A G L Y S R E L Q K D H I L L Q Q ACACTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTTATGgtgagttgcgctttccacgccaaaacaaggcttcaaggaccctaacaggtgtc 3500 H Y A A T E E E R L H L A S Y A cgacactaaagctctgtcaaaaatatccatgatgtcctttgaaaaaatccattgaacttaatggtgaaatataattattatttccttaattctgtgttta 3600 atagaattttttgtctttttttagCATCATCACGAGATCCTGACAGTCCATCAAGGGGAGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAG 3700 S S R D P D S P S R G G N S R S K R I R T A Y T S CATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAG 3800 M Q L L E L E K E F S Q N R Y L S R L R R I Q I A A L L D L S E K CAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGT 3900 Q V K I W F Q N R R V K W K K D K K A A Q H G T T T E T S S C P S S CGCCAGCAAGCACCGGTAGAATGGATGGTGTATGAACACTAAAATTGAACCATAATTGTACAGTTTGTATATAGTTTAATGTACTATATTCGGGGCAACC 4000 P A S T G R M D G V * ttgttttcataatttgtatagaatctatagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtt 4100 tgtgagggataaattgtaaaacaaaaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaact 4200 Ax2R1 tttctgagaattttaccatgcatttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtc 4300 acttat 4306
FIGURE S2: The Anthox2 genomic sequence (Ax2-4300). The forward and reverse primers used in this work are indicated in light green and blue respectively. Genomic sequences are lowercase, cDNA ones uppercase; the start and stop codons are uppercase and bold; the putative transcription start sites that were predicted with the HCtata program (Hamming-Clustering Method for TATA Signal Prediction in Eukaryotic Genes) are lowercase, bold. Putative binding sites for transcription factors are written white and highlighted dark green. The HD in the deduced protein sequence is boxed. The Anthox2 morpholino (MO) binding site immediately upstream to the start codon is boxed.
- 7 - Preliminary version
FIGURE S3: The Tyr-tubulin pattern in the developing Nematostella from 64 cells stage (6 hpf) to post-metamorphosis (11 dpf). A) 64 cells stage embryo (fixed 6 hpf), B) 9th cell cycle (fixed 7h30 pf), C) 12th cell cycle (fixed 11 hpf) D) gastrula (fixed 22 hpf), E) post-gastrula (fixed 30 hpf), F) planula (fixed 37 hpf), G) pre-metamorphosed polyp (fixed 8 dpf), H) metamorphosed polyp (fixed 11 dpf). A, C, D, E) Arrows and arrowhead marks cells in metaphase and anaphase respectively. D) blastopore is indicated with an asterisk. G-H) (future) oral pole is indicated with an asterisk. F, H) arrows mark the apical tuft. Scale bars: A- H) 50 µm, magnifications) 20 µm. The anti-Tyrosine tubulin antibody labels dividing cells up to the gastrula stage where it starts to detect cell membranes and ciliary epithelium too. As soon as the apical tuft appears the anti-Tyrosine-tubulin detects it. hpf: hours post-fertilization, dpf: days post-fertilization.
- 8 - Preliminary version
RFamide nerve not injected animals H2O injected animals net formation in Stage Stage Stage Stage Stage Stage Stage Stage nt nt planulae 0 1 2 3 0 1 2 3
1 dpf (31 hpf) 8 8 0 0 0 9 9 0 0 0 % 100 0 0 0 100 0 0 0 2 dpf (47 hpf) 9 4 5 0 0 11 2 9 0 0 % 44 56 0 0 18 82 0 0 2,5 dpf (57 hpf) 7 3 4 0 0 6 4 2 0 0 % 43 57 0 0 67 33 0 0 3 dpf (72 hpf) 10 2 6 2 0 6 2 3 1 0 % 20 60 20 0 33 50 17 0 3,5 dpf (82 hpf) 9 1 5 3 0 9 5 3 1 0 % 11 56 33 0 56 33 11 0 4 dpf (95 hpf) 8 0 5 3 0 9 1 5 3 0 % 0 63 38 0 11 56 33 0 4,5 dpf (104 hpf) 9 0 0 5 4 9 0 1 7 1 % 0 0 56 44 0 11 78 11 5 dpf (118 hpf) 13 0 3 10 0 % 0 31 108 0 5,5 dpf (128 hpf) 8 0 3 2 3 10 0 2 2 6 % 0 38 25 38 0 20 20 60 6 dpf (142 hpf) 8 0 0 0 8 9 0 1 2 6 % 0 0 0 100 0 11 22 67 6,5 dpf (153hpf) 7 0 0 1 6 7 0 1 3 3 % 0 0 14 86 0 14 43 43
TABLE S4: Staging of nerve net formation in developing Nematostella. Neve net formation was detected by RFamide immunostaining in wt animals either not injected or water injected immediately after fertilization. nt: total number of animals in each condition. Data plotted on Figure 6. hpf: hours post-fertilization, dpf: days post-fertilization.
- 9 - Preliminary version
A
Survival rate N Fl+
Anthox2 MO 0.25 mM 1 34 Control MO 0.25 mM 1 57
Anthox2 MO 0.5 mM 6 313 Control MO 0.5 mM 6 320
Anthox2 MO 0.75 mM 1 26 Control MO 0.75 mM 1 29
Anthox2 MO 1 mM 1 26 Control MO 1 mM 1 18
B
Survival rate (Anthox2 / Control) dpi 0.25 mM 0.5 mM 0.75 mM 1 mM 1 0.95 1.13 1.07 0.94 2 0.78 1.07 0.49 0.73 3 0.77 0.69 0.14 0.43 4 0.51 0.39 0.09 0.40 5 0.51 0.16 0.09 0.30 6 0.12 0.00 0.14
TABLE S5: Survival rates in animals injected with Anthox2 and control morpholinos (MOs) from 1 to 6 days post- injection (dpi). Data plotted in Figure 7B. N= Number of independent injection experiments, Fl+=number of fluorescein positive eggs 1 dpi.
ADDENDUM 2: Survival rate calculation. Survival rate was calculated each day, over the number of fluorescein positive animals. All sample treated in the same condition (Anthox2 or control MO at four different concentrations) in one given injection experiment were pooled allowing to calculate one survival rate for each condition per injection experiment. A mean value of the survival rate of Anthox2 and control MO injected animals was calculated over all injection experiments as well as a standard deviation. The ratio plotted corresponds to (mean of the Anthox2 MO survival rate / mean of the control MO survival rate) (Table S5, Fig. 7B).
- 10 - Preliminary version
A !-tubulin patterns class I class II class III H2O n= 5 3 2 0 % 60 40 0 Fluo- n= 5 5 0 0 % 100 0 0 Control (MO) n= 22 12 7 3 % 54.5 31.8 13.6 Anthox2 (MO) n= 16 5 4 7 % 31.3 25 43.8
B Stage Stage Stage Stage RFamide stages @ 3 dpi nt 0 1 2 3 H2O, 3 dpf (71 hpf) n= 6 2 3 1 0 % 33.3 50 16.7 0 H2O, 3,5 dpf (82 hpf) n= 9 5 3 1 0 % 55.6 33.3 11.1 0 H2O -> Mean values 44.4 41.7 13.9 0 uninj, 3 dpf (71 hpf) n= 10 2 6 2 0 % 20 60 20 0 uninj, 3,5 dpf (82 hpf) n= 9 1 5 3 0 % 11.1 55.6 33.3 0 uninj -> Mean values 15.6 57.8 26.7 0 Fluorescein (-) n= 12 0 5 5 2 % 0 41.7 41.7 16.7 Control (MO) 0.5 mM n= 40 11 23 6 0 % 27.5 57.5 15 0 Anthox2 (MO) 0.5 mM n= 29 16 13 0 0 % 55.2 44.8 0 0 Stage Stage Stage Stage RFamide stages @ 5 dpi nt 0 1 2 3 H2O-injected n= 10 0 2 2 6 % 0 20 20 60 uninjected n= 8 0 3 2 3 % 0 37.5 25 37.5 Fluorescein (-) n= 9 0 2 4 3 % 0 22.2 44.4 33.3 Control (MO) 0.25 mM n= 19 2 9 7 1 % 10.5 47.4 36.8 5.3 Control (MO) 0.5 mM n= 45 4 24 17 0 % 8.9 53.3 37.8 0 Anthox2 (MO) 0.25 mM n= 6 0 1 5 0 % 0 16.7 83.3 0 Anthox2 (MO) 0.5 mM n= 6 2 4 0 0 % 33.3 66.7 0 0
TABLE S6: Table showing the distribution of the MO induced phenotypes. The phenotypes of MO injected planulae were detected either with anti-btubulin immunostaining at 4 dpi (A) or with anti RFamide immunostaining at 3 and 5 dpi (B). The data are shown in Figure 7F.
ADDENDUM 3: Calculation of MO injected animal phenotypes.
To calculate the percentage of animals belonging to a given class of !-tubulin phenotype as well to a given stage of RFamide neuronal network progression all samples coming from different injection experiments were pooled together. Hence, no standard deviation is available (Table S4, S6, Fig. 7E-F).
- 11 - Preliminary version
DsRed2 Expression Rate (%) Ni= 5 Ns= 11 Ni= 2 Ns= 4 Ni= 2 Ns= 4 Ni= 14 Ns= 22 Ni= 4 Ns= 8 Ax2-3000 Ax2-2000 Ax2-1000 CMV empty DPI mean SD mean SD mean SD mean SD mean SD
1 11 10 40 28 55 26 7 10 0 0 2 41 24 79 23 85 6 37 27 4 7 3 39 21 86 14 87 10 40 24 3 9 4 41 19 83 15 85 15 38 24 2 5 5 26 18 95 6 74 11 37 24 7 10 6 25 20 95 7 80 3 28 24 0 0 7 27 24 95 7 74 11 22 23 0 0 8 22 22 88 3 69 17 30 23 0 0 9 43 20 81 13 65 11 25 35 0 NO
TABLE S7: DsRed2 expression rate recorded over from 1 to 9 dpi among the survivors. All DsRed2 constructs were injected at the one cell stage in 30 to 40 embryos per sample. Ni: Number of independent injection experiments. Ns: Number of independent samples. Data plotted in Figure 8B.
ADDENDUM 4: Expression rate calculation.
Positive animals were screened under the rhodamin fliter. The expression rate was calculated over the number of living animals at each day. Each sample treated in the same condition in one given experiment was considered independently. Hence one expression rate was calculated at each day for each sample of each injection experiment. A mean value of the expression rate was calculated in between all similar samples of different injection experiments as well as a standard deviation (Table S7, Fig. 8B).
- 12 - II.3. CHAPITRE 3 : Evolution de la neurogénèse
Les bilatériens partagent des cascades communes de régulation à l’origine des processus développementaux comme la spécification des axes. Les processus de différentiation cellulaire associés sont eux aussi conservés entre les différents phyla et utilisent des gènes orthologues pour la myogénèse (Yun and Wold, 1996), la neurogénèse (Bertrand et al., 2002; Denes et al., 2007) ou la gamétogénèse (Cox et al., 1998) par exemple.
Chez les bilatériens, la spécification des territoires neuronaux selon les axes se met en place au moyen de cascades de signalisation et de facteurs de transcription conservés, comme par exemple la cascade BMP et les gènes Hox. Par ailleurs, la différentiation des progéniteurs neuronaux utilise un code moléculaire faisant intervenir des protéines à homéodomaines et de la famille bHLH. Comme nous l’avons déjà discuté, les cnidaires sont les premiers animaux à présenter une polarité et un système nerveux développé. Pour cette raison, ils constituent un modèle expérimental d’intérêt pour retracer la fonction ancestrale des gènes régulateurs, intervenant dans ces mécanismes et qui sont conservés chez les bilatériens. Les cnidaires pourraient partager avec les bilatériens des processus de différentiation cellulaire communs, comme la neurogénèse. En effet, de nombreux gènes connus des bilatériens pour leur fonction neurogénique sont également représentés chez les cnidaires chez qui les analyses de patron d’expression et les analyses fonctionnelles suggèrent une fonction similaire. Nous avons présenté dans le chapitre précédent l’étude détaillée du gène Anthox2 chez Nematostella qui, comme ses orthologues chez les bilatériens, semble être un bon candidat au soutien des processus de neurogénèse.
Dans ce chapitre, nous avons recensé les processus cellulaires connus menant à la mise en place du système nerveux chez les cnidaires ainsi que les acteurs potentiellement à l’origine de ce processus. Nous discutons les scénarios évolutifs soutenant l’apparition de la neurogénèse d’une manière unique. Par ailleurs, nous discutons le cas des éponges qui, bien que ne présentant pas de système nerveux différencié, ont dans leur génome au moins certains composants des cascades de signalisation et certains des facteurs de transcription qui sont associés à la neurogénèse chez les bilatériens. L’étude des mécanismes de régulation de la neurogénèse chez les cnidaires permettrait de mettre en évidence le pool d’outils moléculaires dont aurait hérité l’ancêtre commun aux bilatériens.
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126 Developmental Biology 332 (2009) 2–24
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Developmental Biology
journal homepage: www.elsevier.com/developmentalbiology
Review Origins of neurogenesis, a cnidarian view
Brigitte Galliot ⁎, Manon Quiquand, Luiza Ghila, Renaud de Rosa1, Marijana Miljkovic-Licina 2, Simona Chera 2
Department of Zoology and Animal Biology, Faculty of Science, University of Geneva, Sciences III, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland article info abstract
Article history: New perspectives on the origin of neurogenesis emerged with the identification of genes encoding post- Received for publication 23 March 2009 synaptic proteins as well as many “neurogenic” regulators as the NK, Six, Pax, bHLH proteins in the Revised 14 May 2009 Demosponge genome, a species that might differentiate sensory cells but no neurons. However, poriferans Accepted 16 May 2009 seem to miss some key regulators of the neurogenic circuitry as the Hox/paraHox and Otx-like gene families. Available online 22 May 2009 Moreover as a general feature, many gene families encoding evolutionarily-conserved signaling proteins and Keywords: transcription factors were submitted to a wave of gene duplication in the last common eumetazoan ancestor, Neurogenesis after Porifera divergence. In contrast gene duplications in the last common bilaterian ancestor, Urbilateria, Nematogenesis are limited, except for the bHLH Atonal-class. Hence Cnidaria share with Bilateria a large number of genetic Nervous system tools. The expression and functional analyses currently available suggest a neurogenic function for numerous Cnidarians orthologs in developing or adult cnidarians where neurogenesis takes place continuously. As an example, in Poriferans the Hydra polyp, the Clytia medusa and the Acropora coral, the Gsx/cnox2/Anthox-2 ParaHox gene likely Urbilateria supports neurogenesis. Also neurons and nematocytes (mechanosensory cells) share in hydrozoans a Evolution common stem cell and several regulatory genes indicating that they can be considered as sister cells. Regulatory genes Performed in anthozoan and medusozoan species, these studies should tell us more about the way(s) Hydra Clytia evolution hazards achieved the transition from epithelial to neuronal cell fate, and about the robustness of Podocoryne the genetic circuitry that allowed neuromuscular transmission to arise and be maintained across evolution. Nematostella © 2009 Elsevier Inc. All rights reserved. Aurelia
Introduction below) but also in poriferans (Larroux et al., 2006, 2008) and some could even be traced in choanoflagellates (King et al., 2008). Urbilateria and its older sisters Cnidaria and Ctenophora The Zootype hypothesis proposed first that a same set of regulatory genes, namely homeobox genes, define the anterior to posterior (AP) In 1978, Ed Lewis in his seminal Nature paper (Lewis, 1978) axis in all animal species at an early and transient developmental predicted the evolutionary conservation of DNA-binding regulatory stage (Slack et al., 1993). Subsequently the Urbilateria hypothesis proteins that would control patterning along the anterior–posterior proposed that, beside the AP axis, deuterostomes and protostomes axis through cis-regulatory elements. Since then, the accumulation of also received from a common putative ancestor, named Urbilateria a molecular and genetic data indeed proved the wide conservation of genetic toolkit that specifies their dorso-ventral axis, including their the genetic networks regulating shared developmental processes neural tube (De Robertis, 2008). In the absence of extant Urbilaterian among bilaterians, not only for the specification of the anterior to species, the Ctenophora and Cnidaria that diverged earlier in animal posterior axis but also the dorso-ventral axis, the head patterning and evolution but display anatomical polarities and differentiate a nervous the eye specification (De Robertis, 2008). As anticipated, the main system, are obvious candidates to test these hypotheses (Fig. 1). In fact cellular differentiation processes in bilaterians also make use of the initial expression analyses performed at the cellular level evolutionarily-conserved genetic circuitries as those used for myo- supported the hypothesis of a common origin for neurogenesis and genesis (Yun and Wold, 1996), neurogenesis (Bertrand et al., 2002; also for the specification of the apical nervous system in cnidarians Acampora et al., 2005; Denes et al., 2007; Tessmar-Raible et al., 2007), and anterior nervous system in bilaterians (Gauchat et al., 1998; gametogenesis (Cox et al., 1998). Since 1991, orthologs of these Galliot and Miller, 2000). However this simple rule of the universal bilaterian regulatory genes were identified not only in cnidarians (see conservation of developmental genetic toolkits between animal phyla received some assault when it appeared that the zootype hypothesis ⁎ Corresponding author. Fax: +41 22 379 33 40. could not be verified in cnidarians (Gauchat et al., 2000; Schierwater E-mail address: [email protected] (B. Galliot). and Desalle, 2001; Chourrout et al., 2006; Kamm et al., 2006; Lee et al., 1 Present address: Ecole Normale Supérieure, Département de Biologie, 45 rue d’Ulm, 45, rue d'Ulm F-75230 Paris, France. 2006; Ryan et al., 2007; Chiori et al., 2009; Quiquand et al., 2009), and 2 Present address: Faculty of Medicine, University of Geneva, 1, rue Michel-Servet, it is nowadays admitted that the specification of the embryonic AP CH-1211 Geneva 4, Switzerland. axis by the Hox gene families only arose after Cnidaria divergence.
0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.05.563 B. Galliot et al. / Developmental Biology 332 (2009) 2–24 3
Fig. 1. Origin of neurogenesis and progressive acquisition of a central nervous system along animal evolution. The differentiation of cells with synaptic transmission can be traced back to the last common ancestor of eumetazoans, whereas the differentiation of sensory cells possibly emerged in the last common ancestor of choanoflagellates and metazoans; in Porifera choanocytes are proposed to correspond to sensory cells. Both Ctenophora and Cnidaria differentiate a nervous system; they diverged prior to Bilateria but their respective positions are controversial. Similarly the position of Placozoa in Metazoa is debated. ⁎Indicate species with sequenced genome; especies that differentiate eyes, ospecies that have lost the medusa stage.
However what is true for the AP axis might not be true for the named Urbilateria (Fig. 1). Cnidarians are most often marine animals specification of the nervous systems. Alain Ghysen wrote about the that commonly display a radial symmetry and are made up of two-cell Origin and Evolution of the Nervous System: “The extreme variability layers, the ectoderm and the endoderm, separated by an extracellular of behaviors and survival strategies among triploblasts would be matrix named mesoglea (Bouillon, 1994b). However this “diploblas- subordinate on the previous attainment by the urbilaterians of a high tic” criterion is disputed as numerous cnidarian species actually level of developmental stability in the building of elementary differentiate “mesodermal” derivatives as striated muscle at one or the functional circuits. According to this view, the initial triploblast radia- other stage of their life cycle (Seipel and Schmid, 2006). Cnidarian tion may have been contingent upon reaching this highly evolved species cluster in two distinct classes (Bridge et al., 1995; Collins et al., stage of neural development” (Ghysen, 2003). In other words, the 2006): the anthozoans that live exclusively as polyps (sea pens as neurogenic circuitry was already established in a very stable way in Renilla, stony corals as Acropora, sea anemones as Aiptasia, Antho- Urbilateria (Arendt et al., 2008), suggesting that it might be possible to pleura, Nematostella) and the medusozoans that display a complex life trace back some features of this ancestral nervous system in cycle with a parental medusa stage and a sessile polyp stage. Among cnidarians that differentiate a rather sophisticated nervous system those, the cubozoans (Tripedalia cystophora) and scyphozoans (Aurelia with numerous cellular and functional similarities to bilaterian ones. aurita, Cassiopea xamachana) predominantly live as medusae, whereas In bilaterians, homologous tasks such as differentiating nerve cells the hydrozoans (Podocoryne, Clytia, Cladonema, Eleutheria) usually (Simionato et al., 2008) and mechanosensory organs (Ghysen, 2003), follow a life cycle where they alternate between these two forms. developing eyes (Pichaud and Desplan, 2002; Gehring, 2004), However some hydrozoan species have lost the medusa stage as the regionalizing the neural tube along the dorso-ventral axis (Denes et marine Hydractinia and the freshwater Hydra polyps (Galliot and al., 2007; Mieko Mizutani and Bier, 2008) or patterning the tripartite Schmid, 2002). Similarly the staurozoans that were only recently brain (Lichtneckert and Reichert, 2005) rely on a shared set of characterized as a group (Collins et al., 2006), live exclusively as transcription factors. We propose here to review the current know- polyps. Cnidarian polyps are basically a tube with a single opening ledge about the molecular mechanisms that support neurogenesis in circled by a ring of tentacles, which has a mouth–anus function. cnidarians and discuss some scenario that led to this unique evolu- Cnidarians together with ctenophores (combjellies) are the first phyla tionary transition. where movements are governed by a neuromuscular system, as exemplified by their active feeding behavior that requires coordinated The complex life cycle of cnidarians movements of their tentacles (Westfall and Kinnamon, 1984; Westfall, 1996). Therefore, cnidarians and ctenophores provide appropriate Cnidaria is supposed to have diverged about 650 million years ago, model systems to trace back the first-evolved nervous systems preceding the Cambrian explosion, the period when ancestors to most (Anderson and Spencer, 1989). In contrast, poriferans (sponges), extant bilaterian phyla arose from a common hypothetical ancestor which diverged earlier during evolution and are capable of chemical 4 B. Galliot et al. / Developmental Biology 332 (2009) 2–24
Fig. 2. Schematic views of neurogenesis and nematogenesis during the cnidarian life cycle. (A) Neurogenesis in the developingPodocoryne hydrozoan. As for all medusozoan species with a medusa stage, the mature jellyfish release the gametes. At mid-gastrula stage (b) the precursors of nerve cells and nematocytes (pr) arise in the endoderm, rapidly differentiate andmigrate to the ectoderm, forming a diffuse network throughout the swimming planula larva (c). At this stage the nerve cells (nv), detected here with an anti-tyrosine tubulin antibody, show laterally oriented neurites that form a ladder (Groger and Schmid, 2001). The anterior pole contains RFamide+ neurons (nv) and the posterior pole large mature nematocytes (ne). Upon metamorphosis, the larval anterior pole becomes the aboral region of the polyp (also named foot) and the larval posterior pole provides the oral region(also named head). (B) In polyps the nerve net is much denser in oral and aboral regions than in the body column. In intactHydra (a), neurogenesis takes place in the body column where interstitial stem cells provide neuronal progenitors that migrate and differentiate in the upper and lower regions of the body column. In head-regeneratingHydra (b), de novo neurogenesis takes place at the tip to reform in two days the apical nerve net. Progenitors are detected in the tip at 24 hpa and neurons after 32 hpa. (C) In the adult medusa (a) neurogenesis takes place in three regions: the manubrium (b), the tentacle bulb (c) and the sensory organs, which may contain eyes (d) and statocysts. b) Closer view of aClytia manubrium with the mouth opening directed to the bottom and the nerve net detected with the anti-RFamide antibody; cell bodies (c.b) and neuronal projections (n.p). (c) Staggered nematogenesis in tentacle bulbs: stemcells located in the most proximal position (α) initiate nematocyte differentiation less proximally (β), until nematocytes migrate distally in the maturation area (γ) and finally reach the tentacle when mature (δ) as shown by (Denker et al., 2008b). Tentacle bulbs are also the site of intense neurogenesis, as depicted on the right with RFamide nerve cells that project from the bulb to the tentacle.Neuronal precursors can also be found in theα zone as suggested by theGsx expression in Clytia (see Fig. 5). (d) Drawing of a Cladonema eye after (Weber,1981) with the tripartite lens, the ciliated photoreceptor cells (ph.c) and the pigment cells (pi.c). B. Galliot et al. / Developmental Biology 332 (2009) 2–24 5 conduction (Leys et al., 1999), do not display any cell types exhibiting immobilizes the prey by releasing large droplets of venom through an synaptic conduction and usually feed by passive filtration. everting tubule (Tardent, 1995). The prey then releases the peptide glutathione, which induces the feeding response, i.e. tentacle bending Anatomy of the cnidarian nervous systems and mouth opening (Loomis, 1955; Lenhoff et al., 1982; Shimizu, 2002). The cnidarian neurons form nerve nets and nerve rings Although electrical activity could be recorded in nematocytes (Anderson and McKay, 1987; Brinkmann et al., 1996), it is not clear In textbooks the organization of the cnidarian nervous system is how the information sensed by the cnidocil apparatus is transduced to described as a “diffuse nerve net” homogenously distributed along the target the discharge function. In fact, nematocyst discharge can occur polyps, which can be visualized by neuron-specific immunostaining. in the absence of neuronal control indicating that nematocytes can However in adults, this nerve net is certainly not homogenous as behave as autonomous mechanoreceptor–effector units (Aerne et al., the distribution of neurons is not uniform, neither at the qualitative 1991). However ultrastructural studies showed the presence of two- nor at the quantitative levels. For example, in Hydra, distinct subsets cell as well as three-cell synaptic pathways in the tentacle epidermis of neurons with specific spatial distribution could be identified of a sea anemone, including synaptic connections between nemato- (Grimmelikhuijzen et al., 1989; Koizumi et al., 1990) and the nerve cytes and surrounding neurons (Holtmann and Thurm, 2001; Westfall cell density is at least six fold higher in the head region than in the et al., 2002). This neuronal control is supposed to pace down the body column (Fig. 2). Similarly in medusa the RFamide neurons are spontaneous firing activity of nematocytes. clearly more abundant in the manubrium and the tentacle bulbs (Grimmelikhuijzen and Spencer, 1984)(Fig. 5). In addition to the Neurogenesis and nematogenesis in cnidarians nerve net, a dense anatomical structure, named the nerve ring was identified at the base of tentacles in some Hydra species (Koizumi Neurogenesis and nematogenesis in the planula (swimming larva) et al., 1992), following the bell margin in jellyfish (Mackie, 2004), around the oral opening in Nematostella (Marlow et al., 2009). Nerve In developing hydrozoans, scyphozoans and anthozoans, nemato- rings are considered as annular forms of central nervous system, genesis and neurogenesis are initiated in late gastrula, as soon as the involved in the coordination of behaviors (Grimmelikhuijzen and ectodermal and endodermal cell layers are established (Fig. 2A). In Westfall, 1995; Mackie, 2004; Garm et al., 2007; Koizumi, 2007). hydrozoans and anthozoans, cells located in the endoderm, named Although the sensory systems and the behavioral repertoire are interstitial stem cells in hydrozoans, give rise to nematoblasts and more elaborate in medusae than in polyps, the analysis of the neuroblasts, which migrate towards the ectodermal layer (Fig. 3). In differentiation of the nervous system is so far the most achieved in the Podocoryne larva, the nematocytes appear in the endoderm at 24 h Hydra polyp (Koizumi, 2002). Nonetheless the current emergence of post-fertilization (hpf), homogenously distributed before migrating to new experimental cnidarian model systems (e.g. Acropora, Nematos- the ectodermal layer, while a subset of larger nematocytes accumu- tella, Clytia, Cladonema, Hydractinia, Cassiopea, Aurelia, Tripedalia,) lates at the posterior end, the future oral pole (Groger and Schmid, should soon complete the picture. In Hydra, neurons, which represent 2001). At 24 hpf the first RFamide sensory neurons are detected in the about 3% of the total cell number (David, 1973) are either sensory cells mid-body region with neurites oriented along the anterior posterior or ganglion neurons. Cell bodies of most sensory neurons are located axis. Few hours later, tyrosin–tubulin nerve cells are detected in the within the ectodermal layer, their processes reaching the surface anterior region, with lateral neurites forming rings. Progressively (Fig. 4F), whereas the bipolar and multipolar ganglion neurons novel tyrosin–tubulin neurons with lateral neurites differentiate (Fig. 4G–I), which are the most common type of neuronal cells, are towards the posterior pole forming repetitive units along the anterior spread in both cell layers, along the mesoglea and function as posterior axis, while anterior and posterior connections also appear. interneurons. In jellyfish sensory neurons can actually function as This anterior to posterior development of the nervous system with sensory-motoneurons, establishing bidirectional synapses with their repetitive units is highly reminiscent of the formation of the central target cells, namely myoepithelial cells and nematocytes (Anderson, nervous system in bilaterians (Groger and Schmid, 2001). 1985; Garm et al., 2006). In sea anemones, sensory neurons are In the scyphozoan Aurelia planula where all neurons are associated with smooth muscle fibers, suggesting that they also ectodermal, the RFamide neurons differentiate first in the vicinity of behave as sensory-motoneurons (Grimmelikhuijzen et al., 1989). the aboral pole and progressively form a dense graded plexus along Synaptic transmission in cnidarians relies on fast neurotransmit- the aboral half (Nakanishi et al., 2008). Similarly the Acropora planula ters. (glutamate, GABA, glycine) as well as slow ones (catechola- develops asymmetrically, with the sensory nerve cells expressing mines, serotonine) and neuropeptides (see in Table 1). For a recent RFamide, Pax-C or Emx that appear denser at the aboral pole but rare update about neurotransmission in cnidarian nervous systems, see or absent from the oral pole (de Jong et al., 2006; Miller et al., 2000), (Kass-Simon and Pierobon, 2007). and the ganglion and sensory neurons expressing cnox-2Am (Gsx ortholog) restricted to the ectoderm of the mid-body region (Hayward The nematocytes (or cnidocytes) are phylum-specific et al., 2001). Therefore the diffuse larval nerve net is already highly mechanoreceptor cells regionalized. In addition, some anthozoan planula as Nematostella develop at the anterior/aboral pole a transient sensory organ, named Besides neurons, cnidarians differentiate highly specialized me- the apical tuft, which senses the signals that will induce settlement of chanoreceptor cells that play a key role in the capture of preys and the larva and its subsequent metamorphosis. The high density of defense — see in Bouillon, (1994a) and Tardent, (1995). These sensory neurons at the aboral pole of the hydrozoan and scyphozoan phylum-specific stinging cells, named nematocytes (or cnidocytes, planulae are supposed to play a role similar to the apical tuft. At the giving their name to the phylum), are abundant, representing 35% of time of metamorphosis, part of the larval nervous system degenerates the cells in Hydra (David, 1973). They display variable morphologies as observed in the hydrozoan and scyphozoan larvae where the aboral and functions; in anthozoans, spirocytes (Fig. 3) are mechanosensory RFamide neurons disappear to reappear at the oral pole of the polyp. cells involved in adhesion to prey and non-prey (Kass-Simon and Thus a complex reorganization of the nervous system is linked to the Scappaticci, 2002). Mature nematocytes are stimulated by chemicals metamorphosis process, with complex migration patterns (Kroiher or preys that contact their cnidocil, they then respond in nanoseconds et al., 1990; Martin, 2000; Nakanishi et al., 2008). A similar process by discharging the toxic content of a thick-wall capsule named nema- also probably occurs in metamorphosing anthozoans (de Jong et al., tocyst (Fig. 4)(Nuchter et al., 2006). The nematocyst discharge 2006). 6 B. Galliot et al. / Developmental Biology 332 (2009) 2–24
Table 1 Putative regulators of cnidarian neurogenesis identified in analyses performed at the cellular expression level and/or functional level.
Signaling Anthozoans Medusozoans References FGF (FGFa1, Nv-pl: apical tuft (lof) ? Rentzsch et al. (2008) FGFRa, FGFa2) Wnt3 Nv: 11 Wnt families, neurogenesis ? Hv, Hm (Wnt3): apical organizer, terminal Kusserow et al. (2005) Hobmayer et al. (2000); differentiation of nematoblast Hs: Guder et al. (2006a); Khalturin et al. (2007) i-cells differentiation, apical organizer Muller et al. (2007); Teo et al. (2006) Dkk1/2/4 ? Hm: expressed in body column, gland cells, Guder et al. (2006b) putative neurogenic Dkk3 ? Hm: differentiating nematocytes Ch-med: Fedders et al. (2004) Denker et al. (2008b) TeBu differentiating nematocytes BMP2/4 Am larva: oral region Nv-ga: Hm: neurogenesis ?? Pc larva: Hayward et al. (2002); Finnerty et al. (2004); asymmetric expression, Nv-pl: apical tuft aboral, neurogenesis ? Reber-Muller et al. (2006); BMP5/8 Nv-pl: pan-endodermal, apical tuft Hv: (2x) endoderm peduncle Matus et al. (2006) Reinhardt et al. (2004) and tentacle zone, neurogenesis? Chordin Nv-ga: blastopore, asymmetric Hv: endoderm, up-regulated Matus et al. (2006); Rentzsch et al. (2006) ectodermal oral–aboral expression during organizer formation Rentzsch et al. (2007) Follistatin Nv-pl: circumoral, neurogenesis ? ? Matus et al. (2006) Gremlin, GDF5 Nv-ga: asymmetric endo. oral–aboral, ? Matus et al. (2006); Rentzsch et al. (2006) Nv-pl: apical tuft Noggin Nv-pl (Noggin1): endo. ? Matus et al. (2006) facing the apical tuft; pharyngeal asymmetrical Jun kinase ? Hv: differentiating nematocytes Philipp et al. (2005) RSK 2 ? Hv: i-cells, neurons, nematoblasts, Kaloulis et al. (2004); Chera et al. (2006); epithelial cells apical organizer (lof) Chera et al. (2007) Notch Nv-pl: similar to Musashi Hm: nematocyte differentiation Marlow et al. (2009), Kasbauer et al. (2007); Khalturin et al. (2007) Hedgehog Nv-pl (Int1, Int2, Int3) neural precursors ? Matus et al. (2008) Opsins ? Cr (20x): eyes, gonads Suga et al. (2008)
Peptides GLWamides (NP) (I–VIII) Af: Hym-54, Hm: Hym-53, -54, -248, -249, Grimmelikhuijzen et al. (2002); Hym-370→muscle contraction -331, -338, -370 peptides in sensory Hansen et al. (2002); Takahashi et al. (2003), neurons→- muscle contraction He-pl: Leitz et al. (1994); sensory neurons stimulating migration Gajewski et al. (1996); Plickert et al. (2003); and metamorphosis (lof) Katsukura et al. (2003); Katsukura et al. (2004) KAamide (NP) Ae: inhibits muscle contraction ? Grimmelikhuijzen et al. (2002) KVamides (NP) ? Hm: Hym-176→ muscle contraction Yum et al. (1998); Hansen et al. (2002) PWamide (EP) ? Hm: Hym-33H inhibits neural differentiation Takahashi et al. (1997); Takahashi et al. (2009); Lentz (1965) RFamides (NP) (I, II, III) Ae: endo. neurons Ps: (A, B, C) Hm, Hv: ecto. Sensory neurons He-pl: Grimmelikhuijzen et al. (2002); slow muscle contraction Rk: ecto. sensory neurons inhibiting Hansen et al. (2002); Pernet et al. (2004), and endo. sensory neurons, migration and metamorphosis Katsukura et al. (2003); pharyngeal nerve net Katsukura et al. (2004) RGamides (NP) ? Hm: Hym-355, positively regulates Takahashi et al. (2000); Hansen et al. (2002) neural differentiation RIamide (NP) (I,II) Ae: inhibit muscle contraction ? Grimmelikhuijzen et al. (2002) RNamides (NP) (I, II) Ae: antagonistic action on ? Grimmelikhuijzen et al. (2002) longitudinal and circular muscles RPamides (NP) (I–V) Ae: tentacle contractions Grimmelikhuijzen et al. (2002) RWamides (NP) (I, II) Ab, Ps: slow contraction of endo. ? Grimmelikhuijzen et al. (2002) muscles; Cp: sphincter contraction
ANTP-class HPs Emx Am: aboral larval sensory neurons Hs: neurogenic ? Hydra: ? de Jong et al. (2006) Mokady et al. (1998) Not ? Hv: tentacle root sensory neurons This work Msx Am: oral larval ectoderm (neurons ?) Hv: body column neurons Pc: de Jong et al. (2006), Miljkovic-Licina et al. (2004), progenitor maintenance Galle et al. (2005) Gsx/Anthox2/ Am-pl (cnox-2): ecto. bipolar and Hv: apical neurogenesis (lof), Hayward et al. (2001); de Jong et al. (2006), cnox-2 (ParaHox) multipolar neurons except aboral nematogenesis along body Pc-pl: endo., Finnerty et al. (2003), Nv-pl (Anthox2): ecto. neural precursors, aboral; Pc-po:+pattern ? Ch-pl: endo. Miljkovic-Licina et al. (2007), oral region Nv-po (Anthox2): ecto. progenitors ? Ch-med: ecto. Yanze et al. (2001), Chiori et al. (2009); neural precursors in body column, TeBus progenitors, neurons Ed-pl, Quiquand et al. (2009) pharynx, tentacles Ed-po: ecto, oral Ed-med: endo, Jakob and Schierwater (2007) early buds (lof) Hs: ecto. Cartwright et al. (2006) body column (antibody) Pdx/Xlox (ParaHox) Nv-pl, Nv-po (Xlox/Cdx): endo. Ch-pl: endo. progenitors ? Ryan et al. (2007) Quiquand et al. ventral midline stripes (2009), this work Cdx (ParaHox) ? Ch-pl: ecto. oral/aboral Ch-med: TeBu. Chiori et al. (2009) nematogenesis Ed-po: ecto, aboral (Jakob and Schierwater (2007) PG-1 (Hox) Nv-pl (Ax6): endo. pharyngeal ring (Ax6a): Ch-med (Hox1): mechanosensory Finnerty et al. (2004); Ryan et al. (2007) body wall Nv-po (Ax6): oral, cells in statocysts Ed-pl (Cnox5): ecto., Chiori et al. (2009) Kamm et al. (2006); mouth opening; endo. base and aboral Ed-po (Cnox5): ecto/endo, Jakob and Schierwater (2007) tips of tentacles oral/aboral (lof) Pc-pl (Cnox1): Yanze et al. (2001) Gauchat et al. (2000) aboral ecto/endo Hv (cnox-1): endo/ecto.hypostome B. Galliot et al. / Developmental Biology 332 (2009) 2–24 7
Table 1 (continued) Signaling Anthozoans Medusozoans References PG-2 (Hox) Nv-pl (Ax7): body wall endoderm No ortholog ? Finnerty et al. (2004); Matus et al. (2006); Nv-po (Ax7): pair of mesenteries Ryan et al. (2007) Nv-pl (Ax8a): pharyngeal endo. Nv-pl (Ax8b): ventral midline Nv-po (Ax8a-Ax8b): ventral pair mesente0ries, endo. tentacle base PG-9 like (Hox) Nv-pl (Ax1): ecto. apical tuft Ch-pl (Hox9–14A,-B): oral pole Ch-med (Hox9–14A): Finnerty et al. (2004); Ryan et al. (2007) Nv-pl (Ax1a): endo. asymmetric body ecto. TeBu, ecto. manubrium Ch-pl (Hox9–14C): Chiori et al. (2009) Kamm et al. (2006); wall Nv-po (Ax1a): ventral ecto. aboral Ed-med (Cnox-1): ecto. Jakob and Schierwater (2007) mesenteries, endo. tentacle base oral ring (lof) Ed-med (Cnox-3): ecto. Gauchat et al. (2000) oral ring (lof) Hv (Cnox-3): ecto. hypostome Orphan Hox-like No ortholog ? Pc-pl (cnox2): ecto/endo aboral Pc-po (cnox2): Masuda-Nakagawa et al. (2000) apical tip Pc-med (cnox2): endo. gastrovascular
PRD-class HPs prdl-a ? Hv: apical neurons and precursors, Gauchat et al. (1998); organizer during head formation Miljkovic-Licina et al. (2004) prdl-b ? Hv: body neural cells, proliferating nematoblasts Gauchat et al. (2004); Miljkovic-Licina et al. (2004) Homeobrain Nv-pl: oral neural-like cells; Nv-po: ? Marlow et al. (2009) restricted to tentacles Gsc Nv-pl: endo. pharyngeal, apical tuft, Hm: apical sensory neurons Pang et al. (2004); Matus et al. (2006) asymmetric directive axis Broun et al. (1999) Otp Nv-po: ecto. oral nerve ring ? Marlow et al. (2009) Rx Nv: neural subsets ? Matus et al. (2007a) Repo Nv: nerve ring ? Marlow et al. (2009) Otx Nv (3x): nerve ring ? Am (2x) nerve ring ? Hm, Pc: cell migration (budding) but de Jong et al. (2006); Mazza et al. (2007), not detected in nervous system Muller et al. (1999); Smith et al. (1999) Pax-A/C (pox neuro) Nv: putative neural, spirocyte ? Matus et al. (2007a) Miller et al. (2000; precursors and neural cell types Am: Plaza et al. (2003) neural cell types Pax-B (Pax2/5/8) Nv: patterning of the nerve ring ? Tc-med: rhopalia Matus et al. (2007a), Kozmik et al. (2003), Pc-pl, -med: neurogenesis Groger et al. (2000) Pax-D (Pax3/7) Am: stripes around the embryo; No ortholog de Jong et al. (2006); Matus et al. (2007a) neurogenesis ? Nv-pl (PaxD1): stripe, i-cells ? Nv-po (PaxD3): tentacle ecto.
SIN-class HPs Six1/2 ? Pc-med, Cr-med: neurogenesis Cr-med: eye Stierwald et al. (2004) Six3/6 ? Pc-med, Cr-med: neurogenesis, Stierwald et al. (2004) nematogenesis; Cr-med: eye Six4/5 ? Pc-med, Cr-med: neurogenesis Stierwald et al. (2004) b HLH TFs Achaete-scute (A type) ? Hm: late nematogenesis, sensory neurons Pc-pl: Grens et al. (1995); Hayakawa et al. (2004); neural precursors, Pc-med: Lindgens et al. (2004), Muller et al. (2003); neural and muscle precursors, nematocytes Seipel et al. (2004c) COE (collier-type) Nv-pl: apical tuft ? Pang et al. (2004)
HMG TFs SoxB NvSox2-pl: ecto. neural-like cells ? Magie et al. (2005) NvSox2-po: restricted to tentacles NvSoxB1-pl: apical tuft, pharyngeal ? Magie et al. (2005) AmSoxB1-pl: presumptive ecto. NvSoxB2-pl: ecto. aboral half AmSoxBa-pl: ? Magie et al. (2005) Shinzato et al. (2008) ecto. aboral half SoxC Am-pl: ecto. sensory neurons Nv-pl: ecto, ? Shinzato et al. (2008) sensory neurons Nv-po: developing tentacles
MADS-box TFs FoxB (Fkh3) Nv-pl: pharyngeal ecto. Nv-po: oral ring Ch-pl: nematogenesis, endo. ganglion cells; Magie et al. (2005) Chevalier et al. (2006) Ch-med: ecto. TeBu, statocysts FoxD1 Nv-pl: broad aboral domain Nv-po: base of ? Magie et al. (2005) tentacles Mef2 Nv: ecto. Precursors, nematocytes, neurons Pc-po: apex, Pc-med: manubrium Martindale et al. (2004) Spring et al. (2002) SRF ? Hv, He: undifferentiated i-cells Hoffmann and Kroiher (2001)
Zinc-containing TFs COUP-TF Am: 10x orphan, neurogenesis ? Hv: differentiating nematoblasts; Grasso et al. (2001), Gauchat et al. (2004) (nuclear receptors) body column neurons RXR (nuclear receptors) ? Tc: putative regulator of crystallin genes Kostrouch et al. (1998) Zic ? Hv: early dividing nematoblasts Lindgens et al. (2004) Gli Nv-pl, Nv-po (Gli3): endo. body wall Hv: ubiquitous expression Matus et al. (2008) (MM-L, BG, unpublished) GCM (Glial Nv-pl: oral ecto. scattered cells ? Marlow et al. (2009) cells missing) Nv-po: endo. oral nerve ring
(continued on next page) 8 B. Galliot et al. / Developmental Biology 332 (2009) 2–24
Table 1 (continued) Signaling Anthozoans Medusozoans References Various TFs C/EBP (bZIP TF) ? Pc-pl: ecto. aboral; Pc-po: endo. bud Pc-med: Seipel et al. (2004b) muscles, tentacle, TeBu, CREB (bZIP TF) ? Hv: i-cells, neurons and nematoblasts, Kaloulis et al. (2004); epithelial cells apical organizer (lof) Chera et al. (2007); (LG, SC, BG, unpublished) Mafl (bZIP TF) ? Pc-pl: endo. aboral; Pc-po: endo. Seipel et al. (2004b) bud Pc-med: muscles, tentacle, TeBu, CBFβ (Runx TF) Nv-po: ecto. tentacle, nematocytes ? Sullivan et al. (2008) Runx (Runx TF) Nv-po: ecto. tentacle, nematocytes ? Sullivan et al. (2008) Smad ? Hv, ubiquitously expressed, stronger in i-cells, Hobmayer et al. (2001) nematocyte differentiation
NEURAL stem cell markers ELAV1 (RNA-binding Nv-pl: scattered ecto. neural precursors ? Marlow et al. (2009) protein) Nv-po: ecto/endo, body wall, tentacles Musashi (Msi) Nv-pl: ecto. oral, tentacle bud ? Marlow et al. (2009) (RNA-binding protein) Nv-po: ecto, pharyngeal, tentacles
Chromatin regulators Polycomb related ? Hy-AEP, Hm: HyEED i-cells, nematoblasts (gof) Genikhovich et al. (2006); Khalturin et al. (2007) CBP/p300 ? Hv: i-cells, neurons and nematoblasts, (LG, SC, BG, unpublished) epithelial cells apical organizer (lof)
Abbreviations: Ax: anthox (anthozoan Hox/ParaHox gene); Cx: cnox (Hox/ParaHox gene); ecto.: ectodermal, endo.: endodermal; i-cells: interstitial cells; EP: epitheliopeptide; ga: gastrula; gof: gain of function assay; i-cells: interstitial cells; lof: loss of function assay; med: medusa; NP: neuropeptide; pl: planula; po: polyp; TeBu: tentacle bulb; x: copy number for a given gene family. Species code: Ab: Anthopleura ballii; Ae: A. elegantissima; Af: A. fuscoviridis; Am: Acropora millipora; Ch: Clytia hemispherica; Ch-pl: Ch planula; Ch-med: Ch medusa; Cp: Calliactis parasitica; Cr: Cladonema radiata; Cr-med: Cr medusa; Hy-AEP: Hydra sexual strain; He: Hydractinia equinata; He-pl: He planula; Hm: Hydra magnipapillata; Hs: Hydractinia symbiolongicarpus; Hv: Hydra vulgaris; Nv: Nematostella vectensis; Nv-ga: Nv gastrula; Nv-pl: Nv planula; Nv-po: Nv polyp; Pc: Podocoryne carnea; Pc-po: Pc polyp; Pc-med: Pc medusa; Ps: Protanthea simplex; Rk: Renilla koellikeri; Tc: Tripedalia cystophora.
Neurogenesis and nematogenesis in the adult polyp differentiate as a sensory or ganglion neuron (Schaller et al., 1989; Bode, 1996). Neuronal differentiation is more intense in the upper The cnidarian polyps display an oral–aboral polarity, with body column and peduncle region than in the central body column differentiated tissues at the extremities but no sensory organs as and mature neurons receive signals from the head and foot regions to recognized in medusae. In Hydra three distinct stem cells populations migrate, explaining the higher neuronal densities recorded at the provide all cell types, the ectodermal epithelial cells, the endodermal extremities. One striking finding was the high level of neuronal epithelial cells and the interstitial cells, which are multipotent stem plasticity observed in adult Hydra polyps (Bode, 1992) with changes in cells restricted to the ectoderm of the body column, continuously neuropeptide phenotype according to the position of the neurons providing neurons, mechanoreceptor cells (nematocytes), gland cells along the body column (Koizumi and Bode, 1986), but also trans- and gametes when the animals follow the sexual cycle (Bode, 1996; differentiation from ganglion to sensory neurons (Koizumi et al., Bosch, 2009). The epithelial stem cells divide every three to four days 1988). This plasticity was also observed in the nematocyte lineage when the interstitial stem cells divide faster, once a day. Surprisingly (Fujisawa et al., 1986). enough, interstitial stem cells seem to be lacking in non-hydrozoan Besides the highly dynamic adult homeostatic context, the species, where it was proposed that neurons differentiate directly regulation of neurogenesis can also be investigated in developmental from epithelial cells. However cell lineage tracing analyses are contexts in Hydra as regeneration of the head and foot regions after required in non-hydrozoan model systems to clarify this question. bisection, asexual reproduction through budding when animals are In Hydra, the nematocyte and neuronal differentiation pathways well fed, reaggregation after tissue dissociation. After bisection, appear to share a common bipotent progenitor (Holstein and David, nematocytes and neurons disappear from the head-regenerating tip 1990; Miljkovic-Licina et al., 2007) before following distinct regula- and a wave of de novo neurogenesis occurs in the presumptive head tions: interstitial cells committed to the nematocyte lineage that are region on the second day (Figs. 2B, 4J–N), preceding the emergence of located in the ectodermal layer of the body column, undergo up to five the regenerated head (Lentz,1965; Yaross and Bode,1978a; Venugopal synchronous cell cycle divisions, forming clusters of syncitial nema- and David, 1981; Koizumi et al., 1990; Miljkovic-Licina et al., 2007). toblasts (Fig. 4). Once they stop proliferating, the nematoblasts start differentiating their nematocyst vacuole, which can be of four distinct The nerve-free Hydra paradigm types (Holstein and Emschermann, 1995). Differentiated nematocytes In Hydra, neurogenesis can be disconnected from patterning by then migrate to their definitive location, namely the tentacles, accor- producing « nerve-free » polyps, which lack the interstitial lineage ding to a process that relies on contact guidance from surrounding derivatives, namely nematocytes, sensory and ganglion neurons, and tentacles (Campbell and Marcum, 1980). In the tentacles, nematocytes are thus named “nerve-free” or “epithelial” hydra. Such animals can be are embedded within large epithelial cells named battery cells, each obtained by different means: either chemically, upon colcemid, battery cell containing several nematocytes, themselves connected to colchicine (Campbell, 1976) and hydroxyurea treatments (Yaross sensory neurons by synapses. After discharge of their capsule, and Bode, 1978b), or genetically as in the nf-1 Hydra magnipapillata nematocytes are eliminated and replaced by new ones. mutant that completely lacks the interstitial lineage (Sugiyama and In contrast, the differentiation of nerve cells appears more direct: Fujisawa, 1978), or in the temperature sensitive sf-1 mutant (Terada interstitial cells committed to this pathway are found predominantly et al., 1988). It is also possible to maintain “pseudo-epithelial” hydra, along the body column, possibly in the head region but neither in the which are depleted of all somatic interstitial lineage derivatives, but tentacles nor in the foot region. These progenitors go through S phase, still contain stem cells restricted to the germ cell lineages (Nishimiya- get arrested in G2 until a signal will let them divide and terminally Fujisawa and Sugiyama, 1995). As anticipated nerve-free animals B. Galliot et al. / Developmental Biology 332 (2009) 2–24 9
Fig. 3. Nematogenesis and neurogenesis in the developing Nematostella (A–C) Spirocytes (green) detected in Nematostella late planula thanks to their peroxidase activity. (D–I) The RFamide sensory nerve net in juvenile (D, 31 days old) and developing Nematostella. Early neurons expressing the neuropeptide (arrowheads) appear in the endodermal layer (E), then migrate to the ectoderm (F, arrowheads) and form a net (G) in the mesoglea. In the newly metamorphosed polyp (H, here contracted), the RFamide nerve net is denser in the oral region (arrow) as observed in the fully metamorphosized polyp (I). Scale bars: 2 mm (C), 10 μm (B), 50 μm (A, E–H), 600 μm (I). completely lose their autonomous feeding behavior and can only and Campbell, 1978a) although head regeneration in such hydra is be maintained by force-feeding (manual introduction of the food significantly slower and less efficient (Miljkovic-Licina et al., 2007). through the mouth opening with a pipette and subsequently washing The manipulation of such animals turned out to be very of the gastric cavity). Nevertheless, epithelial hydra exhibit develop- informative, showing that the differentiation of interstitial cells into mental patterning processes, like budding and regeneration (Marcum nematocytes was not position-dependent, whereas that of nerve cells
Fig. 4. Nematogenesis and neurogenesis in theHydra polyp. (A–E) In Hydra the interstitial stem cells (A) provide precursors (named nematoblasts, B) for the mature nematocytes. Those are characterized by a typical capsule, the nematocyst that discharges its content upon stimulation (arrowheads, C, D). (E) Bright-field view of a tentacle with nematocytes either undischarged (arrow) or discharged (arrowhead) embedded in large epithelial battery cells. (F–I) In Hydra the neurons detected here after tissue maceration can be sensory (F), bipolar (G) or multipolar (H, I) also named ganglion cells. In the ectoderm the sensory neurons are regularly distributed among epithelial and interstitial cells(F, arrowheads). The anti β-tubulin (I) more easily detects the ganglion than the sensory cells. (J–N) De novo neurogenesis in head-regenerating Hydra. Following mid-gastric section, the tip of the head-regenerating half is immediately depleted of neurons (J, outline), progressively repopulated with neuronal progenitors (K) and mature neurons (L, M). However the apicalnervous system at 40 h post- amputation (hpa) is still less dense than in adult polyps (N, topview). mo: mouth opening; te: tentacles). Scale bars: 2μm (A, B, D), 5 μm (C, F–I), 50 μm (J–N). 10 B. Galliot et al. / Developmental Biology 332 (2009) 2–24 was indeed position-dependent, i.e. enhanced in the upper and lower Neurogenesis and nematogenesis in the adult medusa parts of the body column (Yaross and Bode, 1978b). This position- dependent regulation of neurogenesis seems to be largely under the The manubrium and the tentacle bulbs control of epithelial cells (Koizumi et al., 1990; Minobe et al., 1995). In the mature medusa, the manubrium and the tentacle bulbs are Together with experiments performed on chimeras formed between the sites of intense production of neurons and nematocytes as mophologically-distinct strains (Marcum and Campbell, 1978b), these observed in the hydrozoan jellyfish (Figs. 2C and 5). In contrast to data suggested that the interstitial lineage, and more specifically the Hydra polyps where nematogenesis and neurogenesis overlap along neurons do not play a significant role in hydra morphogenesis the body column, the expression analysis of neuronal and nematocyte (Fujisawa, 2003). However the situation is probably more complex markers coupled to in vivo cell labeling and morphological analyses as in the absence of nerve cells, the genetic circuitry is likely repro- revealed that the differentiation stages follow a proximo-distal grammed in the epithelial cells as already reported (Hornberger and gradient along the tentacle bulbs (Denker et al., 2008b) as depicted Hassel, 1997). Moreover interstitial cells and their derivatives appear in Fig. 2Cc. Moreover the tentacle bulb isolated from the medusa has involved in the fine tuning of the morphogenetic processes driven by the capacity to survive for several days in culture, opening the the epithelial cells, as for instance in the reg-16 mutant where head possibility for manipulations and functional studies. regeneration that is strongly deficient, can be reestablished upon depletion of the interstitial lineage (Sugiyama and Wanek, 1993). The medusa-specific sensory organs: the ocellus, the camera eye and Similarly the dramatic apoptosis of the neuronal and nematocyte the rhopalia lineages in head-regenerating tips immediately after mid-gastric Light sensing is widely spread in non-metazoan species but the amputation leads to the activation of the head regeneration program clustering of photoreceptor cells to form sensory organs was a major (Chera et al., 2009). innovation in animal evolution, an innovation that took place in the
Fig. 5. Neurogenesis in hydrozoan and scyphozoan medusae. (A–C) Bottom view of the Clytia medusa nervous system with the manubrium (m) and the tentacle bulbs (tb) containing numerous RFamide sensory neurons (purple-pink). Note the delicate nerve net in the velum (B) and the endogeneous bioluminescence in the tentacle bulbs (green, A, C). (D) The proliferative (arrow) and differentiating (arrowheads) zones of the tentacle bulbs express the ParaHox gene Gsx (red; blue: DAPI staining). (E) In the Podocoryne medusa, the differentiating neurons in the tentacle bulbs strongly express the CREB transcription factor detected with the anti-hydra CREB antibody (red). Scale bars: 500 μm (A), 50 μm (B, C) 10 μm (D, E). (F–I) Rhopalia in the immature Aurelia medusa (ephyra). Most of the adult features can be already observed: the mouth (m), the developing stomach (s), three types of radial canals: adradial, perradial and interradial (ic) and the rhopalia (r; arrows), each of them guarded by a pair of lappets that contain a diffuse nerve net (F, G). At the base of the rhopalium, a stratified epithelium includes columnar ciliated cells with basal axons and cells with intra-epithelial flagella stained with α-tubulin (G, H, arrowhead). In the center of the rhopalium the elongated ovoidal lithostyle (I) contains the photoreception organs named ocelli that contain pigmented cells, and a terminal statocyst (st) that senses gravitation. B. Galliot et al. / Developmental Biology 332 (2009) 2–24 11
Cnidaria–Bilateria ancestor (Gehring, 2004). In cnidarians, both the the aboral pole of medusozoan planulae remains to be shown. The medusa and the polyp can sense light in non-visual photosensitive canonical Wnt pathway appears to regulate the self-renewal of structures (Santillo et al., 2006) but only the jellyfish can differentiate stem cells in Hydractinia polyp, where its pharmacological activation photoreception organs (Martin, 2002). These can be either simple leads to a burst of neurons and nematocytes together with a ocelli as in Aurelia that are composed of photosensitive cells inter- decrease in the pool of interstitial cells (Teo et al., 2006). In contrast mingled with pigment cells, or more complex as camera eyes with a in the adult Hydra polyp, the Wnt3 ligand is expressed at highest lens as observed in Cladonema (Fig. 2Cd) and Tripedalia. The most levels in the most apical region where neurons are already complex eyes with a cornea, lens and ciliated photoreceptor cells for- differentiated, whereas its inactivation by Dkk1/2/4 in the body ming retina are found in cubomedusae. In scyphozoan and cubozoan column might promote neurogenesis (Guder et al., 2006b). In the medusae, eyes associate with pressure sensing organs named stato- Hydra polyp the Dickkopf-3 related gene (HyDkk3) is expressed in cysts to form complex sensory organs named rhopalia (Fig. 5F–I), nematocytes at late stages of differentiation, suggesting some role in connected to the nerve ring (Garm et al., 2006). Therefore rhopalia the maturation or the maintenance of nematocytes (Fedders et al., were proposed to be part of the central nervous system. Behaviors can 2004); similarly in the tentacle bulb of the Clytia medusa Dickkopf-3 in fact be regulated through visual input, as the observed modulations is expressed in the differentiation zone of nematogenesis (Denker et of the swim pacemaker according to the light intensity in Tripedalia al., 2008b). (Garm and Bielecki, 2008). Non-visual photosensitive structures also The TGFβ/BMP pathway is a putative regulator of neurogenesis in regulate animal behaviors (see in Santillo et al. (2006)) as the Hydra where a BMP5–8 ortholog is expressed asymmetrically along pacemakers that regulate the periodic contractions of the Hydra body the body axis, predominantly in the basal half (Reinhardt et al., 2004) (Passano and McCullough, 1962, 1963), the locomotion of eyeless and the chordin antagonist is highly expressed in neurogenic regions pelagic species (Plickert and Schneider, 2004) or the triggering of as the growing bud (Rentzsch et al., 2007). However, in the Podo- spawning supported by the expression of opsins in gonads (Suga et al., coryne larva where the expression of BMP2/4 coincides with the 2008). activation of the Atonal-like gene Atl1 (Reber-Muller et al., 2006) and Besides adult eyes, pigmented photoreceptor cells were identified in anthozoans the regulatory role of the TGFβ/BMP pathway on in the Tripedalia larva, which does not contain any nervous system neurogenesis remains unknown (Hayward et al., 2002; Finnerty et al., (Nordstrom et al., 2003). These single cell ocelli appear quite original 2004). The Notch pathway supports the differentiation of nemato- since they most probably function completely autonomously, sensing cytes in Hydra (Kasbauer et al., 2007; Khalturin et al., 2007) but data the light through their photoreceptors and regulating the animal concerning the anthozoan Notch pathway are not published yet. behavior thanks to the motor-cilium they differentiate. Moreover Finally the complete Hedgehog pathway was characterized in Ne- these photoreceptors are rhabdomeric (microvilli) as observed in matostella where the two Hh ligands are expressed in the pharyngeal most invertebrates and not ciliated as in adult cnidarian eyes and ectoderm and along the endodermal body wall respectively, and vertebrates. It would be of interest to identify other cases of cnidarian the patched and Gli3 genes are restricted to the endoderm in planula larval eyes. In some species as Cladonema, adult eyes can fully and polyps. This pattern does not support a neurogenic role for the regenerate (Stierwald et al., 2004). Given the great variety of eye Hh pathway (Matus et al., 2008). However three intein-containing morphology, the question of a unique origin for all animal eyes or a genes are expressed in ectodermal neural precursors and ganglion repeatedly convergent evolution is a long-standing one (Arendt, 2003; neurons during development. In Hydra, several components of Nilsson, 2004), still debated after the discovery of shared regulators of the Hedgehog pathway are expressed (MM-L, BG, unpublished), eye differentiation as the Pax and Six genes (Kozmik et al., 2003; but the gene encoding the Hedgehog ligand is apparently missing Stierwald et al., 2004) and shared effectors as opsins (Suga et al., from the genome. Therefore the Hedgehog pathway should achieve 2008; Kozmik et al., 2008). neurogenic tasks in cnidarians in a less conventional fashion than in bilaterians. Elements of the cnidarian neurogenic circuitry The transcription factors in cnidarian neurogenesis Growth factor signaling pathways in cnidarian neurogenesis In bilaterians, homeoproteins in combination with the bHLH The deep conservation of the signaling machinery that supports proteins bring a major contribution to neurogenesis during develop- developmental processes in bilaterians came as a surprise in cnida- ment and adulthood (Guillemot, 2007). According to the sequence of rians, when components of the insulin-like (Steele, 2002), Wnts their homeodomain, homeogenes fall into classes, which do have (Hobmayer et al., 2000; Wikramanayake et al., 2003; Kusserow et al., cnidarian representatives (Galliot et al., 1999; Holland and Takahashi, 2005; Teo et al., 2006; Momose and Houliston, 2007), Notch 2005; Ryan et al., 2006). Genes from the ANTP, PRD, SIN, POU and LIM (Kasbauer et al., 2007), VEGF (Seipel et al., 2004a), FGF (Matus et classes perform neurogenic tasks in bilaterians, but in cnidarians, al., 2007b; Sudhop et al., 2004; Rentzsch et al., 2008) and Hedgehog expression and in few cases functional data are only available for the (Matus et al., 2008) pathways were uncovered (Fig. 6). Not only the ANTP, PRD and SIN gene families. We will review here what is ligands, receptors and intra-cellular components were identified but currently known about the neurogenic function of those gene families also the antagonists as the Dickkopf3 and Dickkopf1/2/4 Wnt- in Cnidaria. antagonists (Fedders et al., 2004; Guder et al., 2006b) and the Gremlin, Noggin and Follistatin BMP-antagonists (Matus et al., 2006; The neurogenic function of the non-HOX (NK-like) ANTP-class Rentzsch et al., 2006). This amazing conservation was actually homeogenes confirmed by the even more surprising presence of these pathways The ANTP-class of homeogenes contains numerous gene fami- in sponges (Nichols et al., 2006; Adamska et al., 2007) and partially in lies that distribute into two sub-classes: the non-Hox (also named choanoflagellates (King et al., 2008). NK-type) and the Hox/paraHox families (Gauchat et al., 2000; However the experimental evidences concerning the contribu- Holland, 2001). The non-Hox families are highly conserved from tion of these pathways to neurogenesis in cnidarians are still cnidarians to bilaterians and thus form well defined sister groups limited, although four of them are likely involved in neurogenesis (Gauchat et al., 2000; Schierwater and Desalle, 2001; Chourrout et al., (Table 1). The FGF pathway supports the differentiation of the 2006; Kamm et al., 2006; Quiquand et al., 2009). We will consider apical sensory organ in Nematostella planula as demonstrated by here only those that are putative regulators of the cnidarian nervous loss-of-function assays (Rentzsch et al., 2008), but a similar role at systems. 12 B. Galliot et al. / Developmental Biology 332 (2009) 2–24
Fig. 6. Early diversification of the regulatory gene families involved in neurogenesis in bilaterians. The candidate regulatory genes of the cnidarian nervous system are underlined and the cell lineage is indicated when expression and/or functional data are available: neurogenesis (⁎), nematogenesis (°), eye differentiation (€). The signaling pathways and transcription factors present in poriferans and/or placozoans (Trichoplax with gene names followed by a T when not detected in Porifera) represent gene families that emerged in the last common ancestor of metazoans or even earlier. The gene families that emerged later, either in eumetazoans or in urbilaterians are written bold in the respective columns; the gene families possibly lost in cnidarians or in urbilaterians are stricken. Note that, except for the Atonal-class, the wave of duplications from which arose all modern gene families occurred in the last common ancestor of cnidarians and bilaterians. For references see the text. The cnidarian apical pole surrounding the mouth opening can be considered as a primitive head as the nerve net is denser in that region and forms in some species the nerve ring. In urbilaterians the neuronal cell populations diversified and a new cell type appeared: the glial cells (Reichenbach and Pannicke, 2008). These cell types became highly connected, organized in hierarchical networks, forming the central and peripheral nervous systems. The acquisition of a myelin sheath is a vertebrate innovation that allowed a faster speed for synaptic transmission in large size animals (Zalc et al., 2008).
Divergent roles for the empty spiracle/emx gene family in hydrozoans The Hydra Not ortholog, a marker for apical sensory neurons. Not and anthozoans. The emx genes are involved in forebrain formation in homeobox genes are involved in neurogenesis in bilaterians: inDroso- vertebrates with a special emphasis on the cytoarchitecture of the phila, the Not-like 90Bre gene participates in the differentiation of the cerebral cortex (Cecchi et al., 2000), whereas mutation of theDrosophila neuroblasts of the posterior brain (Dessain and McGinnis, 1993), in homologue, Ems, eliminates the deutero- and tritocerebrum (Hirth Xenopus Xnot-2 promotes notochord formation (Gont et al., 1996), in et al., 1995). In the hydrozoan Hydractinia, the Emx homologue is chicken Cnot1 and Cnot2 are expressed in the early neurectoderm expressed in endodermal epithelial cells of the hypostome (head region) (Stein et al., 1996), in zebrafish the Not-related floating head gene is and up-regulated in posterior regions of the planula larva experimen- required for neurogenesis of the epiphysis (Masai et al., 1997). In Hydra, tally converted to anterior fate (Mokady et al., 1998). However in the the cnot gene is expressed in sensory neurons at the root of the coral Acropora, Emx is expressed in sensory neurons of the aboral half of tentacles (Fig. 7A). During head formation, either budding or the larva until their density drastically decreases at the time of regeneration, cnot transcripts start to be expressed in a limited number metamorphosis (de Jong et al., 2006). Therefore Emx might belong to of neuronal cells at the place where tentacle rudiments will emerge. the ancestral neurogenic genetic circuitry but more studies, particularly Hence the Hydra cnot gene appears to be restricted to the differentiation in developing medusozoans, are needed to strengthen this conclusion. of a limited subset of neurons. B. Galliot et al. / Developmental Biology 332 (2009) 2–24 13
The msh/msx gene, a candidate regulator of neurogenesis. The msh/ ortholog named ind is expressed as a longitudinal band in the inter- msx homeogene family is highly conserved from Porifera to bilaterians mediate neuroectoderm where it promotes activation of proneural (Larroux et al., 2007). Besides the homeodomain, msx genes also genes in the specific set of neuroblasts (Weiss et al., 1998). The encode some Groucho-interacting domains that are conserved in cnox-2/Gsx gene family is currently the most widely studied in cnida- Nematostella but not in hydrozoans (Takahashi et al., 2008). In Dro- rians (Schierwater et al., 2002; Finnerty et al., 2003) and its regulation sophila, the msh gene is involved in both dorso-ventral patterning and has been documented in Hydra (Schummer et al., 1992; Gauchat et al., neurogenesis, specifying neuroblasts in the dorsal neuroectoderm. In 2000; Miljkovic-Licina et al., 2007), Hydractinia (Cartwright et al., the leech, Le-msx transcripts are present in embryonic stem cells, and 1999), Podocoryne (Yanze et al., 2001), Clytia (Chiori et al., 2009; subsequently restricted to the neural tissue. In amphioxus, msx is Quiquand et al., 2009); Acropora (Hayward et al., 2001; de Jong et al., expressed in dorsal cells of the neural tube, similarly to the msx3 2006) and Nematostella (Finnerty et al., 2003). In anthozoans, cnox-2 expression pattern observed in mice embryos. From these results, it Am expressing cells display a neuronal morphology and are restricted was proposed that the msh/msx genes specify the differentiation of to the oral pole of the larva; in the developing Nematostella planula the dorso/lateral neural tube in an evolutionarily-conserved manner (swimming larva), Gsx is expressed in the future head region. In the (Cornell and Ohlen, 2000). In the coral Acropora, msx3-Am is ex- Podocoryne and Clytia larvae, early zygotic Gsx transcripts are initially pressed in the ectoderm of the oral region but no cell-type specificity localised in the anterior endoderm before extending towards the was noted (de Jong et al., 2006). In Hydra Msx is expressed exclusively posterior pole, i.e. the future head region. in neurons along the body column (Fig. 7A), forming a nerve net in the In the Hydra adult polyp, cnox-2 is expressed in the head region central region of the polyp (Miljkovic-Licina et al., 2004). In contrast and along the body column in a subset of neuronal cells in the apical in the jellyfish Podocoryne, msx appears involved in the maintenance region (Fig. 7A) and in dividing interstitial cells and clusters of of progenitors during medusa budding and transdifferentiation (Galle nematoblasts in the body column (Fig. 7B). During head regeneration, et al., 2005). As above more studies are required to conclude about a these two types of cnox-2 expression are submitted to opposite conserved neurogenic fonction for msx in cnidarians. regulations: in head-regenerating tips induction of cnox-2 expression is observed from 24 h post-amputation (hpa), first in prolifera- The EHG gene family appears missing in cnidarians. The evolutiona- ting neuronal cells then in de novo differentiated neurons, whereas rily-conserved neurogenic function of homeogenes was first reported cnox2 expression in nematoblasts vanishes soon after amputation with the engrailed homeoprotein in arthropods, annelids and (Miljkovic-Licina et al., 2007). Hence the two cnox-2 expressing cell chordates (Patel et al., 1989). In vertebrates, two engrailed-related populations respond differently to the signals propagated during head genes (en-1 and en-2) specify the cerebellar territory (Wassef and regeneration. Interestingly, the regulation detected in the neuronal Joyner, 1997), whereas in Drosophila, engrailed exhibits a dual apical cells during head formation in Hydra correlates well with that function, during segmentation and neurogenesis, the latter one observed during larval development in Podocoryne, Clytia, Acropora being considered as ancestral (Gibert, 2002). However, a cnidarian and Nematostella. Moreover in cnox-2(RNAi) knocked-down Hydra, engrailed ortholog was not identified so far, suggesting that this the apical nerve net is not maintained in adult polyps and head gene family arose later during evolution or was lost in cnidarians, regeneration is significantly delayed (Fig. 7C), suggesting a contribu- implying thus that it was not essential at the origins of neurogenesis. tion of cnox-2 progenitors and/or neurons in the head patterning process (Miljkovic-Licina et al., 2007). In the Clytia medusa, Gsx is The ParaHox and Hox-like cnidarian genes expressed in the tentacle bulbs, proximally in clustered interstitial The Hox/ParaHox gene families, which are highly conserved cells and more distally in neurons (Fig. 5D) as reported by (Chiori among bilaterians, exhibit a much lower level of conservation from et al., 2009). The same group also reported about a Cdx ortholog cnidarians to bilaterians that the ANTP non-Hox families (Gauchat expressed in differentiating nematoblasts in the tentacle bulb. These et al., 2000; Schierwater and Desalle, 2001; Chourrout et al., 2006; data definitely support a role for the cnidarian ParaHox genes in the Kamm et al., 2006; Quiquand et al., 2009). Recent analyses showed regulation of the nervous system. that the three ParaHox families (cnox2/Anthox2/Gsx, Pdx/Xlox, Cdx/ Cnox4-Ed) exhibit a much higher level of conservation than the Hox- The PRD-class genes as regulators of nematogenesis, neurogenesis and like ones, which belong to paralogous groups (PG) in only two cases eye differentiation (Anthox6/Cnox1/PG1, Anthox7/Anthox8/PG2), whereas the others The PAIRED-class (PRD-class) gene families distribute into three display limited conserved features (Anthox1/cnox-3/PG9) or are main sub-classes: the paired-like genes, the Otx-related genes and the highly derived (Chiori et al., 2009; Quiquand et al., 2009). It was Pax genes (Galliot et al., 1999). Most PRD-class gene families carry out proposed that the absence of Hox/ParaHox genes in poriferans neurogenic functions in bilaterians. Twenty bilaterian PRD-class gene together with the higher level of conservation of the ParaHox genes families do have representatives in cnidarians (Galliot et al., 1999; would correlate with cellular innovations that took place in the last Ryan et al., 2006) whereas the sponge Amphimedon genome contains common Cnidaria Bilateria ancestor (CBA). However, the expression eight paired-like genes and a single Pax gene but no Otx-related genes of these Hox/ParaHox genes appears tightly regulated during de- (Larroux et al., 2008). In developing and adult cnidarians, most velopmental processes in hydrozoans and anthozoans, suggesting paired-like and Pax gene families exhibit regulations that suggest a that they act as developmental genes. Hox genes likely participate in specific role during neurogenesis. the development or the maintenace of the cnidarian nervous system as the Clytia Hox1 (PG1) in statocysts, the Nematostella Anthox1 The aristaless-like paired-like gene, prdl-a is expressed as a regulator of (PG9-like) in the apical tuft, or the Eleutheria Cnox-3 (PG9-like) in neurogenesis. In intact Hydra polyps, Prdl-a is predominantly ex- the oral ring (see in Table 1). However cellular and functional pressed in neuronal precursors and sensory neurons located in the analyses are required to confirm this statement. Evidences for a most apical region (Fig. 7A), being overexpressed in multiheaded neurogenic function were only obtained for the cnox2/Anthox2/Gsx mutants (Gauchat et al., 1998). However, during the early stages of paraHox gene. head regeneration and budding, prdl-a is transiently expressed in a distinct cell lineage, the endodermal myoepithelial cells located in the Gsx, a regulator of nematogenic and neurogenic precursors. Gsx genes presumptive head region. This transient wave of endodermal expres- belong to the ParaHox gene cluster (Brooke et al., 1998) and in phy- sion occurs concomittantly with the raise in organiser activity logenetic analyses group together with the Pdx/PG2/PG3 gene detected in the regenerating stump by transplantation experiments families (Quiquand et al., 2009). In Drosophila embryos, the Gsx (MacWilliams, 1983). Subsequently prdl-a is reexpressed in the 14 B. Galliot et al. / Developmental Biology 332 (2009) 2–24 differentiating neurons of the presumptive head region (Gauchat and Stern, 2001). This similarity suggested an ancient commitment of et al., 1998; Miljkovic-Licina et al., 2004). This biphasic mode of « neurogenic » paired-like genes in apical/anterior nervous system expression observed during patterning processes is highly reminis- patterning (Galliot and Miller, 2000). cent of that displayed by the vertebrate paired-like genes Hesx1/Rpx, Otx2 and Gsc during early mouse development (Thomas and Bed- The aristaless-like paired-like gene prdl-b. In contrast to prdl-a, prdl- dington, 1996; Rhinn et al., 1998): these genes that support early head b is expressed in proliferating nematoblasts and in a subset of neurons patterning in the embryo, are expressed as two successive waves, a in the gastric region but is not expressed during patterning processes first one in the anterior visceral endoderm/hypoblast that induces a (Gauchat et al., 2004; Miljkovic-Licina et al., 2004). These data suggest second one in the sus-jacent neurectoderm of the rostral region (Foley that some cnidarian paired-like genes like prdl-a already exhibit two B. Galliot et al. / Developmental Biology 332 (2009) 2–24 15 separate functions with distinct regulations, one during maintenance common trait for the Otx anthozoan genes would be their expression of apical neurogenesis in homeostatic conditions and another during at a place and at a time when the nerve ring forms. If confirmed, this patterning processes, whereas others as prdl-b would be restricted to would suggest that the Otx function in apical/anterior neuronal neuronal differentiation in homeostatic conditions. patterning emerged in the CBA.
Goosecoid (Gsc) in Hydra apical sensory neurons. The Hydra goosecoid The Pax genes as regulators of neurogenesis and eye differentiation in homolog, CnGsc, is expressed in the adult polyp in sensory neurons of cnidarians. In bilaterians the Pax gene families that likely derive the hypostome but also in endodermal epithelial cells at the base of from five urbilaterian ancestors, play a critical role in neurogenesis, tentacles and along the body column (Broun et al., 1999). During eye development as well as myogenesis, segmentation and organo- patterning processes, CnGsc is first repressed in the regenerating genesis. The identification of a single Pax gene in Porifera and of three stump or growing bud, and reexpressed at later stages in the presump- Pax families in Cnidaria proved that Pax genes were submitted to an tive head region, suggesting that it is not involved in the organizer early wave of gene duplications likely after the divergence of Porifera activity that drives head regeneration. However, when expressed in (Hoshiyama et al., 2007; Matus et al., 2007a; Larroux et al., 2008). The Xenopus embryos, CnGsc exhibits organiser activity (Broun et al., anthozoans express four Pax gene families that represent these three 1999). A single Gsc ortholog is present in Nematostella (Ryan et al., ancestral families: Pax-A and Pax-C for Pox neuro, Pax-B for Pax2/5/8, 2006) but its regulation and function are currently unknown. and Pax-D for Pax3/7 (Catmull et al., 1998; Miller et al., 2000). By contrast hydrozoans have lost the Pax3/7 ortholog and only express The Rx and repo-related genes. In Nematostella NvRx1 the ortholog of Pax-A as Pox neuro ortholog and Pax-B related to Pax2/5/8. Therefore the Retinal homeobox gene Rx that is expressed upstream of Pax6 a definitive Pax4/6 ortholog has not been found in cnidarians. during eye development in vertebrates, is expressed in scattered However these cnidarian Pax proteins bind the consensus Paired- ectodermal neuronal-like cells in planula and polyps, suggesting a role response elements with broader specificity than the mammalian ones, in the specification of a neuronal subset (Matus et al., 2007a). likely allowing more flexibility (Miller et al., 2000; Sun et al., 2001; Similarly, NvRepo the ortholog of the glial-specific paired-like gene Plaza et al., 2003). Repo is expressed at the oral nerve ring in planula and polyps (Marlow The expression data suggests that Pax genes are already involved et al., 2009). in neurogenesis in cnidarians as in Nematostella, the Pax-A/C genes (pox neuro related) are expressed in putative neuronal/spirocyte The cnidarian Otx-related genes are expressed as putative regulators of precursors and neural cell types (Matus et al., 2007a) similarly to the morphogenetic movements and of the nerve ring. Among PRD-class Acropora PaxC gene (Miller et al., 2000). The Nematostella Pax-B is genes, Otx/Otd orthologs were identified in Podocoryne, Hydra and expressed in scattered ectodermal cells and around the oral region Nematostella however their neurogenic function is currently doubtful. suggesting a role in the patterning of the nerve ring (Matus et al., In Podocoryne and Hydra Otx genes are likely involved in cell mi- 2007a). The Pax-D genes are present as a single copy in Acropora but gration, like that observed during the budding process (Muller et al., four in Nematostella where expression of only two could be detected. 1999; Smith et al., 1999). However in the Hydra polyp Otx is also In both species the Pax-D genes are expressed as stripes around the expressed in the neurogenic zones, i.e. in the tentacle zone and along circumference of the embryo. In Acropora the PaxDam domain is first the body column, but was not detected in neurons (Smith et al., 1999). aboral and then oral after settlement (de Jong et al., 2006); in Ne- In Podocoryne, Otx expression is actually initiated during the budding matostella the NvPaxD1 domain corresponds to the upper body process and maintained in the striated muscle cells of the medusa but column region where interstitial stem cells generate specific cells for was not detected during planula development (Muller et al., 1999). the oral region and the NvPaxD3 domain is restricted to the tentacles In anthozoans two Otx genes were identified in the coral Acropora (Matus et al., 2007a). Further studies should confirm the neurogenic and three clustered ones in the Nematostella although with unclear function of Pax genes in anthozoans. phylogenetic relationships between these paralogs (de Jong et al., In medusozoans, the studies mostly focused on the role of Pax 2006; Mazza et al., 2007). The three Nematostella Otx genes show a genes on eye differentiation in homologous (medusa) and hetero- very similar biphasic expression pattern, with a first wave during logous (Drosophila) contexts. Interestingly the adult jellyfish Tripe- gastrulation, restricted to the earliest involuting endoderm that dalia expresses Pax-B exclusively in the rhopalia (Kozmik et al., 2003). rapidly occupies the aboral region, and a later wave at the oral pole, In cell culture Pax-B efficiently transactivates the Tripedalia crystallin detected as an endodermal pharyngeal ring surrounding the pre- promoters and the Drosophila rh6 rhodopsin; moreover Pax-B can sumptive mouth and in the first developing tentacles (Mazza et al., partially rescue the spa (Pax-2) phenotype and induce ectopic eye 2007). In Acropora, the two Otx genes exhibit distinct regulations, formation in Drosophila (Kozmik et al., 2003). These data suggest that with OtxA predominantly ectodermal, also detected as a ring at the cnidarian Pax genes can already regulate eye differentiation and oral pole and along the body column in scattered cells, and OtxB, possibly in scattered photosensing cells in cnidarian species that do endodermal throughout development (de Jong et al., 2006). Thus a not differentiate eyes. The coral Pax-B and Pax-D genes cannot induce
Fig. 7. Regulatory genes involved in neurogenesis in bilaterians likely support neurogenesis and nematogenesis in Hydra polyps. (A) Markers of neurogenesis: Cnot) Neurons (nv) located at the root of tentacles in the adult polyp (left) and in the spots where tentacle rudiments (t.r.) emerge during budding (middle) and head regeneration (right, here at 52 hpa), express the ANTP-class homeogene cnot. Prdl-a) Sensory neurons and their progenitors located at the apical pole (top view) express the paired-like homeogenes prdl-a (white arrows). Right panel: macerated head tissues stained with Hoechst (blue), anti-prdl-a (red) and anti α-tubulin (green). Gsx/cnox-2) Progenitors and apical neurons (nv, white arrow) located at the base of the head region express the ParaHox homeogene Gsx/cnox-2. During head regeneration, cnox-2 is up-regulated in proliferating precursors and neurons that differentiate in the regenerating tip, shown here at 32 hpa (arrow). Right panel: Apical neurons co-expressing cnox-2 transcripts (green) and β-tubulin (red). Msx) Neurons of the body column express the ANTP-class homeobox msx gene. Msx+ neurons are denser in the budding zone and restricted to the ectodermal layer (black arrow). HyCOUP-TF, prdl-b) A subset of sensory neurons in the body column express Hy-COUP-TF and the paired-like homeogene prdl- b (not shown). (B) Markers of nematogenesis: Gsx/cnox-2, hyCOUP-TF and prdl-b are expressed in synchronously dividing nematoblasts (nb, thin black arrows) along the body column. Both hyCOUP-TF and prdl-b genes are repressed in the adult apical and basal regions (brackets) but also in the presumptive head region during budding (arrowheads) and head regeneration (large arrow). These genes are expressed at distinct stages along the nematocyte pathway, with Gsx/cnox2 transcripts detected in precursors and hyCOUP-TF expressed in nematoblast clusters that start differentiating. Scale bars: 100 μm and 10 μm. For references see in the text. (C) Silencing of Gsx/cnox-2 through RNAi leads to alterations of neurogenesis after repeated exposures to dsRNAs: In intact Hydra the apical nerve net is no longer visible (upper panels); after amputation (lower panels) the de novo neurogenesis normally observed in head-regenerating tips (left, here at 40 hpa) is drastically reduced (outline, right). Scale bars: 50 μm. (D) Putative epistatic relationships in Hydra nematogenesis deduced from studies performed by (Lindgens et al., 2004; Miljkovic-Licina et al., 2007). Gsx/cnox-2 regulates directly or indirectly HyZIC expression in proliferating nematoblasts: note the complete disappearance of HyZIC transcripts (red) in cnox-2(RNAi) silenced cells (green). Scale bar: 10 μm. 16 B. Galliot et al. / Developmental Biology 332 (2009) 2–24 by themselves eye formation when expressed in Drosophila imaginal al., 2004c). In Hydra the type A ortholog, CnASH, is expressed in discs but can achieve such task when chimeric (Plaza et al., 2003). In clusters of differentiating nematoblasts (Grens et al.,1995; Lindgens et the jellyfish Podocoryne that does not differentiate eyes, Pax-B is al., 2004) and in sensory neurons at the base of tentacles (Hayakawa expressed in the early steps of neuronal cell differentiation (Groger et al., 2004). When ectopically expressed in Drosophila instar larvae, et al., 2000). These data strongly speak for a neurogenic function for CnASH led to the formation of ectopic sensory organs similarly to the Pax genes that arose in the CBA, and further studies in Trichoplax Drosophila cognate genes when ectopically expressed; moreover a (Hadrys et al., 2005) and Porifera (Larroux et al., 2006) should trace partial rescue was noted when CnASH was expressed in achaete/scute the ancestral function of Pax genes in cell specification. However the double mutants (Grens et al., 1995). In the jellyfish Podocoryne carnea, expression of Pax genes is clearly not restricted to the nervous system two Achaete/Scute genes were analyzed: the first one, Ash1, also in cnidarians as NvPaxC appears to be expressed also in gland cells, related to class A, consistently showed an expression in differentiating NvPax-B at the endodermal/ectodermal boundary of the pharynx, nematocytes (Muller et al., 2003) whereas the second, Ash2, related and PcPax-B in the entocodon during medusa formation. to the class B, is likely involved in the differentiation of secretory cells (Seipel et al., 2004c). These data suggest that the neurogenic function The Six genes and the eye differentiation in jellyfish. In contrast to the of type A ASH genes is ancestral and conserved from cnidarians to Pax genes, the Six gene families were already established in the CBA bilaterians. and remained highly conserved along cnidarian and bilaterian evo- lutions. A single Six gene was identified in Porifera, related to the Six1/ The Atonal-like gene Atl1 is a candidate proneural gene in the jellyfish 2-so family (Hoshiyama et al., 2007) whereas three distinct families Podocoryne. In the developing jellyfish Podocoryne, an Atonal-like were identified in cnidarians: Six1/2, Six3/6 and Six4/5, suggesting a gene (Atl1) was found expressed in endodermal neuronal precursors, wave of gene duplication that took place between Porifera and and in adulthood, in mechanosensory cells and neuronal precursors eumetazoans. In two hydrozoan medusae, Podocoryne (no eyes) and located in the tentacle bulbs and the manubrium. Moreover, when in Cladonema (with eyes) these three families are likely involved in vitro transdifferentiation is induced, Atl1 is up-regulated in proliferat- neurogenesis being expressed along the manubrium, in the nerve ring ing neuronal precursors arising from adult striated muscle cells and/or the tentacle bulbs (Stierwald et al., 2004). Moreover the Six1/ (Seipel et al., 2004c). Interestingly this study also mentions that 2 and Six3/6 genes are expressed in the eye cup, at low and high levels during medusa budding, the striated muscle precursors in the respectively (Stierwald et al., 2004). During eye regeneration Six1/2 entocodon express Atl1, highlighting the fact that neurogenesis and and Six3/6 but not Six4/5 are up-regulated very early, Six1/2 myogenesis that are supposed to share a common origin, indeed make preceding Six3/6 suggesting that Six1/2 is acting rather upstream in use of common regulators. the cascade directing eye formation but is probably not required for eye maintenance. In the scyphozoan jellyfish Aurelia the Six1/2 The putative neurogenic function of the nuclear receptors RXR and ortholog is also expressed in the rhopalia (Bebenek et al., 2004). COUP-TF. Nuclear receptors (NRs) are ligand-dependent transcrip- Finally given the genetic interactions that occur between the Pax and tion factors activated by steroid hormones and non-steroid molecules Six genes during eye specification (but also for muscle specification such as retinoic acid, thyroid hormone and vitamin D (Moras and and kidney differentiation) in bilaterians, one can speculate that this Gronemeyer, 1998). However, some of the NRs are considered as interaction was already at work in cnidarians (Hoshiyama et al., 2007). “orphan”, i.e. lack a well-identified ligand (Benoit et al., 2006). In Hence several key components of the genetic circuitry driving eye cnidarians, a variety of nuclear receptors were characterized including specification in bilaterians are already available and properly a unique COUP-TF gene in Hydra but six in Acropora,aFTZ-F1 gene in regulated in cnidarians. However the same question remains disputed Nematostella and a RXR gene in Nematostella and Tripedalia (Kostrouch (Fernald, 2004; Kozmik et al., 2008): How to explain that the same et al., 1998). Therefore, the NRs gene families diversified very early gene regulatory network supports eye differentiation in a large variety during evolution, before divergence of Cnidaria (Fig. 6). of phyla? Does it reflect a common origin for all the eyes across the phyla or rather a reiterated recruitment of the same regulatory A putative function in eye differentiation for the nuclear receptor RXR. network in distinct contexts? As in vertebrates, the Tripedalia RXR ortholog might also regulate the expression of the crystallin genes as it is predominantly expressed at The bHLH genes as candidate regulators of neurogenesis and myogenesis the medusa stages and it specifically recognizes in vitro direct repeats in hydrozoans identified in the crystallin gene promoter of this cubozoan jellyfish As for many gene classes involved in developmental processes, the (Kostrouch et al., 1998). Moreover, similarly to its vertebrate cognates, complement of basic Helix–loop–Helix (bHLH) genes was already the Tripedalia RXR transcription factor is potentially regulated established when Cnidaria arose and remained strikingly stable over by retinoic acid as it binds the 9-cis retinoic acid as a ligand. Fur- the evolution (Simionato et al., 2007). The bHLH genes were initially ther studies in hydrozoan and scyphozoan jellyfish should esta- characterized in genetic analyses as proneural genes, i.e. directing the blish whether the RXR function is a common trait in cnidarian eye ectodermal cells towards a neuronal fate: In Drosophila, the achaete differentiation. and scute genes exhibit a proneural function in sensory organ for- mation (Jan and Jan, 1994), and the vertebrate orthologs play a similar The neurogenic and nematogenic function of the nuclear receptor COUP-TF. proneural function during development (Bertrand et al., 2002). In all bilaterian species the orphanCOUP-TF genes were clearly associated Surprisingly a sponge bHLH gene was recently shown to display pro- with neurogenesis (Park et al., 2003). In mice,COUP-TFI disruption results neuronal properties when expressed in Xenopus or Drosophila in multiple defects of the central nervous system (Qiu et al., 1997) and (Richards et al., 2008). Hence pieces of the metazoan neurogenic together with Pax6 and Emx2, it acts as an early intrinsic factor for early circuitry predated the emergence of a nervous system. regionalisation of the neocortex (Zhou et al., 2001). Hence COUP-TF genes, which in most contexts behave as potent negative transcriptional The type A achaete-scute ortholog appears restricted to the nervous regulators (Achatz et al., 1997), bring a major contribution to both system in hydrozoans. The Achaete-Scute (ASH) genes distribute in neurogenesis and the CNS patterning during the embryonic life, as well as two distinct classes, A and B. The A class is represented by four genes in neurophysiology of the adult nervous system (Pereira et al., 2000; in Drosophila and C. elegans, two in mouse whereas the class B is Cooney et al., 2001). According to these data, neurogenesis is considered absent in Drosophila but present in mouse, C. elegans and Podocoryne as the ancestral developmental function ofCOUP-TF genes whereas the indicating an ancient duplication event (Ledent et al., 2002; Seipel et vertebrate COUP-TFII gene seems to be devoted to mesenchymal- B. Galliot et al. / Developmental Biology 332 (2009) 2–24 17 epithelial interactions during organogenesis (Pereira et al., 1999). In Hy- The Fox and MADS-box transcription factors. The Fox genes encode dra, hyCOUP-TF is expressed in few interstitial cells, in proliferating and transcription factors that bind DNA thanks to their winged-helix differentiating nematoblasts, as well as in neurons of the body column domain and are involved in the development of the nervous system (Gauchat et al., 2004). In the nematocyte pathway hyCOUP-TF actually in deuterostomes (Mazet and Shimeld, 2002; Mazet et al., 2005). seems to be turned on later than prdl-b, at a time when nematoblasts Among the 20 families identified in bilaterians, at least 6 families enter the differentiation phase (Fig. 4). In the neuronal cell lineage,hy- already diversified in sponges whereas 9 new families appeared in COUP-TF expressing cells correspond to a subset of small bipolar neurons. cnidarians and only 3 in urbilaterians (Magie et al., 2005; Larroux When animals were rendered “nerve-free”, hyCOUP-TF expressing cells et al., 2006; Chevalier et al., 2006; Larroux et al., 2008). In Clytia two disappeared in few days. During budding and regeneration,hyCOUP-TF of these gene families already diversified, the FoxB gene being expression vanished in regions where either apical or basal differentiation expressed in numerous places where neurogenesis takes place in- occurred (Gauchat et al., 2004). Moreover the Hydra hyCOUP-TF cluding in the sensory organs named statocysts that develop along expressed in mammalian cell cultures can repress the transactivation the bell rim (Chevalier et al., 2006). As FoxB genes are implicated in induced by the RAR:RXR nuclear receptors. In summary, the Hydra neurogenesis in bilaterians, these data suggest an evolutionarily- hyCOUP-TF is supposed to promote the differentiation of both nemato- conserved function in neurogenesis for some of the Fox families. In cytes and neurons, reflecting hence an ancestral neurogenic function for Hydra, Budhead, a fork head/HNF3 ortholog rather appears involved the COUP-TF NR family. in apical specification (Martinez et al., 1997). Concerning the MADS- box transcription factors, the expression of the Nematostella Mef2 Various transcription factor families involved in neurogenesis gene is consistent with a role in the differentiation of ectodermal cell types including nematocytes and neurons (Martindale et al., 2004) The neurogenic function of the C2H2 zinc-finger transcription factors, whereas the Hydra SRF ortholog possibly plays a similar function Zic, Gli. The Zic and Gli transcription factors form two highly related in the interstitial cell precursors and nematoblasts (Hoffmann and evolutionarily-conserved families that interact with each other and Kroiher, 2001). bind their target sequences thanks to their C2H2 zinc-finger domains (Aruga et al., 2006). In vertebrates, ascidians and nematode, Zic genes The basic leucin zipper (b-ZIP) CREB transcription factor. The bZIP exert multiple functions during neuronal development including early transcription factors, defined by the presence of a basic domain neural patterning in vertebrates as evidenced by the region-specific followed by a leucine zipper domain involved in DNA-binding and morphogenetic alterations of the central nervous system induced upon dimerization respectively, form a large class of transcription factors inactivation of the different Zic genes in mice (Aruga, 2004; Merzdorf, that can be traced in fungi, plants and animals. In bilaterians, this 2007). However, they also specify mesodermal derivatives as in class is formed of 19 families, 13 of them being already expressed in amphioxus and ascidians (Gostling and Shimeld, 2003) and the Dro- cnidarians (Fig. 4). Phylogenetic analyses including cnidarian sophila Zic ortholog odd-paired is a segmentation gene not involved in sequences indeed concluded that an early wave of gene duplications neuronal differentiation. In Hydra a HyZic gene is expressed in the took place in the last common-CBA (Amoutzias et al., 2007). In bila- nematocyte lineage where it is turned on during the first synchronous terians, the CREB transcription factor that is targetted by a wide divisions of nematoblasts and off at the final runs of division, before variety of stimulus, regulates multiple developmental and physiolo- differentiation of mature nematocytes occurs (Lindgens et al., 2004). In gical processes including neuron survival and neuron degeneration nerve-free animals Hyzic expression is rapidly turned off supporting (Mantamadiotis et al., 2002), nervous development, learning and the hypothesis that Hyzic function is restricted to the early stages of memory (Lonze and Ginty, 2002). In Drosophila, Aplysia, rats and nematocyte differentiation; moreover in cnox-2(RNAi) hydra Hyzic mice, CREB-dependent transcription is required for synaptic plasti- expression is abolished (Fig. 7D), suggesting that Hyzic is directly or city and learning and memory processes, more specifically for the indirectly regulated by the Gsx ortholog (Miljkovic-Licina et al., 2007). transition from short-term to long-term memory, suggesting that Further studies in anthozoan and medusozoan species should confirm CREB is an universal modulator of memory in bilaterians (Barco and refine the neurogenic function of Zic genes, i.e. restricted or not to et al., 2006). In Hydra, the CREB transcription factor was initially nematocyte differentiation, and involved or not in patterning of the identified as a key regulator of the early stage of head regeneration cnidarian nervous system. (Galliot et al., 1995; Kaloulis et al., 2004). However, the CREB protein was also detected at strong levels in proliferating progenitors, The Sox/TCF transcription factors in cnidarians. The Sox genes encode including progenitors for the nematocyte and neuronal cell lineages, High Mobility Group (HMG) transcription factors that are already as well as mature nematocytes, ganglion and bipolar sensory present in choanoflagellates and sponges (King et al., 2008; Larroux neurons (Chera et al., 2007). In hydrozoan medusae CREB might et al., 2008) and diversified early in metazoan evolution with play a similar role, being strongly expressed in differentiating representatives of the groups B, C and F in sponges, B, C, E and F in neurons in the tentacle bulbs (Fig. 5E, here Podocoryne). Future cnidarians and ctenophores (Magie et al., 2005; Jager et al., 2006, work should tell us more about the various functions of CREB in 2008; Shinzato et al., 2008). In bilaterians Sox genes are involved in cnidarian nervous systems. germ cell specification, mesendodermal patterning, neural induction, development of the central and peripheral nervous systems and The Runx and CBPβ genes in Nematostella neurogenesis. The DNA- organogenesis (Guth and Wegner, 2008). In Nematostella, Acropora binding of the Runx transcription factors is enhanced upon and Clytia, 14, 6 and 10 Sox genes respectively were identified (Fig. 6); heterodimerization, especially with CBPβ when co-expressed. among those, the expression patterns of the Acropora and Nematos- These two gene families appear to form a rather stable old couple, tella SoxC orthologs suggest some role in the specification of the ecto- already present in Porifera and Cnidaria, which did not diversify dermal sensory neurons during development (Table 1). Nematostella prior to the emergence of bilaterians (Sullivan et al., 2008). In the and SoxB2 genes in ectodermal neuronal-like cells in developing Ne- adult Nematostella, Runx and CBPβ are expressed in putative matostella suggest some ancestral neurogenic function. Similarly the neurons and neural precursors in the tentacles, in scattered ecto- expression of the SoxB and SoxE genes in the neurosensory structures dermal cells along the body column and in case of CBPβ, also in the of the adult ctenophore combjelly Pleurobrachia evokes some role in mouth and upper pharynx. Detailed histological analyses suggest the maintenance of the nervous system (Jager et al., 2008). Conse- that these two genes are often co-expressed and participate in quently some Sox genes might have been recruited at the time of the the differentiation and maintenance of the apical nervous system emergence of the nervous system. (Sullivan et al., 2008). 18 B. Galliot et al. / Developmental Biology 332 (2009) 2–24
What role for the phylum-specific genes in neurogenesis and SoxB, SoxE), winged-helix group (FoxB), MADS-box class (Mef2), zinc nematogenesis? fingers (zic), nuclear receptors (COUP-TF, RXR), Runx/CBPβ and bZIP (CREB) transcription factors likely regulate neuronal differentiation Two types of molecules are considered as putative phylum-specific since early eumetazoan evolution (for references see above), similarly actors, first the bioactive peptides, neuropeptides or epitheliopeptides to the bHLH family members in myogenesis (Muller et al., 2003; Seipel that are often evolutionarily-conserved but most probably play et al., 2004c). The reiterated and independent recruitment of phylum-specific roles in differentiation and developmental processes orthologous genes and signaling pathways to perform similar func- (Table 1), and second the phylum-specific genes. Recently a peptide- tions in various phyla cannot be excluded and is actually discussed gated Na-channel was found expressed at the root of Hydra tentacles, concerning eye evolution (Kozmik et al., 2008; Suga et al., 2008), but suggesting that fast transmission through neuropeptides already does not represent the most parsimonious scenario. existed in ancient nervous systems (Golubovic et al., 2007). The neurogenic function of peptides is well known in cnidarians. What roles for the “neurogenic” genes in Porifera, a non-neuronal Neuropeptides can modulate muscle activity (Grimmelikhuijzen et phylum? al., 1996) as hym-176 that triggers contraction of the ectodermal myoepithelial cells in Hydra (Yum et al., 1998). In fact the (eyeless) The evolutionarily-conserved regulatory genes expressed in Hydractinia planulae exhibit dramatically increased phototaxis when nematocytes and neuronal cells were in most cases identified in exposed to RFamide indicating that RFamides, expressed by the poriferans: the genes for the ANTP, Pax, POU, LIM-HD, Sox, nuclear neurosecretory cells in all cnidarian species, whatever the stage of the receptor, Fox (forkhead), T-box, Mef2, Ets and bHLH transcription life cycle, can modulate the non-visual behavior (Plickert and factors emerged and in many instances already diversified prior to the Schneider, 2004). The authors propose that the RFamide cells act as Porifera/eumetazoan split (Larroux et al., 2006; Jager et al., 2006; interneurons between photosensing cells and myoepithelial cells. Richards et al., 2008). Similarly the main signaling pathways Wnt, Besides physiological activity, neuropeptides can trigger or en- TGF-β, RTK, Notch, Hedgehog, and Jak–Stat as well as the adhesion hance neuronal differentiation as hym-355 (Takahashi et al., 2000), or molecules are present in sponges (Nichols et al., 2006). Moreover the head activator that also affects cell proliferation and head patterning modulated expression of the bHLH (Richards et al., 2008) and NK-type processes (Schaller et al., 1989; Hobmayer et al., 1997). Similarly the (Gazave et al., 2008) genes during embryogenesis suggest a possible neuropeptides LW-amides play a pivotal role in developmental role in cell differentiation and region specification. This surprising processes as larval metamorphosis in Hydractinia (Plickert et al., finding according to which the origin of the neuronal genetic circuitry 2003). Interestingly neuronal differentiation is also under the control predated the occurrence of nerve cell differentiation, is intriguing. In of epithelial cells as among peptides that were identified in the fact the choanocytes were proposed to exert some sensory function, systematic Hydra peptide project (Fujisawa, 2008), the epitheliopep- and as such might represent a proto-neuronal cell type (Gazave et al., tides belonging to the LPW family can inhibit nerve cell differentiation 2008). Therefore, these gene families, which likely constitute the (Koizumi, 2002). In bilaterians, peptides are best known as neuro- hallmark of metazoans, might already be committed to a “proto- transmitters or hormonal regulators involved in physiological pro- neuronal” function. Alternatively, they might specify cell fate cesses (Boonen et al., 2009); hence homologous functions in cell independently of their ability to differentiate mechanoreceptor and/ differentiation or developmental processes remain to be deciphered. or nerve cells. Two types of arguments can be proposed to explain the As nematogenesis is a cnidarian-specific process, genes that trigger absence of neurogenic “success” for the poriferan “neurogenic” genes: nematocyte differentiation are frequently phylum-specific. A micro- firstly the absence of some essential neurogenic genes explaining that array analysis identified 51 genes as nematocyte-specific, most of the genetic circuitry cannot be mounted properly; among those them encoding putative secreted proteins expressed at distinct stages missing genes the ParaHox/Hox-like genes were never identified in of the pathway (Hwang et al., 2007). Out of these 82% do not have Poriferans and we saw that Gsx/cnox-2 plays an essential role in the bilaterian orthologs implying that beside conserved regulatory genes, regulation of neuronal precursors in Hydra as well as in bilaterians, nematocyte differentiation makes use of a large proportion of genes, secondly the absence of some target structures, i.e. myofibers, might which were not retained in bilaterians and whose origin could not be make the organization of a rudimentary genetic circuitry useless. traced back so far. However one cannot rule out the possibility that these genes represent a genuine neurogenic program originally acquired by the A tentative integrative view of the early evolution of neurogenesis metazoan ancestor and secondarily lost in poriferans.
Common molecular tools for the specification of the cnidarian and The early diversification of the regulatory gene families in eumetazoans bilaterian nervous systems and the emergence of neurogenesis in the Cnidaria–Bilateria ancestor
Several criteria support a common origin for neurogenesis in Interestingly most of these putative neurogenic gene families that cnidarians, ctenophores and bilaterians: first the presence in non- encode transcription factors underwent an early wave of amplification bilaterian phyla of gene families orthologous to those that encode in the last common Cnidaria-Bilateria ancestor (Fig. 6). This event transcription factors with neurogenic functions shared by protostomes likely preceded the emergence of the nervous system as evidenced by and deuterostomes, second their consistent cellular expression pat- the few representatives present in sponges versus the large number of terns in the cnidarian nervous system or during its differentiation, cnidarian–bilaterian orthologous families belonging to the Homeo- third the loss and gain of function assays that affect the maintenance or box, bHLH, bZIP, Wnt classes. Moreover the comparative analysis of the differentiation of the cnidarian nervous system (even though only the transcription factor classes between one poriferan and two cni- few gene families were tested so far) and fourth the heterologous darian genomes confirmed that several classes that exhibit an assays that proved that the cnidarian genes can affect neurogenesis evolutionarily-conserved role in neurogenesis in bilaterians have when expressed in bilaterian developmental contexts. The functional emerged after the divergence of Porifera. This is the case of the Hox/ dissection of the genetic cascades regulating the differentiation of the ParaHox, Otx-like, Atonal/Twist gene families that are obvious nervous system in cnidarians and ctenophores was only recently candidates for having conducted the emergence of the nervous launched but the expression analyses currently available indicate that system. As the bilaterian orthologs also regulate neurogenic functions, the ANTP-class (not, msx, Gsx), PRD-class (Pax, paired-like, Rx, repo, we speculate that the combination of transcription factors that drove gsc, six), bHLH-class (Achaete-scute, atonal), HMG-group (Sox2, SoxB2, neurogenesis in the Cnidaria–Bilateria ancestor was iteratively used B. Galliot et al. / Developmental Biology 332 (2009) 2–24 19 along evolution. In a limited number of cases, these “eumetazoan” sister cells in cnidarians. In fact members of the Six, Pax, bHLH, Sox, families underwent a secondary wave of duplications after the diver- Pou gene classes are involved in both neurogenesis and myogenesis gence of cnidarians, in urbilaterians, suggesting that the duplication of in bilaterians. In the Podocoryne jellyfish where both differentiation genes previously recruited for neurogenesis, led to the complexi- pathways were monitored during medusa budding and induced fication of the neuronal structures thanks to paralogous genes. transdifferentiation, the analysis of the Six, C/EBP, MafL, Atonal-like 1, Achaete-scute 2 genes (Table 1) as well as the observation of the Symbiosis might have contributed to the emergence of sophisticated transient expression of neuronal markers during myogenesis (Seipel sensing tools et al., 2004b,c; Stierwald et al., 2004) suggested that muscle cells and nerve cells derive from common myoepithelial cells. These molecular Our current knowledge about the molecular tools supporting the data actually fit with the three steps model proposed by George various cellular differentiation pathways in distinct animal phyla Mackie for the origin of neuromuscular transmission (Mackie, 1970) tremendously increased over the past ten years with genomic sequen- whereby muscle cells and nerve cells would have diverged from myo- cing, extended phylogenetic analyses of gene classes, cellular expres- epithelial cells (see Fig. 5a in Arendt, 2008). The scenario is as sion and functional analyses. This vast bag of informations allowed us follows: Starting from a primordial myoepithelium capable of to see emerging principles for understanding cell type specification. “neuroid” conduction, the protomyocytes progressively detached Detlev Arendt recently proposed three common principles that from the basal side of the myoepithelium to sink into the interior; at applied all along animal evolution to homologous cell types and sister the second step protoneurons evolve from the myoepithelium to cells (Arendt, 2008), 1) the multifunctionality of ancestral cells as the connect the myocytes to the outside forming a group of electrically myoepithelial cnidarian cells that carry out epithelial functions but interconnected cells; at the third step, neurosensory cells and also muscular contraction and electric conduction (Mackie and neurons evolved from the protoneurons, developing long processes Passano, 1968), 2) the progressive segregation of the ancestral functions that connected them to each other and to the myocytes by chemical in more specialized cell types that abandon some of the ancestral polarized junctions. This scenario that is largely driven by the functions by silencing the corresponding genetic functions, to carry electrophysiological properties of the different cell types, is coherent out a more limited number of functions, 3) the divergence of some and attractive. However it predicts that the origin of the neurosen- functions thanks to the duplication of the molecular tools such as the sory cells followed the emergence of protoneurons. In fact sensory- amplification of gene families within a given class. like cells appear to have predated by far the origin of the nervous The current set of data concerning neurogenesis in non-bilaterian system, already present in early metazoans as poriferans where they species certainly obey these principles of linear diversification of cell seem to use a proto-neural program, the Notch/Delta and bHLH types but the analysis of the origin and early evolution of neurogenesis pathway to differentiate (Richards et al., 2008). These results suggest also requires to take into account decisive processes that might that the program leading to the emergence of neurogenesis was constitute a fourth principle: the incorporation of foreign genetic multilayered, a pre-program being already available in the different material, either through horizontal gene transfer or through symbio- cell types of the last common metazoan ancestor, therefore the sis. Few cases are currently documented but these certainly deserve differentiation of neurons could have arisen from myoepithelia as attention. The analysis of the respective behaviors of the different well as from sensory cells. cellular contingents in Hydra actually led to the proposal that the venom capsule (named nematocyst or cnidocyst) in nematocytes Conclusions and perspectives would result from a symbiogenetic process (Shostak, 1993). More recently Denker et al. showed that the horizontal transfer of a bacterial Most if not all the pieces of the puzzle that regulates the nervous gene encoding a subunit of bacterial poly-γ-glutamate (PGA) synthase system in bilaterians are present in cnidarians, but given the almost in the genome of the cnidarian ancestor might have been decisive for complete absence of functional analysis, the question of how these the specification of the nematocyte weapon, the cnidocyst (Denker different pieces interact in cnidarians remains completely open. There et al., 2008a). In fact only the receptor part of this highly sophisticated are nevertheless clear differences between neurogenesis in cnidarians cell, the cnidocil, might be retained along evolution, sharing typical and bilaterians as for instance the origin of the neuronal precursors features with other mechanosensory cells (Holstein and Hausmann, during early development: in cnidarians those were identified in the 1988), while structures homologous to the nematocyst capsule were endoderm, from where they migrate towards the ectodermal layer, not identified in other animal phyla so far, indicating that phylum- showing hence a major difference with bilaterians where partitioning specific innovations might also stand alone, even when they contri- of the ectoderm into neural and non-neural portions during early buted to the sustained evolutionary success of the phylum where they embryogenesis is the first event. However, this cnidarian specificity arose. might be revisited as such migration was not observed in developing The second case where the importation of foreign material might scyphozoans (Nakanishi et al., 2008). The fact that in some hydrozoan have been decisive, concerns the specification of the eyes that com- species the destruction or removal of the interstitial stem cells results bine photoreceptor and pigment cells since their origin (Gehring, in the elimination of the sensory-motor, ganglion and nematocyte cell 2004). As photoreception is widely distributed among living organ- lineages but not of the ectodermal sensory cells would be consistent isms, including in bacteria, Walter Gehring proposed that photo- with this ectodermal neurogenic potential (Martin and Thomas, 1981; reception in the cnidarian ancestor might result from a series of Thomas, 1987). symbiotic transfers, a cyanobacteria into a red algae, a red algae into a Also some genes that were expected to be universal regulators of dinoflagellate, the transformation of the chloroplast into an eye inside neurogenesis appear missing; the best example is engrailed that is the dinoflagellate and finally the transfer of the dinoglagellate into a apparently not expressed in cnidarian genomes. Also some key genes cnidarian. A long way to go before seeing, but that certainly highlights for brain patterning in protostomes and deuterostomes appear to be the potency of such mechanisms to bring novelties in organisms that submitted to looser constraints in cnidarians, as Otx that might sup- were less constrained than most bilaterian species. port cell migration in developmental processes in hydrozoans, but be required for the formation of the nerve ring in Nematostella. Also some What was the proto-neuronal cell from which nerve cells evolved? genes provide inconsistent expression patterns between different cnidarian species, making difficult to identify the common themes If we assume that sharing molecular signatures signify a common among these developmental variations. Sampling more taxa in Cnida- history, then the nerve and muscle cells should be considered as ria and Ctenophora will help unravel the core genetic mechanisms 20 B. 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DISCUSSION
À l’issue d’un travail entièrement consacré à l’étude de des familles de gènes Hox/ParaHox de cnidaires, en combinant des approches phylogénétiques pour tenter d’identifier les paramètres d’un scénario évolutif le plus parsimonieux possible, et des approches cellulaires et moléculaires pour caractériser le rôle de l’un d’entre eux au cours de la neurogénèse, nous discuterons ici les résultats les plus saillants de ce travail ainsi que certains aspects inachevés mais que nous espérons prometteurs.
Organisation ancestrale des gènes Hox et ParaHox
III.1.1. Une méthode originale pour reconstruire l’évolution des gènes Hox et ParaHox
La méthode que nous avons employée pour retracer l’histoire évolutive des gènes Hox et ParaHox diffère des précédentes de plusieurs points de vue. Tout d’abord, nous avons fait attention d’utiliser des séquences provenant d’une grande variété d’espèces, représentante de nombreux phyla, en sélectionnant dans la mesure du possible des espèces à évolution lente, ce qui n’est pas le cas dans les autres analyses. Le jeu de données utilisé dans notre analyse est le plus diversifié depuis le séquençage du génome de Nematostella. Par ailleurs, nous avons testé le soutien des nœuds de chaque famille combinant les résultats de deux méthodes d’analyse phylogénétique et ceci répété sur un grand nombre de jeu de données, différant dans leur composition en séquences hautement dérivées caractérisées comme perturbatrices. Enfin, les relations entre les familles, intitulées « metagrouping » ont été mises en évidence en s’attachant à la fréquence d’apparition des nœuds plus qu’à leur soutien. Cette méthode systématique paraît bien adaptée pour tirer de l’information des séquences courtes comme les homéodomaines. Quatre types de résultats nouveaux et originaux ont découlé de l’application de cette méthode (Quiquand et al., 2009) : 1) l’identification de véritables orthologues Xlox/Pdx chez les cnidaires, 2) la confirmation qu’un certain nombre de séquences de cnidaires que nous appelons « PG9-like », représentent effectivement les groupes « postérieurs » des gènes Hox/ParaHox, 3) l’état orphelin du groupe paralogue PG1 due à l’absence de regroupement des séquences « antérieures » PG1, PG2 et PG3 dans nos analyses phylogénétiques alors que ce regroupement était admis « de fait » depuis la découverte des gènes Hox, et 4) enfin la meilleure conservation au sein des eumétazoaires des familles de gènes ParaHox par rapport aux gènes Hox.
151 Concernant la diversification des gènes Hox postérieurs chez les cnidaires, nos résultats sont en accord avec ceux présentés par Chiori et al., à l’exception du fait que nous les avons classifiés en quatre groupes (B-E) quand eux les classifiaient en trois groupes (A-C). En comparaison avec les autres séquences Hox et ParaHox de cnidaires, les séquences postérieures Hox sont en effet très diversifiées et forment différents groupes distincts. De plus, leur affiliation aux séquences Hox postérieures de bilatériens, qui forment un groupe unique, est peu soutenue, mais est évidente en appliquant notre méthode de fréquence d’apparition d’évènements. Ceci prouve que les gènes Hox postérieurs de cnidaires sont très divergents.
La méthode que nous avons mise au point pour pallier l’effet perturbant des séquences de cnidaires hautement dérivées en faisant varier systématiquement le jeu de données, nous a permis d’une part de mettre en évidence pour la première fois des homologues Xlox/Pdx chez les cnidaires et de suggérer que la séquence divergente de Nematostella Xlox/Cdx serait en fait plus proche de la famille Pdx. Cette séquence était précédemment proposée comme affiliée à la fois aux deux familles Pdx et Cdx (Chourrout et al., 2006). Dans l’étude de Chiori et al., elle est proposée comme appartenant à la famille Cdx, mais sans aucun appui statistique. Notre analyse inclut des séquences Pdx d’hydrozoaires qui ont probablement eu un effet attracteur sur la séquence Xlox/Cdx. Cette différence pourrait expliquer ces résultats divergents. Par ailleurs la famille Pdx fournit donc un exemple solide d’une famille de gènes clairement mieux conservée chez les médusozoaires, en l’occurrence chez les hydrozoaires, que chez les anthozoaires. Or depuis la publication des ESTs d’Acropora et du génome de Nematostella, les anthozoaires étaient le plus souvent présentés comme les meilleurs représentants de l’état ancestral (Collins et al., 2006).
Notre analyse corrobore celle de Chiori et al. par le fait que nous avons également noté que certaines familles de gènes Hox/ParaHox ne sont pas représentées dans toutes les classes de cnidaires. Alors que la famille Hox PG2 ne se retrouve que chez Nematostella, d’autres familles ont clairement divergées entre anthozoaires et medusozoaires comme la famille PG1, mais aussi les familles relatées au groupe PG9/Cdx.
Le gène Gsx/Cnox2/Anthox2 a été identifié chez 15 espèces de cnidaires différentes. En revanche, les familles Pdx et Cdx sont représentées par seulement deux séquences d’hydrozoaires chacunes (II.1-2. Chapitre 1-2) (Kamm et al., 2006; Chiori et al., 2009; Quiquand et al., 2009). De plus, on peut constater que pour les
152 espèces de cnidaires chez qui le génome est disponible (Nematostella et Hydra magnipapillata) la totalité des gènes ParaHox n’a pas été retrouvée. Nematostella et l’hydre possèdent toutes deux le gène ParaHox Gsx et Nematostella présente un deuxième gène ParaHox (Xlox/Cdx) affilié selon les analyses à la famille Pdx (Quiquand et al., 2009), Cdx (Chiori et al., 2009) ou aux deux (Chourrout et al., 2006). Les gènes ParaHox étaient donc présents chez l’ancêtre commun des eumétazoaires, mais ceux-ci n’ont pas été systématiquement conservés chez les cnidaires à l’exception de Gsx. La conservation de ce gène chez de nombreux cnidaires dans trois classes différentes, suggère une fonction fondamentale et ancestrale qui, comme nous l’avons confirmée (II.2. Chapitre 2), est probablement liée à la détermination des précurseurs neuronaux et nécessaire à la mise en place correcte du système nerveux.
Ceci met en évidence l’importance fondamentale d’utiliser plusieurs modèles de cnidaires pour les études évolutives comparées. Bien que représentant un modèle très utile à l’approche expérimentale, Nematostella appartient à la classe des anthozoaires qui a divergé des hydrozoaires il y a environ 500 millions d’années (Peterson and Butterfield, 2005). Il s’avère donc très intéressant que des modèles de médusozoaires ayant un cycle de vie complet comme Clytia soit utilisé, d’autant plus que les études de perte de fonction sont maintenant bien maitrisées chez cette espèce (Momose and Houliston, 2007).
Enfin, nous défendons l’hypothèse que les familles ParaHox sont mieux soutenues que les familles Hox et nous obtenons ce résultat en incluant ou non la séquence Cdx d’hydrozoaire récemment identifiée (Chiori et al., 2009) (II.1-2. Chapitre 1-2). Ce résultat contraste avec ceux de Chiori et al. qui proposent que les familles Hox et ParaHox antérieures sont plus soutenues que les familles Hox et ParaHox postérieures. Pour ce point en particulier, la raison pour laquelle nos conclusions diffèrent est principalement dû au fait que les séquences Pdx n’avaient pas été identifiées chez les cnidaires (en particulier chez Clytia) lors de leurs analyses. Enfin, d’une manière étonnante, le support qu’ils obtiennent pour la famille Cdx (en excluant les séquences hautement dérivées) est également bas alors que nous obtenons un très bon soutien dans notre deuxième analyse présentée au chapitre 2 (II.2.).
153 III.1.2. Duplication en tandem d’un complexe ProtoHOX versus duplications multiples de gènes de type Hox
Les analyses phylogénétiques présentées aux chapitres 1 et 2 (II.1-2.) nous ont amené à la conclusion que les cnidaires possèdent des gènes Hox antérieurs (PG1, PG2) et postérieurs (PG9-like) ainsi que les trois familles de gènes ParaHox, Gsx, Pdx/Xlox et Cdx, favorisant l’hypothèse d’un complexe ProtoHox à trois gènes. En ce qui concerne l’émergence des complexes HOX et ParaHOX, de nombreuses hypothèses ont été présentées (voir discussion dans Quiquand et al., 2009), mais nous avons décidé de n’en confronter ici que deux. L’hypothèse initiale de Brooke et al. reposait sur l’affiliation respective des familles ParaHox à différentes familles Hox : Gsx et PG1, Xlox/Pdx et PG3/4, Cdx et PG9. Selon eux ces deux clusters résultaient de la duplication d’un complexe ancestral nommé ProtoHOX (Brooke et al., 1998). Avec des variations concernant la composition de ce complexe, cette hypothèse a été soutenue à plusieurs reprises (Finnerty and Martindale, 1999; Gauchat et al., 2000; Ferrier and Holland, 2001b; Ferrier and Minguillon, 2003; Garcia-Fernandez, 2005a; Chourrout et al., 2006), et nous la soutenons également (II.1. Chapitre 1) (Quiquand et al., 2009).
En revanche, Ryan et al. ayant mis en avant le lignage indépendant de la famille Gsx, sans orthologie avec la famille PG1, proposent une hypothèse alternative favorisant la duplication de gènes en tandem plutôt qu’un complexe ancestral entier (Ryan et al., 2007). Dans cette hypothèse, les gènes du complexe ParaHOX ne représentent pas un complexe frère au complexe HOX mais des gènes Hox dispersés dans le génome. Chiori et al. ont renforcé ce point de vue puisque dans leur analyse les familles Gsx et Cdx sont des groupes frères mais ne révèlent pas d’orthologie avec les familles PG1 et PG9. Eux aussi favorisent l’hypothèse de la duplication de gènes (Chiori et al., 2009).
À l’opposé, nous montrons que les quatre familles PG2-PG3-Gsx-Pdx se groupent entre elles, en excluant PG1 et ceci sans affinité plus particulière d’une famille pour l’autre. Notre analyse infirme donc l’hypothèse de Brooke et al. selon laquelle les familles Gsx et PG1 sont orthologues ainsi que Pdx et PG3 (Brooke et al., 1998). Par ailleurs, nous montrons également que les familles Cdx/PG9/Hox postérieures de cnidaires ont statistiquement plus d’affinité entre elles qu’avec n’importe quelles autres familles. Comme Brooke et al. nous soutenons que l’origine des complexes HOX et ParaHOX vient d’un complexe ancestral unique puisque nous détectons des relations phylogénétiques entre les familles Hox et ParaHox. De plus, nous
154 favorisons l’hypothèse la plus parcimonieuse d’un complexe ProtoHOX à trois gènes comprenant les paralogues Gsx/PG2, Pdx/PG3, Cdx/PG9 qui, après une duplication en tandem, aurait donné les complexes HOX et ParaHOX primitifs présents chez l’ancêtre commun des eumétazoaires (voir Fig. 7 dans Chapitre 1 (Quiquand et al., 2009). Précédement, les structures hypothétiques du complexe ProtoHOX reposaient sur l’existence des gènes Hox et ParaHox antérieurs et postérieurs chez les cnidaires. L’hypothèse que nous soutenons présente l’originalité de s’appuyer sur la découverte de la famille Pdx chez les cnidaires et propose d’abandonner la vue « bilatérienne » d’une organisation fondée sur l’opposition entre gènes antérieurs et gènes postérieurs (Zhang and Nei, 1996). D’une part, cette vue n’est pas soutenue par les analyses phylogénétiques, d’autre part les évidences pour la stricte correspondance, c’est-à-dire l’homologie entre l’axe oral-aboral des cnidaires et l’axe antéro-postérieur des bilatériens sont actuellement faibles. Donc l’organisation du complexe ProtoHOX lors de son émergence ne pouvait pas être lié à une contrainte développementale qui n’est vraisemblablement apparue que des centaines de millions d’années plus tard.
III.1.3. Les gènes ParaHox ont-ils un mode d’expression conservé au cours de l’évolution ?
Clytia est donc le premier cnidaire chez qui l’existence des trois gènes ParaHox a été démontrée. Contrairement aux résultats de Chiori et al., nous avons pu détecter l’expression de Gsx au stade larvaire. Mises ensemble, ces deux publications montrent que les trois gènes ParaHox sont exprimés pendant le développement larvaire de Clytia dans des types cellulaires non déterminés, sans évidence pour l’existence d’une colinéarité spatiale. En revanche chez la méduse adulte, Gsx et Cdx sont exprimés dans les bulbes tentaculaires. Les cellules positives pour Gsx sont supposés correspondre à des précurseurs neuronaux ou des neurones différenciés et celles positives pour Cdx à des nématoblastes différenciés (Chiori et al., 2009). Chez le polychaete Nereis et chez l’amphioxus, les trois gènes ParaHox sont exprimés dans le système nerveux ventral (Brooke et al., 1998; Kulakova et al., 2008). En revanche, chez la souris, ils sont tous les trois détectés dans le pancréas (Rosanas-Urgell et al., 2005). L’expression pancréatique de Gsx semblerait être davantage une expression dérivée. En effet comme nous en avons discuté, la fonction de Gsx est conservée dans le développement du tube neural et du neuroectoderme chez la souris et la drosophile respectivement. Ces données s’appuient sur des analyses faites sur des lignées transgéniques. De plus, il existe
155 un important degré de conservation des domaines d’expression de ce gène au sein du règne animal dans les structures nerveuses (II.2-3. Chapitre 2-3). Il n’y a pour le moment aucune analyse fonctionnelle relatant le rôle de Gsx dans le pancréas. Il semble donc peut probable que l’activité des gènes ParaHox soit restreinte à la mise en place du patron endodermique comme cela a pu être suggéré (Garcia-Fernandez, 2005a).
Rôle des gènes ParaHox dans l’apparition de nouveaux types cellulaires
Il est admis désormais que la neurogénèse n’est probablement apparue qu’une seule fois au cours de l’évolution. Cette hypothèse est appuyée par les similarités qui existent entre le répertoire de gènes neurogéniques connus des vertébrés et des invertébrés. De plus, la neurogénèse aurait une origine très ancienne qui remonterait au phylum des cnidaires puisque des gènes orthologues se retrouvent également dans ce phylum (II.3. Chapitre 3). Plus que par leur aspect morphologique, les différents types cellulaires et leur évolution sont désormais abordés par le biais de leurs caractéristiques moléculaires (« fingerprinting », empreintes génétiques) (Arendt, 2008). Les gènes orthologues du répertoire neurogénique conservés des cnidaires aux bilatériens partagent des domaines d’expression dans des types cellulaires supportant une fonction dans la différentiation nerveuse. Enfin, les quelques analyses fonctionnelles qui existent chez les cnidaires corroborent la fonction neurogénique de ces gènes conservés (Miljkovic-Licina et al., 2007; Rentzsch et al., 2008). Gsx fait partie de cette empreinte dans les précurseurs neuronaux et les neurones de cnidaires (II.2-3. Chapitre 2-3) mais aussi de bilatériens et s’intègre sans aucun doute dans un réseau de régulation complexe, probablement différent suivant les phyla, menant à la mise en place d’un système nerveux diffus (II.2. Chapitre 2) ou centralisé.
Les techniques en biologie du développement utilisées chez les cnidaires reposent principalement sur l’étude des patrons d’expression. L’accumulation de ce type de données permet déjà d’avoir une idée des empreintes génétiques des différents types cellulaires constituant les organismes de ce phylum, en particulier les types neuronaux (II.3. Chapitre 3). La comparaison avec les connaissances déjà acquises chez les bilatériens autorise à spéculer sur l’implication évolutive de certains gènes clés dans des processus developpementaux d’intérêts comme la neurogénèse, ainsi que sur l’évolution des différents types cellulaires qui existent dans le règne animal. La combinaison de mes résultats de thèse avec les données déjà existantes nous
156 permet d’envisager l’importance de Gsx dans l’évolution des types cellulaires neuronaux.
Gsx pourrait faire partie des gènes recrutés dans les précurseurs neuronaux ou dans les lignées de cellules neuronales à leur apparition chez l’ancêtre commun aux eumétazoaires. Il est clair que chez les bilatériens, le gène Gsx ne s’exprime pas dans tous les types de cellules nerveuses. Le maintien de la fonction de Gsx dans une sous-population de cellules nerveuses chez les bilatériens pourrait avoir eu lieu lors du partage des fonctions d’une cellule ancestrale à des cellules filles, la fonction de Gsx étant gardée dans l’une et perdue dans les autres par ségrégation de fonction.
Ne connaissant pas les types cellulaires dans lesquels le gène Pdx est exprimé chez les cnidaires, deux hypothèses sont alors possibles. Soit Pdx est exprimé chez les cnidaires et le bilatériens dans des cellules homologues. Dans ce cas, d’une manière similaire à Gsx, la fonction de Pdx aurait pu être recrutée à l’apparition d’un nouveau type cellulaire chez les cnidaires puis ségrégée dans un type de cellule plus spécialisé descendant du type ancestral existant chez les cnidaires. Soit Pdx aurait pu être recruté de manière indépendante chez les cnidaires et les bilatériens dans des types cellulaires non-homologues. Il serait donc informatif de regarder les types cellulaires qui expriment Pdx chez les cnidaires.
Enfin Cdx, qui semble n’exister que chez les hydrozoaires, pourrait quant à lui avoir été recruté pour deux fonctions différentes chez les hydrozoaires et les bilatériens et perdu chez les anthozoaires.
Une lignée spécifique d’hydre appelée « nerve free » est dépourvue de la lignée cellulaire interstitielle. Ces hydres sont donc dans l’incapacité de différencier des nématocytes, des neurones ainsi que des cellules glandulaires et des gamètes qui sont tous des types cellulaires qui descendent de la lignée interstitielle. Hwang et al. ont comparé le profil d’expression des gènes de cette population d’hydre avec celle d’hydre « sauvage » par le biais des puces à ADN (Hwang et al., 2007) et ils ont ainsi mis en évidence les gènes spécifiquement exprimés dans la lignée des cellules intertitielles. Pour raffiner cette approche et connaître l’empreinte génétique spécifique à chaque type de cellules neuronales, l’idéal serait de pouvoir réaliser ce type d’expériences sur des cultures cellulaires de neurones chez les cnidaires et chez les bilatériens. De tels résultats constitueraient un apport primordial pour reconstruire les homologies des types cellulaires neuronaux permettant d’inférer le
157 type cellulaire neuronal ancestral. Il n’existe pour le moment pas de cultures cellulaires chez les cnidaires.
Conclusions
Nous avons montré que les cnidaires possèdent de nombreux outils génétiques utilisés par les bilatériens dans la neurogénèse. Les patrons d’expression et les études fonctionnelles suggèrent une fonction conservée de ces gènes dans la neurogénèse chez ce phylum également. C’est le cas du gène ParaHox Gsx/Anthox2 dont nous avons étudié la fonction dans ce travail et qui paraît être un régulateur ancestral de la neurogénèse.
Gsx est un facteur de transcription représentant un bon candidat régulateur de la neurogénèse des cnidaires aux bilatériens. Mais d’un point de vue plus global, il serait intéressant de savoir précisément dans quelle cascade de régulation il s’intègre et quelle est sa place, par quels gènes il est régulé et quels gènes il régule. L’utilisation des approches modernes d’analyse à grande échelle en particulier au moyen de la technique de l’immunoprécipitation de la chromatine en combinaison des puces à ADN (ChiP on chip) chez les cnidaires serait essentielle à la compréhension de l’évolution de la neurogénèse dans le monde animal.
Ce type de démarche permettrait de voir si les gènes d’identités neurales agissent également en concert chez les cnidaires et si leur régulation par la voie BMP est un mécanisme ancestral. D’une manière plus large, la comparaison des réseaux de régulation intégrant Gsx entre les cnidaires et les bilatériens permettrait de caractériser les changements d’architecture de ces réseaux et d’en déduire les changements génétiques qui ont permis l’acquisition d’un système nerveux centralisé ou des types cellulaires spécialisés. Il sera peut-être possible un jour de changer la hiérarchie des réseaux de régulation de gènes de manière expérimentale et de corréler ces modifications avec des changements morphologiques et, qui sait, de créer ainsi une méduse au système nerveux centralisé ou une souris au système nerveux diffus.
D’une manière plus restrictive, plusieurs points sont encore à développer dans notre projet :
1) Nous avons montré d’une part, qu’Anthox2/Gsx chez Nematostella était exprimé dans des cellules reconnues comme des précurseurs neuronaux et des neurones différenciés et d’autre part que son expression était nécessaire au développement correct de son système nerveux. Les analyses des types cellulaires positifs pour
158 Anthox2 ont été faites sur des reconnaissances morphologiques. À la vue de leur forme et de leur localisation et suivant les descriptions de Marlow et al., les cellules positives pour Anthox2 présentent dans l’ectoderme des tentacules du polype ressembleraient à des neurones sensoriels. Nous aimerions néanmoins confirmer ces résultats au moyen d’un marqueur neuronal comme les anticorps anti-GABA ou anti-(FM)RFamide (Marlow et al., 2009).
2) D’un point de vue fonctionnel, nous aimerions approfondir le rôle d’Anthox2/Gsx chez Nematostella au cours de son développement et dans le maintien de son système nerveux à l’état adulte. À l’aide des anticorps anti-Tyrosine-tubuline ou anti- acetylated tubulin (Rentzsch et al., 2008) nous aimerions regarder si l’inhibition d’Anthox2 affecte la mise en place de la touffe apicale. Enfin nous voudrions approfondir le mode de régulation d’Anthox2 en utilisant la technique d’injection de gène rapporteur. Nous avons déjà montré qu’il existe vraisemblablement un élément répresseur situé dans la région -3kb-2kb en amont de la séquence codante. Nous souhaiterions désormais approfondir la dissection du promoteur en nous focalisant sur les types cellulaires dans lesquels nos constructions, portant des régions différentes de la région régulatrice, sont exprimées. Nous suspectons en effet la présence d’un élément régulateur neurogénique responsable de l’expression du gène Anthox2 dans les cellules neuronales.
3) La revue présentée au chapitre 3 (II.3.) témoigne du grand nombre d’outils génétiques partagés entre les cnidaires et les bilatériens au cours de la neurogénèse. Nous souhaiterions mettre en œuvre une analyse à « moyenne » échelle au moyen de la PCR (Polymerase Chain Reaction) en temps réel (Real Time PCR) pour étudier les niveaux d’expression d’une grande série de ces gènes au cours du développement de Nematostella. Puisque Anthox2/Gsx participe très certainement à la régulation de la mise en place du système nerveux, en synergie avec d’autres acteurs au sein d’un réseau de régulation génétique, nous aimerions mettre en évidence les gènes cibles régulés par Anthox2/Gsx. Pour cela, la même série de gènes pourrait être testée dans un contexte de perte de fonction (injection de morpholinos) ou de surexpression (injection de transgènes dirigeant l’expression d’Anthox2 sous le contrôle d’un promoteur à l’expression ubiquitaire) du gène Anthox2.
Par la suite, le type d’approche que nous avons développé pour l’analyse de la fonction d’Anthox2 (inhibition, gène rapporteur) ainsi que les perspectives que nous
159 souhaiterions suivre pourraient êtres appliqués à certains autres gènes détectés comme potentiellement impliqués dans la même cascade de régulation qu’Anthox2.
Perspectives concernant les cnidaires en tant que modèles de développement et d’évolution
Les techniques en biologie du développement utilisées chez les cnidaires reposent sur l’inhibition des gènes par le biais de l’ARN interférence ou d’injection de morpholinos, depuis peu sur la transgénèse chez l’hydre. Le travail présenté au chapitre 2 (II.2.) ouvre de nouvelles possibilités pour l’étude de la régulation des gènes grâce à l’injection de gènes rapporteurs et représente un premier pas vers l’anémone transgénique.
Selon nous une importance primordiale doit être apportée à trois volets en particulier, pour répondre plus clairement aux questions du type de celles traitées dans cette thèse :
1) Les études descriptives des types cellulaires et de la morphogénèse font défaut en particulier chez Nematostella. Des études du type de celles menées par l’équipe d’Uli Technau sur le développement embryonnaire en microscopie électronique (Kraus and Technau, 2006; Fritzenwanker et al., 2007) ou sur la description du système nerveux de Nematostella dans l’équipe de Mark Martindale (Marlow et al., 2009) sont d’une très grande utilité et méritent d’être complétées.
2) Au niveau qualitatif il faudrait davantage développer les approches à grande échelle comme les puces à ADN avec ou sans l’immunoprécipitation de la chromatine pour dégager des profils d’expression et des modes de régulation relatifs à des conditions spécifiques chez les cnidaires. L’accumulation de ce type d’analyses ainsi que des études fonctionnelles de gènes ciblés faites chez les bilatériens, chez qui les outils d’étude génétique sont mieux développés, donne déjà un répertoire de données très important sur les mécanismes de régulation du développement et les cascades génétiques qui les gouvernent. Ceci représente une base de réflexion importante sur l’évolution de ces processus. Plus il sera possible de confronter des données de ce type entre les cnidaires et les bilatériens ou entre les grands branchements du règne animal représentant des étapes fondamentales en matière d’innovations morphologiques, plus les scénarios dans ce domaine se développeront et seront pertinents.
160 3) Par ailleurs il est désormais primordial de privilégier les études fonctionnelles chez les cnidaires pour approfondir les rôles du nombre important de gènes rapportés ces dernières années depuis le séquençage des génomes d’Hydra magnipapillata et de Nematostella, qui sont conservés et régulateurs du développement chez les bilatériens. Les approches à grande échelle sont un moyen de présélectionner les gènes actifs dans des processus d’intérêts et les études fonctionnelles sont un moyen de raffiner leur étude. Le développement des techniques permettant la perte ou le gain de fonction ou encore la dissection des régions régulatrices des gènes, d’une manière transitoire (injection de morpholinos, d’ARN messager, de gènes rapporteurs ou de transgènes permettant la surexpression) ou stable (transgénèse) est essentiel à la compréhension des processus de développement comme la neurogénèse et à la clarification de leur histoire évolutive.
L’émergence des études fonctionnelles chez les cnidaires ainsi que le développement des approches à grande échelle visant à décrypter les transcriptomes dans de multiples espèces représentant tous les phyla animaux et dans de multiples conditions nous placent au début de la compréhension des processus de développement ancestraux qui agissent chez les cnidaires.
161
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174 ANNEXES
Cartes des plasmides / Plasmid maps pGEM-T easy vector clones 177 1.1. Nv-Ax2gDNA-F1/R1 – Ax2-1131bp 179 1.2. Nv-Ax2gDNAsh – Ax2-585bp 181 1.3. NvAx2gDNApromxNhe1F2/R3 183 1.4. NvAx2gDNApromxEcoRVF6/R5 185 1.5. NvAx2gDNApromxNhe1IIF7/R6 187 1.6. Nv-Ax2gDNApromF8/R7 – Ax2-3127bp 189 1.7. Nv-Ax2gDNApromF8/R1 – Ax2-4300bp 191 1.8. Nv-Ax2cDNA-F1/R1 – Ax2-955bp 193 1.9. Nv-Ax2cDNApromF10/R1 – Ax2-1025bp 195 1.10. Nv-Ax2cDNA-F1/R1xSph1 – Ax2-385bp 199 1.11. Xma1-Ax2CDs-FLAG-Nar1 201 1.12. Nv-ActingDNA-AauactinF/R - Actin-342bp 203 1.13. Nv-SoxB2cDNAF1/R1 – SoxB2-817bp 205 1.14. Nv-Sox2cDNAF1/R1 – Sox2-705bp 207 1.15. Nv-FHcDNAF1/R1 – FH-848bp 209 1.16. Ch-GsxcDNAF1/R1 – Gsx-867bp 211 1.17. Ch-GsxgDNAF1/R1 – Gsx-1335bp 213 1.18. Ch-GsxgDNApromEcoRVF2/R2 215 1.19. Ch-PdxgDNAF3/R2 – Pdx-197bp 217 1.20. Ch-PdxgDNAF3/R3 – Pdx-143bp 219 1.21. Ch-CnoxCcDNAlong – CnoxC-657bp 221 1.22. Ch-CnoxCcDNAsh – CnoxC-384bp 223 1.23. Td-PdxcDNAF5/R4 – Pdx-298bp 225 1.24. Td-PdxcDNAF4/R4 – Pdx-324bp 227 1.25. Td-CnoxAcDNATH5F3/TH5R6 – CnoxA-545bp 229 1.26. Hv-Cnox2gDNACx2Hv-Xmn5’/Cx2Hv-16HB3 – Cnox2-1139bp 231
Reporter constructs: HyactEGFP derived 233 2.1. HyactEGFP-EcoR1 235 2.2. HyactEGFP-Sce1 237 2.3. NvAx2p(-3000)EGFP 239 2.4. EmptyEGFP 243
Reporter constructs: CMVDsRed2 derived 245 3.1. CMVDsRed2 247 3.2. CMVEGFP 251 3.3. NvAx2p(-3000)DsRed2 255 3.4. NvAx2p(-2000)DsRed2 259 3.5. NvAx2p(-1000)DsRed2 263 3.6. CMVAx2CDsFLAG 267 3.7. Ax2(-3000)Ax2CDsFLAG 271
175 The sequences reported correspond to the insert but the restriction sites are indicated for the full vector (except for the reporter constructs derived from HyactEGFP as the backbone sequence is not clear)
176 pGEM-T easy vector clones
XXX: p-GEMT easy vector XXX: forward primer XXX: reverse primer XXX: intron UPPERCASE: coding sequences Lowercase: non coding sequence XXX: START and STOP codons
177
178 1.1. Nv-Ax2gDNA-F1/R1 – Ax2-1131bp
Vector: pGEM-T easy vector Template: NvgDNA Primers: Ax2 F1/R1 PCR product: PCR 85 n°10 Insert length: 1131 bp Plasmid: 3 – retransformation - 9 Orientation: antisens Microsynth: 475020 (T7) Comments: does not contain the ATG
179 INSERT - Reverse complement
SP6gtgattaagtcggtaccaacgagtacttcagagccgcgaCACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGA CCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTAGAGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGAC TCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACAACACTACGCTGCGACAGAGGAGGAGAGACTTCATCCTGCGAGTTATGgtgaggtg cgctttccacgccaaaacaaggcttcaaggaccctaacaggtgtccgacactaaagctctgtcaaaaatatccatgatgtcctttgaaaaaatccatt gaacttaatggtgaaatataattattatttccttaattctgtgtttaatagaattttttgtcttttcttagCATCATCACGAGATCCTGACAGTCCAT CAAGGGGAGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAGCATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTT TCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAGCAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGA CAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCACCGGTAGAATGGATGGTGTATGAacactaaaat tgaaccataattgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcataatttgtatagaatctatagtttggcgaacgaa ctgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgagggataaattgtaaaacaaaaacaatttaaaagcct taaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaacttttctgagaattttaccatgcatttgtataaaacggcaa gagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaaatcgaT7
Retriction sites full clone:
Unique:
Aat2 Acc65 Age1 Ahd1 AlwN1 Apa1 BfuA1 Bgl2 Bpm1 Bsa1 BseY1 BspLU BspM1 BsrG1 BstB1 BstX1 Bsu36 BtgZ1 Cla1 Dra3 Ecl2 EcoK EcoRV Hind3 Kpn1 Nco1 Nde1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 T7Ter Xho1
Not found:
Aar1 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Blp1 BmgB1 Bpu10 BsaB1 Bsg1 BsiW1 BsmB1 BspE1 BssH2 BstAP BstE2 BstZ1 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1
180 1.2. Nv-Ax2gDNAsh – Ax2-585bp
Vector: pGEM-T easy vector Template: Nv-Ax2gDNA-F1/R1 – Ax2-1131bp x SalI + Xho1 Self ligation Insert length: 585 bp Plasmid: 6 Orientation: antisens José: 27722 (SP6) Comments: made for and used in ISH
181 INSERT - Reverse complement:
SP6catatgGTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAA GCAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTT CGTCGCCAGCAAGCACCGGTAGAATGGATGGTGTATGAacactaaaattgaaccataattgtacagtttgtatatagtttaatgtactatattcgggg caaccttgttttcataatttgtatagaatctatagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccattt cgaagtttgtgagggataaattgtaaaacaaaaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttc gtataaacttttctgagaattttaccatgcatttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaat aaaagcttgtcaaatcgaT7
Retriction sites full clone:
Unique:
Aat2 Age1 Ahd1 AlwN1 Apa1 Bgl2 Bpm1 Bsa1 BseY1 Bsm1 BspLU BsrG1 BstB1 BstX1 BtgZ1 Dra3 Ecl2 EcoK EcoR1 EcoRV Hind3 Nco1 Nde1 Not1 Psi1 PspOM Sac1 Sac2 Sap1 Sca1 Sph1 T7Ter
Not found:
Aar1 Acc65 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 BfuA1 Blp1 BmgB1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 BsmB1 BspE1 BspM1 BssH2 BstAP BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 Pst1 R4atB R4atL R4atP R4atR Rsr2 Sal1 SanD1 Sbf1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Spe1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1 Xho1
182 1.3. NvAx2gDNApromxNhe1F2/R3
Vector: pGEM-T easy vector Template: NvgDNA x Nhe1 Primers: Ax2 F2/R3 PCR product: PCR 157 n°1&2 Insert length: 1774 bp Seq unknown: 1639bp Plasmid: pl7 Orientation: antisens José: 31313 (SP6), Microsynth: 534521 (T7), 535459 (Ax2NvF5) Comments: iPCR clone
183 INSERT - Reverse complement: XXX: already known
SP6gtgattCACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCTTATGCCAGCTCTGCATTCCTAC TAGTGCTAGCgtattttgttcgagttcgttaaaattatgtttccggaaagattagagaaacaattattatagtaaatacaggccgttacaatttgata tctttaattgttatctagccgttgtcgtccttttcatttagattagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattcggaacgaa aaatacttgagaatccttttttatatatattctttgagatactaagagattagatcaatcagaagagttaatgaactggtaggttaagaagtttattt tgctcaagttcctgtgatcaagaaatgccgcaaaaatagttgattcttgtttttgttaatgaacgattcattcgtatctttgacgggttaaagttccc cactgggcggcacctaatcttgtgtaactaacaagatagagagcctacgtggtgtaaagatttacctgctacgcacaaactaagcgcatgtaatgtgt tcaccaagacgagaggcattcaatattttatcgctcagttgacgtacaatcgcttcaattgccccccagtgttcactaactctactgttttcttcacg caaaacgcaaaacgatcttttaatattttcctagaaaataattgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcgccggcaaat taaaggtgtttattccatttttgaacagggggtaaaaagaatacctattctccttcggtctagtcgccaggctatttatttttcttgttatatgatta aaacatatttcacattttggaattggggaacaattacccatgacgcgtgcagaacggccattctaacgtcctggaaaagcctaattaacgctctcaat gccataaagctcaatttgaaaaattagtttattacatgaatcctaaacaaataagttccgaaagaaaacttgcaggtttccaaaaacagtttacattg attaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaagaattatggtctacatctggaagtttggaattattggggagagaattggg gagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgagaagacgtcgattgcttacacaggaagcgtggcgcgcattgtagcaa aagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgacaattacgggcgaaaaaaggccgagggattgttaacaattcaaat cggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttatactcaaggggaaaaataattctgtgaaacaggaccaatgaaa tttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaatctccttaaaacatgtcaactacctgtatcagtaattaac cttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttgtgttgcgaacaataggcgacgtcttagacacgtgtgct ctatccagggtggtcaatatatagagccctgcatctgaacccggcagtagaacactcgcagctcccagacggcacacatctcctgatatcATGTCTTC GTCCTTCTACATTGACTCGaatcgaT7
Retriction sites full clone:
Unique:
Aar1 Ahd1 AlwN1 Bcg1a Bcg1b BfrB1 Bpm1 Bsa1 BseY1 Bsg1 BspE1 BssH2 BstX1 BtgZ1 Dra3 Ecl2 EcoK Hind3 Mfe1 Nco1 Nde1 Nhe1 Nsi1 Pml1 Psi1 Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Sph1 Xho1 Xmn1
Not found:
Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsaB1 BseR1 BsiW1 BsmB1 BsrG1 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 I_Ceu Kas1 Kpn1 loxP Msc1 Nar1 Nru1 Pac1 PflF1 PflM1 Pme1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1
184 1.4. NvAx2gDNApromxEcoRVF6/R5
Vector: pGEM-T easy vector Template: NvgDNA x EcoRV Primers: Ax2 F6/R5 PCR product: PCR 159 n°1 Insert length: 532 bp Seq unknown: 458bp Plasmid: pl6 Orientation: sens Microsynth: 541956 (T7) Comments: iPCR clone
185 INSERT: XXX: already known
T7tcgattcaattattatagtaaatacaggccgttacaatttgatatcaaaacgaatcgaaataactttgattttcgatcgtttgtgaggatgatttg tcttttcgttcataaccttcgttcgtttttacggttaatgttcttgacatttttgaataaatgttcttgtattcaatctagttctcaaaacaccaggc ttttatatttattgaagaggctgttcctatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaactccg tgataaaattcagaattagatcatttgctatcggcatataaggaactaaatttaccaggcaatcttttgataaatattacccgatgagaattgtttat tttgtctgcgaaagcaaatcaaagagctgacttctgtatgaagcctatttaccttcaacattcatttcatttccttagtcaagtatttgagaggaaga aacttcggctagcgtattttgttcgagttcgttaaaattatgtttccggaaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 Bcg1a Bcg1b BfrB1 BfuA1 Bpm1 Bsa1 BseY1 BspE1 BspLU BspM1 BstX1 BtgZ1 Dra3 Ecl2 EcoK EcoRV Mlu1 Nae1 Nco1 Nde1 NgoM4 Nhe1 Nsi1 Pme1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 Bsm1 BsmB1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nru1 Pac1 PflF1 PflM1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
186 1.5. NvAx2gDNApromxNhe1IIF7/R6
Vector: pGEM-T easy vector Template: NvgDNA x Nhe1 Primers: Ax2 F7/R6 PCR product: PCR 168 n°1 Insert length: 1477 bp Seq unknown: 1018bp Plasmid: pl6II Orientation: sens Microsynth: 551301 (T7), 551850 (SP6) Comments: iPCR clone
187 INSERT: XXX: already known
T7tcgattgtcttttcgttcataaccttcgttcgtttttacggttaatgttcttgacatttttgaataaatgttcttgtattcaatctagttctcaaa acaccaggcttttatatttattgaagaggctgttcctatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaa ttaactccgtgataaaattcagaattagatcatttgctatcggcatataaggaactaaatttaccaggcaatcttttgataaatattacccgatgaga attgtttattttgtctgcgaaagcaaatcaaagagctgacttctgtatgaagcctatttaccttcaacattcatttcatttccttagtcaagtatttg agaggaagaaacttcggctagcgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtactttgattttctggt gagtcgttttgttttcggtcgccacgtctatgtcccgcagttctttttgtactaccgtcacaaggccctcaaaaatacctgtgaacataaaagccaaa cacaacagcctcaaagtgagcaaaaaacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaactgcaggatcaca aaaagtgtttcatgtttccttttctctcgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggcttgcgacgggt ttgcggaggggcgaatacgctcctggttattgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaatttcaggccaat taaattaattttccatatgtcagagaccttgccatcttactaggacttgatcaatcgttttttttttttctttttggtcaattgtgtttacgctgtta actcgatcatgactttaattaagtcgcgttcgcaagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctattcgcgcgcgattcaaggc tcattggtaatcttttgagctttgacttgcaagactaaatgagctccaactaggtactaaagctttagagatgtttcccgtggaattaaagaggcaca agtttgctttagtgttaccggggtaacttctaagaagaattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgcttgaattttt tgtactgttgttattttttccatactaaaatcgtacatcttctattttacgccttgtttttagtactttgaaaacctttttcacaaataagtcagatt gtttaagagaaatacattccagaagtttaaagataatgtatattgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttgattttcgatc gtttgtgaggatgataatcacSP6
Retriction sites full clone:
Unique:
Aat2 Afe1 Age1 Ahd1 AlwN1 Apa1 Bbs1 BfrB1 BfuA1 Blp1 BmgB1 Bpm1 BsaB1 BseY1 Bsm1 BspLU BspM1 BssH2 BstX1 BtgZ1 Dra3 EcoK EcoRV Hind3 Hpa1 Mfe1 Mlu1 Nae1 Nco1 NgoM4 Nhe1 Nsi1 Pac1 Pml1 Psi1 PspOM Sac2 Sal1 Sap1 Sbf1 Spe1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Bgl2 Bpu10 BseR1 Bsg1 BsiW1 BsmB1 BspE1 BsrG1 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 I_Ceu Kas1 Kpn1 loxP Msc1 Nar1 Nru1 PflF1 PflM1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
188 1.6. Nv-Ax2gDNApromF8/R7 – Ax2-3127bp
Vector: pGEM-T easy vector Template: NvgDNA Primers: Ax2 F8/R7 PCR product: PCR 186 n°2 Insert length: 3127 bp Plasmid: pl6 Orientation: antisens Microsynth: 566874 (SP6), 566873 (T7), 570465 (Ax2F9), 574771 (Ax2F6), José: 37024 (Ax2F7) Comments: amplified with the long expand PCR, Ax2 upstream sequences, does not contain the ATG
189 INSERT - Reverse complement:
SP6gtgattgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtattttgattttctggtgagtcgttttgtt ttcggtcgccacgtctatgtcccgcagttctttttgtactaccgtcacaaggccctcaaaaatacctgtgaacataaaagccaaacacaacagcctca aagtgagcaaaaaacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaactgcaggatcacaaaaagtgtttcat gtttccttttctctcgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggcttgcgacgggtttgcggaggggcg aatacgctcctggttattgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaatttcaggccaattaaattaattttc catatgtcagagaccttgccatcttactaggacttgatcaatcgttttttttttttctttttggtcaattgtgtttacgctgttaactcgatcatgac tttaattaagtcgcgttcgcaagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctatttgcgcgcgattcaaggctcattggtaatct tttgagctttgacttgcaagactaaatgagctccaactaggtactaaagctttagagatgtttcccgtggaattaaagaggcataagtttgctttagt gttaccggggtaacttctaagaagaattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgcttgaattttttgtactgttgtta ttttttccatactaaaatcgtacatcttctgttttacgccttgtttttagtactttgaaaacctttttcacaaataagtcagattgtttaagagaaat acattccagaagtttaaagataatgtatattgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttgattttcgatcgtttgtgaggatg atttgtcttttcgttcataaccttcgttcgtttttacggttaatgttcttgacatttttgaataaatgttcttgtattcaatctagttctcaaaacac caggcttttatatttattgaagaggctgttcctatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaa ctccgtgataaaattcagaattagatcatttgctatcggcatataaggaactaaatttaccaggcaatcttttgataaatattacccgatgagaattg tttattttgtctgcgaaagcaaatcaaagagctgacttctgtatgaagcctatttaccttcaacattcatttcatttccttagtcaagtatttgagag gaagaaacttcggctagcgtattttgttcgagttcgttaaaattatgtttccggaaagattagagaaacaattattatagtaaatacaggccgttaca atttgatatctttaattgttatctagccgttgtagtccttttcatttagattagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattc ggaacgaaaaatacttgagaatccttttttatatatattctttgagatactaagagattagatcaatcagaagagttaatgaactggtaggttaagaa gtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaatagttgattcttgtttttgttaatgaacgattcattcgtatctttgacgggtta aagttccccactgggcggcacctaatcttgtgtaactaacaagatagtgaacctacgtggtgtcatgatttacctgctacgcacaaactaagcgcatg taatgtgttcaccaagacgagaggcattcaatattttatcgctcagttgacgtacaatcgcttcaattgccccccagcgttcactaactctactgttt tcttcacgcaaaacgcaaacacgatcttttaatattttcctagaaaataattgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcg ccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaatacctattctccttcggtctagtcgccaggctatttatttttcttgtt atatgattaaaacatatttcacattttggaattgggaaacaattacccatgacgcgtgcagaacggccattctaacgtcctggaaagcctaattaacg ctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaacaaataagttccgaaagaaaacttgcaggtttccaaaaacagt ttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaagaattatggtctacatctggaagtttggaattattggggaga gaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgagaagacgtcgattgcttacacaggaagcgtggcgcgcat tgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgacaattacggacgaaaaaaggccgagggattgttaaca attcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttatactcaagggggaaaataattctgtgaaacaggac caatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaatctccttaaaacatgtcaactacctgtatcag taattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttgtgttgcgaacaataggcgacgtcttagaca cgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcgcagctcccagacggcacacatctcctgatata atcgaT7
Retriction sites full clone:
Unique:
Aar1 Age1 Ahd1 AlwN1 BfrB1 Blp1 BmgB1 Bpm1 BsaB1 Bsg1 BspE1 BstX1 BtgZ1 Dra3 EcoK Nco1 Nhe1 Nsi1 Pac1 Psi1 Sac2 Sal1 Sap1 Sbf1 Spe1 Sph1 Xho1 Xmn1
Not found:
Acc65 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Bgl2 Bpu10 BseR1 BsiW1 BsmB1 BsrG1 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 I_Ceu Kas1 Kpn1 loxP Msc1 Nar1 Nru1 PflF1 PflM1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1
190 1.7. Nv-Ax2gDNApromF8/R1 – Ax2-4300bp
Vector: pGEM-T easy vector Template: NvgDNA Primers: Ax2 F8/R1 PCR product: PCR 289 n°1 Long expand template PCR Insert length: 4300 bp Plasmid: pl5 Orientation: antisens Microsynth: 798749 (T7), fasteris: BS-046 (SP6), OG-001 (Ax2F9), OG- 002 (Ax2F6) Comments: amplified with the long expand PCR
191 INSERT - Reverse complement:
SP6gtgattgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtattttgattttctggtgagtcgttttgtt ttcggtcgccacgtctatgtcccgcagttctttttgtactaccgtcacaaggccctcaaaaatacctgtgaacataaaagccaaacacaacagcctca aagtgagcaaaaaacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaactgcaggatcacaaaaagtgtttcat gtttccttttctctcgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggcttgcgacgggtttgcggaggggcg aatacgctcctggttattgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaatttcaggccaattaaattaattttc catatgtcagagaccttgccatcttactaggacttgatcaatcgttttttttttttctttttggtcaattgtgtttacgctgttaactcgatcatgac tttaattaagtcgcgttcgcaagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctatttgcgcgcgattcaaggctcattggtaatct tttgagctttgacttgcaagactaaatgagctccaactaggtactaaagctttagagatgtttcccgtggaattaaagaggcataagtttgctttagt gttaccggggtaacttctaagaagaattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgcttgaattttttgtactgttgtta ttttttccatactaaaatcgtacatcttctgttttacgccttgtttttagtactttgaaaacctttttcacaaataagtcagattgtttaagagaaat acattccagaagtttaaagataatgtatattgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttgattttcgatcgtttgtgaggatg atttgtcttttcgttcataaccttcgttcgtttttacggttaatgttcttgacatttttgaataaatgttcttgtattcaatctagttctcaaaacac caggcttttatatttattgaagaggctgttcctatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaa ctccgtgataaaattcagaattagatcatttgctatcggcatataaggaactaaatttaccaggcaatcttttgataaatattacccgatgagaattg tttattttgtctgcgaaagcaaatcaaagagctgacttctgtatgaagcctatttaccttcaacattcatttcatttccttagtcaagtatttgagag gaagaaacttcggctagcgtattttgttcgagttcgttaaaattatgtttccggaaagattagagaaacaattattatagtaaatacaggccgttaca atttgatatctttaattgttatctagccgttgtagtccttttcatttagattagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattc ggaacgaaaaatacttgagaatccttttttatatatattctttgagatactaagagattagatcaatcagaagagttaatgaactggtaggttaagaa gtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaatagttgattcttgtttttgttaatgaacgattcattcgtatctttgacgggtta aagttccccactgggcggcacctaatcttgtgtaactaacaagatagtgaacctacgtggtgtcatgatttacctgctacgcacaaactaagcgcatg taatgtgttcaccaagacgagaggcattcaatattttatcgctcagttgacgtacaatcgcttcaattgccccccagcgttcactaactctactgttt tcttcacgcaaaacgcaaacacgatcttttaatattttcctagaaaataattgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcg ccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaatacctattctccttcggtctagtcgccaggctatttatttttcttgtt atatgattaaaacatatttcacattttggaattgggaaacaattacccatgacgcgtgcagaacggccattctaacgtcctggaaagcctaattaacg ctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaacaaataagttccgaaagaaaacttgcaggtttccaaaaacagt ttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaagaattatggtctacatctggaagtttggaattattggggaga gaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgagaagacgtcgattgcttacacaggaagcgtggcgcgcat tgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgacaattacggacgaaaaaaggccgagggattgttaaca attcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttatactcaagggggaaaataattctgtgaaacaggac caatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaatctccttaaaacatgtcaactacctgtatcag taattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttgtgttgcgaacaataggcgacgtcttagaca cgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcgcagctcccagacggcacacatctcctgatatc ATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGTCGGTACCAACGAGTACTTCAGAGCCGCGACACACTTACGAATCTCCTGTTCC TTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTAGCGTGCACCCGTATATGCATCATG TGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACAACACTACGCTGCGACAGAGGAG GAGAGACTTCATCTTGCGAGTTATGgtgagttgcgctttccacgccaaaacaaggcttcaaggaccctaacaggtgtccgacactaaagctctgtcaa aaatatccatgatgtcctttgaaaaaatccattgaacttaatggtgaaatataattattatttccttaattctgtgtttaatagaattttttgtcttt ttttagCATCATCACGAGATCCTGACAGTCCATCAAGGGGAGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAGCATGCAACTACTCGAG CTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAGCAAGTGAAAATCTG GTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCA CCGGTAGAATGGATGGTGTATGAacactaaaattgaaccataattgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcat aatttgtatagaatctatagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgaggg ataaattgtaaaacaaaaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaacttttctg agaattttaccatgcatttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaaat cgaT7
Retriction sites full clone:
Unique:
Aar1 Acc65 Ahd1 AlwN1 Bgl2 Blp1 BmgB1 Bpm1 Bsg1 BspE1 BsrG1 BstB1 BstX1 Bsu36 BtgZ1 Cla1 Dra3 EcoK Kpn1 Nco1 Pac1 Psi1 Sac2 Sal1 Sap1 Sbf1 T7Ter
Not found:
Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Bpu10 BsaB1 BsiW1 BsmB1 BstAP BstE2 BstZ1 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 I_Ceu Kas1 loxP Msc1 Nar1 Nru1 PflF1 PflM1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1
192 1.8. Nv-Ax2cDNA-F1/R1 – Ax2-955bp
Vector: pGEM-T easy vector Template: NvcDNA6d Primers: Ax2 F1/R1 PCR product: PCR 137 n°5 Insert length: 955 bp Plasmid: 14 Orientation: antisens José: 29810 (2005), microsynth: 725550 (2007) Comments: used in ISH, does not contain the ATG
193 INSERT - Reverse complement
SP6tagtgattAAGTCGGTACCAACGAGTACTTCAGAGCCGCGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCT GACCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTAGCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGG ACTCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACAACACTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTCATGCATCAT CACGAGATCCTGACAGTCCATCAAGGGGAGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAGCATGCAACTACTCGAGCTTGAGAAAGAG TTCAGTCAAAACAGATATCTTTCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTCCCGAGAAGCAAGTGAAAATCTGGTTCCAAAACCG ACGCGTTAAATGGAAGAAGGACAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCACCGGTAGAATGG ATGGTGTATGAacactaaaattgaaccataattgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcataatttgtataga atctatagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgagggataaattgtaaa acaaaaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaacttttctgagaattttacca tgcatttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaaatcgaT7
Retriction sites full clone:
Unique:
Aat2 Acc65 Age1 Ahd1 AlwN1 Apa1 BfuA1 Bgl2 Bpm1 Bsa1 BseY1 BspLU BspM1 BsrG1 BstB1 BstX1 Bsu36 BtgZ1 Cla1 Dra3 Ecl2 EcoK EcoRV Hind3 Kpn1 Nco1 Nde1 Nhe1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 T7Ter Xho1
Not found:
Aar1 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Blp1 BmgB1 Bpu10 BsaB1 Bsg1 BsiW1 BsmB1 BspE1 BssH2 BstAP BstE2 BstZ1 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 loxP Mfe1 Msc1 Nar1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1
194 1.9. Nv-Ax2cDNApromF10/R1 – Ax2-1025bp
Vector: pGEM-T easy vector Template: NvcDNA8d Primers: Ax2 F10/R1 PCR product: PCR 279 n°10 Insert length: 1025 bp Plasmid: 17 Plamid: 48 Orientation: sens Orientation: antisens Fasteris: BS-037 (T7), BS-038 (SP6) Microsynth: 750972 (T7), 76750 (SP6) Comments: made for in vitro translation to test Cnox2 hydra antibody, contains the ATG
195
196
INSERT:
T7tcgattcccagacggcacacatctcctgatatcATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGTCGGTACCAACGAGTACT TCAGAGCCGCGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTATGCCAGCTCTGCATTCC TACTAGTGCTAGCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGAACTGCAGAAAGATCATA TTTTGCTGCAACAACACTACGCTGCGACAGAGGAGGGGAGACTTCATCTTGCGAGTTATGCACCATCACGAGATCCTGACAGTCCATCAAGGGGAGGA AATTCACGGTCAAAGCGGATCAGAACGGCATACACCAGCATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCCTTCG CCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAGCAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAAGCAG CGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCACCGGTAGAATGGATGGTGTATGAacactaaaattgaaccataat tgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcataatttgtatagaatctatagtttggcgaacgaactgtgatcgcc caatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgagggataaattgtaaaacaaaaacaatttaaaagccttaaatggaaag gcgggggggatatacacaaaaaaaattgcatgtaaattttcgtataaacttttctgagaattttaccatgcatttgtataaaacggcaagagatttgc cggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Acc65 Age1 Ahd1 AlwN1 Apa1 Bbs1 BfuA1 Bgl2 Bpm1 Bsa1 BseR1 BseY1 BspLU BspM1 BsrG1 BstB1 BstX1 Bsu36 BtgZ1 Cla1 Dra3 Ecl2 EcoK Hind3 Kpn1 Nco1 Nde1 Nhe1 Psi1 PspOM Sac1 Sac2 Sal1 Sap1 Sbf1 T7Ter Xho1
Not found:
Aar1 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Blp1 BmgB1 Bpu10 BsaB1 Bsg1 BsiW1 BsmB1 BspE1 BssH2 BstAP BstE2 BstZ1 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 loxP Mfe1 Msc1 Nar1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1
197 198 1.10. Nv-Ax2cDNA-F1/R1xSph1 – Ax2-385bp
Vector: pGEM-T easy vector Template: p-NvAx2cDNAF1/R1 - Ax2-955bp x Sph1 Self-ligation Insert length: 358 bp Plasmid: 10 Orientation: antisens Microsynth: 727938 (T7) Comments: made for and used in ISH
199 INSERT - Reverse complement:
SP6gtgattAAGTCGGTACCAACGAGTACTTCAGAGCCGCGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGA CCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTAGCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGAC TCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACAACACTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTCATGCATCATCA CGAGATCCTGACAGTCCATCAAGGGGAGGAAATTCACGGTCAAAGCGGANCAGAACGGCATACACCAGCATGCGACNCGGNNT7
Retriction sites full clone:
Unique:
Aat2 Acc65 Ahd1 AlwN1 Apa1 Bcg1a Bcg1b BfuA1 Bpm1 Bsa1 BseY1 Bsm1 BspLU BspM1 BstX1 Bsu36 BtgZ1 Cla1 Dra3 Ecl2 EcoK Kpn1 Mlu1 Nae1 Nco1 Nde1 NgoM4 Nhe1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Xmn1
Not found:
Aar1 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsaB1 Bsg1 BsiW1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 BxatB BxatL BxatR BxatP _Chi EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 loxP Mfe1 Msc1 Nar1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
200 1.11. Xma1-Ax2CDs-FLAG-Nar1
Vector: pGEM-T easy vector Template: p-Nv-8dcDNA-Ax2F10/R1-ATG pl48 Primers: PCR306: A: 01- Ax2NvF12-Xma1 / 03- Ax2NvR10-FLAG, B: 02- Ax2NvF13-FLAG / 04- Ax2NvR11-Nar1, PCR 306-03 & 04 purif PCR307: 01 306-3 + 306-7 purif x10 02 Ax2NvF12-Xma1 / 04- Ax2NvR11-Nar1 x25 PCR product: PCR 307 n°4 Insert length: 1037 bp Plasmid: 12 Orientation: sens Fasteris: OG12 (T7), OG13 (SP6) Comments:
201 INSERT:
T7tcgattcccgggccaccATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGTCGGTACCAACGAGTACTTCAGAGCCGCGACACA CTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTAGCGTG CACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACAACA CTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTTATGCATCATCACGAGATCCTGACAGTCCATCAAGGGGAGGAAATTCACGGTCAAAGC GGATCAGAACGGCATACACCAGCATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCCTTCGCCGCATTCAAATCGCC GCTTTGCTAGATCTTTCCGAGAAGCAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAAGCAGCGCAACATGGCACAAC AACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCACCGGTAGAATGGATGGTGTAGACTACAAGGACGACGATGACAAGTGAacactaaaattga accataattgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcataatttgtatagaatctatagtttggcgaacgaactg tgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgagggataaattgtaaaacaaaaacaatttaaaagccttaa atggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaacttttctgagaattttaccatgcatttgtataaaacggcaagag atttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaggcgccaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Acc65 Age1 Ahd1 AlwN1 Apa1 Bbs1 BfuA1 Bgl2 Bpm1 Bsa1 BseY1 BspLU BspM1 BsrG1 BstB1 BstX1 Bsu36 BtgZ1 Cla1 Dra3 Ecl2 EcoK EcoRV Hind3 Kas1 Kpn1 Nar1 Nco1 Nde1 Nhe1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sma1 T7Ter Xho1
Not found:
Aar1 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Blp1 BmgB1 Bpu10 BsaB1 Bsg1 BsiW1 BsmB1 BspE1 BssH2 BstAP BstE2 BstZ1 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu loxP Mfe1 Msc1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1
202 1.12. Nv-ActingDNA-AauactinF/R - Actin-342bp
Vector: pGEM-T easy vector Template: NvgDNA Primers: AauactinF/R PCR product: PCR 72 n°5-7 Insert length: 342 bp Plasmid: A2 Orientation: sens Microsynth: 471757 (T7) Comments:
203 INSERT:
T7tcgattGGTCATCACCATAGGAAACGAGCGCTTCAGGTGCCCCGAGGCTATGTTCCAGCCTTCCTTCCTGGGTATGGAATCCGCTGGTATCCATGA GACCACCTACAACTCCATCATGAAGTGCGACGTCGACATCCGTAAAGATTTGTATGCTAACACTGTCCTGTCTGGAGGCTCAACCATGTACCCAGGCA TCGCCGACCGCATGCAAAAAGAAATTACTTCCCTCGCTCCCCCGACCATGAAAATCAAGATCATCGCTCCACCAGAGAGGAAATACTCCGTCTGGATC GGAGGCTCCATCCTCGCTTCCCTGTCCACCTTCCAACAGATGTGGATCTTCAAAaatcacSP6
Retriction sites full clone:
Unique:
Afe1 Ahd1 AlwN1 Apa1 Bcg1a Bcg1b BfrB1 BfuA1 BseY1 BspLU BspM1 BstX1 Dra3 Ecl2 EcoK Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sap1 Sbf1 Sca1 Spe1 T7Ter Xmn1
Not found:
Aar1 Acc65 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 Bsm1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1 Xho1
204 1.13. Nv-SoxB2cDNAF1/R1 – SoxB2-817bp
Vector: pGEM-T easy vector Template: NvcDNA6d Primers: NvSoxB2F1/R1 PCR product: PCR 191 n°5 Insert length: 817bp Plasmid: 21 Orientation: antisens Microsynth: 605775 (T7) Comments:
205 INSERT - Reverse complement:
SP6gtgattATGGGCAAGCAAGAAGACGGGCACGTGAAGAAGACCGATGAATGCGTTCATGGTTTTGGTCACGAGGCAAGAGAAAGCATTTACGCCAG TATTAATCCACGAATGCACAACTCTGAGATCAGCAAGAGGTTAGGAGCGGAGTGGAAAATGTTGACGGGCTGAAGAGAAGGAGCCGTTTATAGCGGAG GCAAAGCGCCTGCAAGCACTTCACATCCAGGAGCACCCGGACTATAAGTACAAACCCGAACGGCGCAAACCCAAATCAGTGCAGAAGAAAGACCTCGC TTCCCCTGTCTTCTCCCCGTACGCTGCATCTATGATGGCGGTTGACAAATTCTCGACGAATCAATTGCCACAAACAATAGCTCATTCAGTTGCTTTGT CCCAGGATCCAATGTATTCGAAAATAAATGGTGGACCGCCGTTTCATCACTCCGTGTCGCCGGGTTACCCCGTGATATACCCGAGTGTCAGTAATGGA GGGAATGTGCACTCAGGCTCGCCTTCTTCCCGCCAAATCTTCGCCGGCGCCATGGACAGTACTCACTCGTTTCGCGCGAGTGATATGATGGCGGGTCG ACCAGTGTATAGCAGTCAGGGCTACCAGGGGGCTCTCCACTCGCAGGTCCAGCAGCGACTATCGCAAGTGGAGGATAGCAGAGGTATGAAGTCCATGA ACGCGACTCCGAGTCCACCCGTGTCAAGTCCCGACCCCATGTCTAAAGCGTACTCGAGCTCCGAGCTGAGTAACCAACGAGTGTGGCAGCCACAGCAG GACCTAACCAGACCCGTCGCTTACGTACCGGTATTGCTCTAGaatcgaT7
Retriction sites full clone:
Unique:
Aat2 Age1 AlwN1 Apa1 BamH1 BfrB1 Bpm1 Bsa1 BseY1 Bsg1 BsiW1 BspLU BstB1 BstE2 BstX1 BtgZ1 EcoK Kas1 Mfe1 Mlu1 Nar1 Nde1 Nsi1 Pml1 Psi1 PspOM Pst1 Sac2 Sap1 Sbf1 SgrA1 SnaB1 Spe1 Sph1 Xba1 Xho1
Not found:
Aar1 Acc65 Afe1 Afl2 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsaB1 BseR1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kpn1 loxP Msc1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 Sma1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xcm1
206 1.14. Nv-Sox2cDNAF1/R1 – Sox2-705bp
Vector: pGEM-T easy vector Template: NvcDNA6d Primers: NvSox2F1/R1 PCR product: PCR 191 n°3 Insert length: 705bp Plasmid: 7 Orientation: sens Microsynth: 605774 (T7) Comments:
207 INSERT:
T7tcgattATGACCAAACCAGGAGATCATATTAAACGGCCCATGAACGCGTATATGGTATGGTCGAGAAAAGAGCGAAGGAGGATAGCAGAGGAATGC CCGCGTATGTTGAACTCGGAGATAAGCAAAAGGCTAGGACTCGAGTGGAATTCGCTCACTTTGGACGAAAAACAGCCTTACGTAGAAGAAGCCAAAAG ACTGCGCGAACTGCATAAAAAGGACCATCCGGATTACAAATATCAACCGAAGCGCAAACCCAAAACAAGTCCAAAACTTAAAACACCGGGACTAAACC CGTTTATGCATGGCTATGGGGAGATGCCAGGGATAGGTATGCCTCCTCCAACGAACCTATGCCAACCAATGGCTTCCCATATGGGTCCTATGGTTAAC ATGGCAAGCTGCCCTGGATCGTGCACACTGCCAGAGCCTCCACCGCCCTACCATTTCTCCCCGCACTACTCCTTCGTGCAGAATATCTCGGACTATAA GAATCGATGTGGTTCGCATCTATCACTGATGAGCAGGGATCTGCCATACCCGTCACCCATCGGATATCCATCGCACGGCGCATCGCACCCGGTGCAGT TTGTACATCGCAGTCTTATTCCAGACTCTGGGTCTGTCGTACACGCGACTTCACCCATAGACGCACGAAGTGGGCTAGCGAGACATCCTTTAGACTGT GTTGTCATGCGGCCGGAGGGTTACTGAaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Apa1 Bcg1a Bcg1b BfuA1 Bpm1 Bsa1 BsaB1 BseR1 BseY1 Bsm1 BspE1 BspLU BspM1 BsrG1 BstAP BstX1 Cla1 Ecl2 EcoK EcoRV Hpa1 Nae1 Nco1 NgoM4 Nhe1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 SnaB1 Spe1 Sph1 Xho1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsiW1 BsmB1 BssH2 BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nru1 Pac1 PflF1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1
208 1.15. Nv-FHcDNAF1/R1 – FH-848bp
Vector: pGEM-T easy vector Template: NvcDNA8d Primers: NvFHF1/R1 PCR product: PCR 230 n°8 Insert length: 848bp Plasmid: 2 Orientation: antisens José: 42001_vg2-p2 (T7) Comments: cloned to use as positive control in ISH
209 INSERT - Reverse complement:
SP6gtgattATGATGGAGCACACGGGGGTGCCTCCAGCGGCCATGCAAGACCCATCACAAAACCCGCACGAGCTCAAGAAATCCAAGGACAAGGAGAA AGCGTATCGCCGGAGCTACACGCACGCCAAGCCGCCATATTCATATATCTCACTCATCACGATGGCCATTCAACAGAGCCCAAACAAGATGCTCACAC TGAGCGAGATCTACCAATTCATCATGGACTTGTTTCCCTACTACAGGCAAAACCAACAGCGCTGGCAGAACTCTATCCGGCACAGTTTATCATTCAAT GATTGCTTCGTGAAGGTGCCGCGCTCTCCTGACCGCCCCGGGAAAGGCAGTTACTGGACTCTCCACCCGGACTGCGGTAATATGTTCGAGAACGGGAG CTACCTTCGCAGGCAGAAGCGCTTCAAAGCCGAGAAAAAAACCGGACCTGAGTCACCTTAGCAAGGTGAGCGGTATGACACACAACCCGGTCACAGTA CAAAGCATGGCGAAGAGCATGGCGGCACAACCTCGCTCAATGGGAACCCCTAGCTTCCTTGCACCGTCTCCGTACGGGACGGCTATGGGCATGGGACA CGTTGGGGGCATGACCGCCATGGGTATGGCAGGTATGCCCATGAATAAGTCGTTTAATCACCCATTCGCTATCAAGAATATCATCGCGCAAGATCACG AAGCTGAGCTTCGAGGCTACGACCCCATGCACTTCAGTCCTTATCATCCATCACTCCAATCCATGGGTTCCCTAGGACTCCCTAAATCCGCCTACGAA TCGCAACCTATCACGACGGATACGAGTCCGTACTACCAGGGTTGCGTCTTCACGCCGTCGAGTTGTGGTATAaatcgaT7
Retriction sites full clone:
Unique:
Aat2 Ahd1 Ale1 Apa1 Avr2 Bbs1 Bcg1a Bcg1b BfrB1 Bgl2 Blp1 Bpu10 Bsa1 BseY1 BsiW1 BsmB1 BspLU Dra3 EcoK Mlu1 Msc1 Nae1 Nde1 NgoM4 Nsi1 PflM1 PshA1 Psi1 PspOM Pst1 Sac2 Sal1 Sbf1 Sca1 Sma1 Spe1 Sph1
Not found:
Aar1 Acc65 Afl2 Age1 Asc1 AsiS1 Bae1a Bae1b BamH1 BbvC1 Bcl1 BmgB1 BsaB1 BseR1 Bsg1 Bsm1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Nar1 Nhe1 Nru1 Pac1 PflF1 Pme1 Pml1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
210 1.16. Ch-GsxcDNAF1/R1 – Gsx-867bp
Vector: pGEM-T easy vector Template: ChcDNA2d Template: medusaecDNA Primers: ChGsxF1/R1 PCR product: PCR 279 n°4 Insert length: 867bp Plasmid: 30 (21, 34) Plasmid: 39 Orientation: sens Microsynth: 750970 (T7) Comments:
211 INSERT:
T7Tcgattgtcacagtacactgcacacgcagaaagtgaagaagaagaagaagaaagcagaaaatagatttaaaaggaattttatttgtgaagaaaaat ttattaaaaaaATGTCGCGATCATTTTTCATCGATACGATTATTCACGAGAAAGAGAAGCTGTTACGACAGACCTCGCCGATCAAACAACAACCGATC GCATCGTCGTCACCATCATCACGCGAACCATCGCCGATCTCCGAACACGGAAATCAATATCAACTTTCACCGATATCCCGATACAATCAATCGCCATC ACCTCGCTCACCTCCCTCACCGACTGGACATCAAGATTATTCAAGATCATCGCAAAATATATCTTCCACATCGTTACACCATGGCTCCGCATTGTGCG GATGTTGTCCACCACAGCCACACTCACGCTTATGCATGTGCTCCTCGTGTGAAAGCGGACCCAGAGAAGGGGAAGCGGCGCCGTTCGGATCACCAAGA GAAGCACCTCATCACACAACAAGATATTTATATGGTGGAAGCGAAAGAGGGCGTATCTTTTCTCTTGCATCTCCGATCAATTCAAGAGCTCGACCACA ATTTCCCGCGCTTTATGTACGAGACTTGGACAGCCGTCGCTTACAACTTCAACAACAGGTACAACACCAACGACAACAACAACTTGAAGAACAACAAG GAGGAGGTAGCAAAAGCAAACGAATCCGTACAGCCTATACTAGTGTCCAACTTCTTGAACTTGAAAAAGAATTCCAAAATAATCGATATCTTTCACGA TTAANGCGTATTCAAATTGCAGCTATGTTAGATCTGACGGAAAAACAAGTGAAGATTTGGTTTCAGAACCGACGTGTGAAATGGAAGAAGGaatcacS P6
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 Bcg1a Bcg1b BfuA1 Bgl2 BmgB1 Bpm1 Bsa1 BseY1 Bsg1 BspLU BspM1 BstX1 Dra3 EcoK Kas1 Mlu1 Nae1 Nar1 Nde1 NgoM4 PshA1 Psi1 PspOM Pst1 Sac2 Sal1 Sap1 Sbf1 Sca1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Blp1 Bpu10 BsaB1 BsiW1 Bsm1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kpn1 loxP Mfe1 Msc1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
212 1.17. Ch-GsxgDNAF1/R1 – Gsx-1335bp
Vector: pGEM-T easy vector Template: gDNA Primers: ChGsxF1/R1 PCR product: PCR 279 n°2 Insert length: 1335bp Plasmid: 11 Orientation: antisens Microsynth: 750969 (T7) Comments:
213 INSERT - Reverse complement:
SP6gtgattgtcacagtacactgcacacgcagaaagtgaagaagaagaagaagaaagcagaaaatagatttaaaaggaattttatttgtgaagaaaaa tttattaaaaaaATGTCGCGATCATTTTTCATCGATACGATCATTCACGAGAAAGAGAAGTTGTTACGACAGACCTCGCCGATCAAACAACAACCGAT CGCATCGTCGTCGCCATCATCACGCGAACCATCGCCGATCTCCGAACACGGAAATCAATATCAACTTTCACCGATATCCCGATACAATCAATCGCCAT CACCTCGCTCACCTCCCTCACCGACTGGACATCAAGATTATTCAAGATCATCGCAAAATATCTCTTCCACATCGTTACACCATGGCTCCGCATTGTGC GGATGTTGTCCACCACAGCCACACTCACGCTTATGCATGTGCTCCTCGTGTGAAAGCGGACCCAGAGAAGGGGAAGCGGCGCCGTTCGGATCACCAAG AGAGGTAACTCATCACACAACAAGATATTTATATGGTGGAAGCGAAAGAGGGCGTATCTTTTCTCTTGCATCTCCGATCAATTCAAGAGCTCGACCAC AATTTCCCGCGCTTTATGgtaaatttcattatctcattattctatcatttttcttgatctcttttcttgactcccaaacaaattttacattggaaggg taattttcttgactttctgcatttttaaaaggacacaaatgcaattttaagaaatttggcgcgcattgaggtaaaagcgcgcaaattgtctttgcatg atattgtaaacatatttatgctgaagaatattgcgcctgaaataatgatattttcgcgccaaaaacacatattacgtgttgcgataattgcaaacaaa gtagatattctgaattgtttgataattatgttttaaacgttcgatcgtttatgaagacgcaaacattaaaaacattcttgttttgacaaaacaatcgc gtgctacagagaagagtactatcaagatgacccctccctttctgtaaagctacaatgaatgaaacttaacattcttcttctttttctttcaacagTAC GAGACTTGGACAGCCGTCGCTTACAACTTCAACAACAGGTACAACACCAACGACAACAACAACTTGAAGAACAACAAGGAGGAGGTAGCAAAAGCAAA CGAGTCCGTACAGCCTATACTAGTATCCAACTTCTTGAACTTGAAAAAGAATTCCAAAATAATCGATATCCTTCACGATTAAGGCGTATTCAAATTGC AGCTATGTTAGATCTGACGGAAAAACAAGTGAAGATTTGGTTTCAGAACCGACGTGTGAAATGGAAGAAGaatcgaT7
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 Bbs1 BfuA1 Bgl2 BmgB1 Bpm1 Bsa1 BseY1 Bsg1 BspLU BspM1 BstX1 Dra3 Kas1 Mlu1 Nae1 Nar1 Nde1 NgoM4 PshA1 Psi1 PspOM Pst1 Sac2 Sal1 Sap1 Sbf1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Blp1 Bpu10 BsaB1 BsiW1 Bsm1 BsmB1 BspE1 BsrG1 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kpn1 loxP Mfe1 Msc1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
214 1.18. Ch-GsxgDNApromEcoRVF2/R2
Vector: pGEM-T easy vector Template: gDNA x EcoRV Primers: ChGsxF2/R2 PCR product: PCR 315 n°7&8 Insert length: 714bp Seq unknown: 454bp Plasmid: 22 Orientation: sens José: 55115_BG_mq22-m13f (M13F) Comments: iPCR clone
215 INSERT:
T7tcgattGACCTCGCCGATCAAACAACAACCGATCGCATCGTCGTCACCATCATCACGCGAACCATCGCCGATCTCCGAACACGGAAATCAATATCA ACTTTCACCGATATCtaatggaacaccttgaatggtaagttattcttcctgaatgcttgttatttgttaaagtttgaaggaacccctggcccttcact ttatttcccctatttcatgattattgagctttgaaacagtccgcataaaaaaacaaaatataaagttcaatttcattaattcacattaattcttaatg attgatttattcttctgtttatagtattaaatttgaatggaaaaaaatcgatatattcatgggagtaaagaaatcaaaagaatttaaatggacgggag agaaaatgaagatgaaaaagaaaaagaacattcttttttaatagcataaatagagagatagaaatgtcaaatagtttaaaatctgtgtcgcaagtgcg catgcccaaagtaagacaacatctataaagaggntgttaaaaaattctgatatatcattaacagaaacgtcgcgtcacagtacactgcacacgcagaa agtgaagaagaagaagaanaaagcggaaaatagatttaaaaggaattttatttgtgaagaaaaatttattaaaaaaATGTCGCGATCATTTTTCATCG ATACGATTATTCACGAGAAAGAGAAGCTGTTACGACaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 Bcg1a Bcg1b BfrB1 BfuA1 Bpm1 Bsa1 BseY1 Bsg1 Bsm1 BspLU BspM1 BstX1 Dra3 Ecl2 EcoK EcoRV FspA1 Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Swa1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 BmgB1 Bpu10 BsaB1 BseR1 BsiW1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
216 1.19. Ch-PdxgDNAF3/R2 – Pdx-197bp
Vector: pGEM-T easy vector Template: ChgDNA Primers: ChPdxF3/R2 PCR product: PCR 309 n°1 Insert length: 197bp Plasmid: 3 (striation pl15) Orientation: sens Fasteris: BS70 (T7) Comments:
217 INSERT:
T7tcgattGCTTATACGCGAGCACAGCAACTAGAACTGGAAAAAGAATATCGATATAATCGATATATATCGAGAGCACGTCGAATCGAATTAGCTAAA AATTTGACGCTAACAGAAAAACATATCAAAATATGGTATCAGAATCGAAGAATGAAGGAGAAAAGAGACGAGGAAGATATTATGAGAGGTACAACTGT TTTGGATCCaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 Bae1a Bae1b BamH1 Bcg1a Bcg1b BfrB1 BfuA1 BmgB1 Bpm1 Bsa1 BsaB1 BseY1 BsmB1 BspLU BspM1 BstX1 BtgZ1 Dra3 Ecl2 EcoK Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 Bpu10 BseR1 Bsg1 BsiW1 Bsm1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
218 1.20. Ch-PdxgDNAF3/R3 – Pdx-143bp
Vector: pGEM-T easy vector Template: ChgDNA Primers: ChPdxF3/R3 PCR product: PCR 309 n°3 Insert length: 143bp Plasmid: 9 (striation pl19) Orientation: sens Fasteris: BS71 (T7) Comments:
219 INSERT:
T7tcgattGCTTATACGCGAGCACAGCAACTAGAACTGGAAAAAGAATATCGATATAATCGATATATATCGAGAGCACGTCGAATCGAATTAGCTAAA AATTTGACACTAACAGAAAAACATATCAAAATATGGTATCAGAATCGAAGAATaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 Bcg1a Bcg1b BfrB1 BfuA1 BmgB1 Bpm1 Bsa1 BsaB1 BseY1 BspLU BspM1 BstX1 BtgZ1 Dra3 Ecl2 EcoK Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 Bpu10 BseR1 Bsg1 BsiW1 Bsm1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
220 1.21. Ch-CnoxCcDNAlong – CnoxC-657bp
Vector: pGEM-T easy vector Template: ChcDNA2d Primers: gsx-clyt-R4 PCR product: PCR 309 n°3 Insert length: 657bp Plasmid: 5 Orientation: sens Microsynth: 519431 (T7) Comments: Cloned using Gsx degenerated primers
221 INSERT:
T7ttcgatTCTGCTATTTTGGAACCAAATTTTCCACAAGGATTAAATAACAACAATACACCACCACCCCTTTCATCACCATCTTACCTTCGAAATGAT GCTAACAGATACGGTGGCACGGGAACCCCAACAACAGACTCTCTCGAACAATCTGCATTCAGTCACATCTCATCGCCAGATACGACAGCTAATTCACC ACCAATGACAGAACCAACTTACAGCACAACAGCAGGAAGCACGTTCGACACCTCCGTGCAATCCTACCTTGCCAACACGTCTTTAGCTAACCCACCAT CAACCTCAGCAATGTCATTTTACAACACTCCCACCTCTCTACTATCCAACCAACACTTATCCACAGATTACTCACAATTTGGATATGCTTCTCCTTCA AACTATTTCTACAGCTCTGGTTACCCATCCATCGGATCTGGGTACAATACTTATCCAGGAATGACCAACACTGGGCCTTTACCAGCTGGTCCATGGAT ATGCCGAGATATCGATACCAAACGAAAACGAATGACTTATTCACGAAAACAACTTTTAGAATTGGAAAAAGAATTTCATTTCAGTCAATTCTTAAAGA AAGAACGAAGATCAGACTTGGCAAAACAACTCAGTTTAACCGAACGACAAATCAAAATTTGGTTTCAAAACAGCCGAatcactSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 Ale1 AlwN1 Apa1 BbvC1 BfrB1 BfuA1 BmgB1 Bpm1 Bpu10 Bsa1 BseY1 Bsm1 BspLU BspM1 BstB1 BstE2 BstX1 Cla1 Dra3 Ecl2 EcoK EcoRV Mlu1 Nae1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 T7Ter Xcm1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 Bcl1 Bgl2 Blp1 BsaB1 BseR1 Bsg1 BsiW1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xho1
222 1.22. Ch-CnoxCcDNAsh – CnoxC-384bp
Vector: pGEM-T easy vector Template: ChcDNAmed Primers: ChCnoxCF/R PCR product: PCR 270 n°8 Insert length: 384bp Plasmid: 59 Orientation: antisens Microsynth: 722128 (T7) Comments:
223 INSERT – Reverse complement:
SP6gtgattGATACGGTGGCACGGGAACCCCAACAACAGACTCTCTCGAACAATCTGCATTCAGTCACATCTCATCGCCAGATACGACAGCTAATTCA CCACCAATGACAGAACCAACTTACAGCACAACAGCAGGAAGCACGTTCGACACCTCCGTGCAATCCTACCTTGCCAACACGTCTTTAGCTAACCCACA ATCAACCTCAGCAATGTCATTTTACAACACTCCCACCTCTCTACTATCCAACCAACACTTATCCACAGATTACTCACAATTTGGATATGCTTCTCCTT CAAACTATTTCTACAGCTCTGGTTACCCATCCATTGGATCTGGGTACAATAATTATCCAGGAATGACCAACACTGGGCCTTTACCAGCTGGTCCATGG AatcgaaT7
Retriction sites full clone:
Unique:
Aat2 Ahd1 Ale1 AlwN1 Apa1 BbvC1 BfrB1 BfuA1 BmgB1 Bpm1 Bpu10 Bsa1 BseY1 Bsm1 BspLU BspM1 BstE2 BstX1 Dra3 Ecl2 EcoK Mlu1 Nae1 Nde1 NgoM4 Nsi1 PflM1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 Xcm1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 Bcl1 Bgl2 Blp1 BsaB1 BseR1 Bsg1 BsiW1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xho1
224 1.23. Td-PdxcDNAF5/R4 – Pdx-298bp
Vector: pGEM-T easy vector Template: TdcDNAmedusae Primers: TuxF5/R4 PCR product: PCR 3 n°24 Insert length: 298bp Plasmid: 20 (51) Orientation: sens Microsynth: 669668 (T7) Comments:
225 INSERT:
T7tcgattCGAACAGAACCAAACTGAAAAGGAGAAAACGAAAGATGACAAACCACCAAAAGCTTGGCAGTCAGCTAAAGTGAAGAAGAGGAATCGCAC GACCTACACACGAGTACAGCAGCTGGAGCTGGAGAAAGAATATCGGTACAGTAAGTACATATCAAGAGCCAGGAGAATCGAGTTAGCTAAAAATTTAA CTCTGACAGAAAAACATATTAAGATCTGGTATCAAAATCGCAGGATGAAAGAAAAGCGGGACGAAGAAGACGCTTTGAGAAATGAGAATAGTCTGGAT GGGCAACTAAGGaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 Apa1 Bbs1 Bcg1a Bcg1b BfrB1 BfuA1 Bgl2 Bsa1 BseY1 BspLU BspM1 BstB1 BtgZ1 Dra3 Ecl2 EcoK Hind3 Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 Xcm1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Blp1 BmgB1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 Bsm1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xho1
226 1.24. Td-PdxcDNAF4/R4 – Pdx-324bp
Vector: pGEM-T easy vector Template: TdcDNAmedusae Primers: TuxF4/R4 PCR product: PCR 4 n°24 Insert length: 324bp Plasmid: 40 (34 polyp cDNA) Orientation: sens Microsynth: 669670 (T7) Comments:
227 INSERT:
T7tcgattGGGAAGTTGAAGACAACCAGCACAAACGAACAGAACCAAACTGAAAAGGAGAAAACGAAAGATGACAAACCACCAAAAGCTTGGCAGTCA GCTAAAGTGAAGAAGAGGAATCGCACGACCTACACACGAGTACAGCAGCTGGAGCTGGAGAAAGAATATCGGTACAGTAAGTACATATCAAGAGCCAG GAGAATCGAGTTAGCTAAAAATTTAACTCTGACAGAAAAACATATTAAGATCTGGTATCAAAATCGCAGGATGAAAGAAAAGCGGGACGAAGAAGACG CTTTGAGAAATGAGAATAGTCTGGATGGGCAACTAAGGaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 Apa1 Bcg1a Bcg1b BfrB1 BfuA1 Bgl2 Bsa1 BseY1 BspLU BspM1 BtgZ1 Dra3 Ecl2 EcoK Hind3 Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 Xcm1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 BbvC1 Bcl1 Blp1 BmgB1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 Bsm1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstB1 BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 Kpn1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xho1
228 1.25. Td-CnoxAcDNATH5F3/TH5R6 – CnoxA-545bp
Vector: pGEM-T easy vector Template: TdcDNAmedusae Primers: TH5F3/TH5R6 PCR product: PCR 3 n°24 Insert length: 545bp Plasmid: 30 Orientation: sens Microsynth: 722127 (T7) Comments:
229 INSERT:
T7tcgattCCATGTGGAGAATGAACTGTTCAACATCGTCTTGTGAAACGTGCTCTTCGCGATGGTCACATGTTATTCCAGCACCCTACCAAAATCGAT ATTGTAATCCAAGAAGAACTGTTGACGATAAATATGGTAAAGTAAGGCCATGGATAGTTGCTCGAAATTCCGTCGACAATAAACATCTGGTCTCAAGG GATTATCGTCCTCCCTGCCATGCCTCAAGAGTTAGTTTGCGTCACTGTCGAAGTTGTTACCTAGCCGCGGAAACGCAATATATCACGTATTTTGCTCG CCAATTTATCCCGAGAGAATGCAATTGTATCGATTGTGATAGGGAAGGGATGAAAAGATTTCGAACAAGTTTCAACACGTCACAACTAACAGAACTGG AAAAAGAATTCCAACAGAACAAATACTTAACACGTCGTCGTCGAGTCGAACTTGCTGTCGGCTTAAAGTTGTCAGAAAAACAGGTCAAGGTGTGGTTT CAAAATAGACGGATGAAGTGGAAAAAGCAGACAAAATTTGAAGAAGAAGAAGAAGAGGGGAGAaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Ahd1 AlwN1 Apa1 BfrB1 BfuA1 Bpm1 BseY1 Bsm1 BspM1 BstB1 BstX1 Dra3 Ecl2 EcoK Mfe1 Mlu1 Nae1 Nde1 NgoM4 Nru1 Nsi1 Psi1 PspOM Pst1 Sac1 Sbf1 Sca1 Spe1 Sph1 Xmn1
Not found:
Aar1 Acc65 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Avr2 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Bgl2 Blp1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 BsmB1 BspE1 BsrG1 BssH2 BstAP BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hind3 Hpa1 I_Ceu Kas1 Kpn1 loxP Msc1 Nar1 Nhe1 Pac1 PflF1 PflM1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
230 1.26. Hv-Cnox2gDNACx2Hv-Xmn5’/Cx2Hv-16HB3 – Cnox2-1139bp
Vector: pGEM-T easy vector Template: HvgDNA Primers: Cx2HvXmn5’/Cx2Hv16HB3 PCR product: PCR 105 n°1 Insert length: 1139bp Plasmid: 13 Orientation: sens Microsynth: 508964 (SP6), 508083 (T7) Comments: does not contain the STOP codon
231 INSERT:
T7tcgattgaacctcttcttaaaacgagttagatttttttatggttgttaaggatatagaATGTCAACTTCGTTTTTAATAGATTCTCTAATACATGA AAAAGAAAAGTATAAGATACGGCAGCAGCCTGGAACATCTTTTTTATTTCGTGAATCATCTCCTCCAGATCGATCGCCGAGTTATTCACCCGGTGCGT CAATGATTAGATATTCCAATTCTTCTTCTCCAAGAAGTTTAGATTCACCTATAAATCCATTGGATCGACACCCTCTTGAACGAGTACATCAAGTAGTT AGTTGTATGAGAGGACCTTCGATGTGTAATTGCTGTCGGCCTCCGACTGTTCAACCTATGTGTACAGTATGTGAACCTAGGGAACCAGGTGAAGGTAC CTCTTCACAATATCCTTATACCCGCGAACCTCATGACCATGCAAGAGGCTTGTATGGAAATGATAGGTCAAGACTTTTTCCAATATTATCTCCTTTAC ACGGGCAAAGAGCGCAGTTTTCCCCAAATTATGgtaagcttaatttttttttttattcataaaaagtttttgttttaactttacaactttattatttg tgaaaaaaataggtagttaaacgtatgtatagaataaaaaaacaaataaaattgctttgttagtaaattcggcattttagtgtataaagttctaacta aaagtttaaaaactgttcacaagttcggcataaatcgcgcactttttttatcactaaatttgagcgcccttttgcactaatggtcgcgtcaatttaag cggtgtataatgtcctgaatgacataagatgaccctagatgaaactctactaaatatattaacaacttaattttttcatttagTTTACGATTTGGAAC TTCGTCATTCCCGTCAACTTCAACTGCAACACCAAGAACACGAAACAGATCTCTACGGAAAATCTAAACGCATTCGAACCGCCTATACTAGCATTCAG TTACTTGAACTTGAAAAAGAGTTTCAAAATAATCGTTATTTATCGAGATTACGGAGAATCCAAATAGCTGCTATTCTTGACTTAACAGAAAAACAAGT TAAAATATGGTTTCAAAATCGACGTGTAAAATGGAAAAAAGATAAGAAAGGATATAGCTATTCCCCTACaatcacSP6
Retriction sites full clone:
Unique:
Aat2 Acc65 Ahd1 AlwN1 Apa1 Avr2 Bcg1a Bcg1b BfrB1 BfuA1 Bgl2 BmgB1 Bsa1 BseR1 BseY1 BspLU BspM1 BsrG1 BstB1 BstX1 BtgZ1 Ecl2 EcoK Hind3 Kpn1 Mlu1 Nae1 Nco1 Nde1 NgoM4 Nsi1 PflM1 Psi1 PspOM Pst1 Sac1 Sac2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1
Not found:
Aar1 Afe1 Afl2 Age1 Ale1 Asc1 AsiS1 Bae1a Bae1b BamH1 Bbs1 BbvC1 Bcl1 Blp1 Bpu10 BsaB1 Bsg1 BsiW1 BsmB1 BspE1 BssH2 BstAP BstE2 BstZ1 Bsu36 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu Kas1 loxP Mfe1 Msc1 Nar1 Nhe1 Nru1 Pac1 PflF1 Pme1 Pml1 PshA1 R4atB R4atL R4atP R4atR Rsr2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SnaB1 Srf1 Stu1 Swa1 T3RNA T7RNA T7Ter PISce Xba1 Xcm1 Xho1
232 Reporter constructs: HyactEGFP derived
233 234 2.1. HyactEGFP-EcoR1
Correspond to the accession number DQ369740 Given by Sequenced with GFP-C-for1 & hy-act-promF1 by Marjana Miljkovic-Licina Comments: efficient by injection at the 1 cell stage of Nv (!20- 100ng/ul). For the restriction sites of the backbone see previous map in the lab
235 INSERT - Reverse complement:
XXX: Hyact promoter XXX: HyactCDs XXX: EGFPCDs XXX: Hyact3’UTR aagcttgcatgcctgcaggtcgactctagaggatcccccatcgatctgactaacctaaccagtgcaaaaaaatttaaaagatttgcattgtgaaagtt agaatattataaaaaatctaaaacgagtattactcgagtaaatgttatacgatctatagattaaatatattaaaaatgtatagcgaatgttaaactaa atatataatataaacttgaaaacttactaaattgcaaaaactcaaaaccgactgtatcatttttacaggaaaccgttattcaagatacttaagttgtt tactacattattataacatcttgcaattagcaagacaatcgttattttaacatcacggtatcgaaaggattttgagaaattttattgaaacattttaa acaaaaaatatcatatttagatgcattttaagccgagatgcaggattctgaatgaaaaagaaaaaaagaagtctcggtagagtaaaagtgatcggttt gcaactgtaaaatttattgaagtaccaataattttatttaaaataaaactgaaatataaagttaaagttgctgttctataagtttactcgaattttaa aaccattgtaacgctagagtaatatttgagtctactaagttagtccccgcactttttaatcaagcaataaatacccaaactttgcttattcaaatcaa taaaccaatatatctcttaaaataaagtaaaaacttctgaaattctataaaaaaaaatttaatttcgaaatatcaaatgtaacttcaacaccgcacta ttttcttttaaacaactgatatagtaattacttctcaaaaacgttatctcaaggtttgtgatgtacttaaaaccactcctattttgttacgcgtttaa aaaagcaaacataagttggtttctattgatgaatgagaacatatttcatttaaagttaaaatcctaccagtggtttcactgtacgtaaacaccgtcaa aaaaacaggaacgtttttaaagattaataattgaagtaaaaaaaatttaataccgggggttaaaaaaatcttttaaaataattataaatatatatatt aaaatttataaatttttaaacacatttaaaatatatattaagtataataaaagtaatattataaaaaaaaatttaattttataattatttttattaaa tttataaataataggtaaaacttacatatccgttttattttttcttaataaaataacgcgtgcaaatttttgtccatataaagaccttttcgaacaat aacttttttgcttagccgttttttttcttatatggtcaaaaaagcgctcaagcgattcaccataaaaagcgcaattagttcagcgttcgttattcaga agcttcagctttgcttgatactcagctcttctctttttaaacaaaacacttaatcaaaATGGCCGATGATGAAGTTGCCGCCCTCGCTGCAGCCCCGG TAGAAAAAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGT GGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTAC TTTCTGTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAA GAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATT GATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAAT CAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCC TTTTACCAGACAACCaTTACCTGTCCaCACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCT GCTGGGATTACACATGGCATGGATGAACTATACAAATAGcattcgtagaattcacaattcgattatatttatactggactatttttacatctgttcgg ttattttcacatttatttttctatatatatcttataaacgttttaaaacccatgtaatttttgttaagctgtaatataaaagacgtcctaacaaactt cttttattactgaatttcctttaattataataaataacaagttttaaaataaattcaggcaattaaggcgctcctgaggtactaaaattaatgtaaac atttaaaattaacttggatggtcttaagtactgtactcgtgattttgttatactttattattagaaaagtcgtctattaactttttgttccttaattt acttgattaaattgtcgcttaatttatcaaatcaggttttgcgcgttattttagagaaaaacttattagaaaaatgaataagcaaagtttaggctaac atgtttttttattattttaaatagttcaagtcaatgacgtataaaatgcatttgcaaaaaattttaagtaaccctataaacttagcaatagtagatac tggatgcaagcattcagtagcagcattgcatatctgctgtctttacgtacaaataacagcaaaaatggacctttattggcttcacatcgtcgtaaaac atgtgttattggacttgtcacaaatgtgttaagtatacagagcttagctcttgatgttgatcactagT
Retriction sites without the backbone:
Unique:
Aat2 Afe1 Ale1 AlwN1 BamH1 Bcg1a Bcg1b BfuA1 Bpm1 Bsa1 BseY1 BspM1 BsrD1 BsrG1 BssS1 Bsu36 BtgZ1 Drd1 Ear1 EcoR1 Hpa1 Mfe1 Msc1 Nco1 Nde1 Pml1 Pvu2 Sal1 Sap1 Sbf1 Sca1 Spe1 Sph1 T7Ter Xba1 Xho1
Not found:
Aar1 Acc65 Age1 Ahd1 Apa1 ApaL1 Asc1 AsiS1 Avr2 Bbs1 BbvC1 BciV1 Bcl1 Bgl1 Bgl2 BmgB1 Bmr1 Bpu10 BsaB1 BsaXa BsaXb BseR1 Bsg1 BsiW1 BsmB1 BspE1 BspH1 BsrB1 BssH2 BstAP BstE2 BstX1 Bts1 BxatB BxatL BxatR BxatP _Chi Cla1 Dra3 Eag1 Eci1 Ecl2 EcoK EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Fsp1 I_Ceu Kas1 Kpn1 loxP Nae1 Nar1 NgoM4 Nhe1 Not1 Nru1 Pac1 PflF1 PflM1 Pme1 PshA1 PspOM Pvu1 R4atB R4atL R4atP R4atR Rsr2 Sac1 Sac2 SanD1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xcm1 Xmn1
236 2.2. HyactEGFP-Sce1
Vector: pBSK12 x Sma1 + Spe1 Insert: HyactEGFPEcoR1 x Sph1 (blunting) + Pvu1 + Spe1 (Hyactprom – EGFP – 3’UTRactterm) Plasmid: 1 Microsynth 602801 (TermR1promF2), 600783 (M13r), 600782 (M13 -40) Comments: made to inject along the meganuclease restriction enzyme (Cf Grabher & al, 2004), not more efficient than HyactEGFP in our hands
237 INSERT: XXX: Hyact promoter XXX: HyactCDs XXX: EGFPCDs XXX: Hyact3’UTR XXX: Sce1 tagggataacagggtaatctcgagaagcttgatatcgcctgcagcccgactctagaggatcccccatcgatctgactaacctaaccagtgcaaaaaaa tttaaaagatttgcattgtgaaagttagaatattataaaaaatctaaaacgagtattactcgagtaaatgttatacgatctatagattaaatatatta aaaatgtatagcgaatgttaaactaaatatataatataaacttgaaaacttactaaattgcaaaaactcaaaaccgactgtatcatttttacaggaaa ccgttattcaagatacttaagttgtttactacattattataacatcttgcaattagcaagacaatcgttattttaacatcacggtatcgaaaggattt tgagaaattttattgaaacattttaaacaaaaaatatcatatttagatgcattttaagccgagatgcaggattctgaatgaaaaagaaaaaaagaagt ctcggtagagtaaaagtgatcggtttgcaactgtaaaatttattgaagtaccaataattttatttaaaataaaactgaaatataaagttaaagttgct gttctataagtttactcgaattttaaaaccattgtaacgctagagtaatatttgagtctactaagttagtccccgcactttttaatcaagcaataaat acccaaactttgcttattcaaatcaataaaccaatatatctcttaaaataaagtaaaaacttctgaaattctataaaaaaaaatttaatttcgaaata tcaaatgtaacttcaacaccgcactattttcttttaaacaactgatatagtaattacttctcaaaaacgttatctcaaggtttgtgatgtacttaaaa ccactcctattttgttacgcgtttaaaaaagcaaacataagttggtttctattgatgaatgagaacatatttcatttaaagttaaaatcctaccagtg gtttcactgtacgtaaacaccgtcaaaaaaacaggaacgtttttaaagattaataattgaagtaaaaaaaatttaataccgggggttaaaaaaatctt ttaaaataattataaatatatatattaaaatttataaatttttaaacacatttaaaatatatattaagtataataaaagtaatattataaaaaaaaat ttaattttataattatttttattaaatttataaataataggtaaaacttacatatccgttttattttttcttaataaaataacgcgtgcaaatttttg tccatataaagaccttttcgaacaataacttttttgcttagccgttttttttcttatatggtcaaaaaagcgctcaagcgattcaccataaaaagcgc aattagttcagcgttcgttattcagaagcttcagctttgcttgatactcagctcttctctttttaaacaaaacacttaatcaaaATGGCCGATGATGA AGTTGCCGCCCTCGCTGCAGCCCCGGTAGAAAAAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATG TTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCT GTTCCATGGCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGC CATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG TTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATC ATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAA TACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCaTTACCTGTCCaCACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACA TGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGcattcgtagaattcacaattcgattatatttat actggactatttttacatctgttcggttattttcacatttatttttctatatatatcttataaacgttttaaaacccatgtaatttttgttaagctgt aatataaaagacgtcctaacaaacttcttttattactgaatttcctttaattataataaataacaagttttaaaataaattcaggcaattaaggcgct cctgaggtactaaaattaatgtaaacatttaaaattaacttggatggtcttaagtactgtactcgtgattttgttatactttattattagaaaagtcg tctattaactttttgttccttaatttacttgattaaattgtcgcttaatttatcaaatcaggttttgcgcgttattttagagaaaaacttattagaaa aatgaataagcaaagtttaggctaacatgtttttttattattttaaatagttcaagtcaatgacgtataaaatgcatttgcaaaaaattttaagtaac cctataaacttagcaatagtagatactggatgcaagcattcagtagcagcattgcatatctgctgtctttacgtacaaataacagcaaaaatggacct ttattggcttcacatcgtcgtaaaacatgtgttattggacttgtcacaaatgtgttaagtatacagagcttagctcttgatgttgatcactagttcta gagcggccgcgacttaagattaccctgttatcccta
Retriction sites full clone:
Unique:
Aat2 Afe1 Ahd1 Ale1 BamH1 BsrG1 Bsu36 Dra3 Eag1 Ecl2 EcoK EcoR1 EcoRV Hpa1 Mfe1 Msc1 Nae1 Nco1 Nde1 NgoM4 Not1 Pml1 Sac1 Spe1 T3RNA T7RNA T7Ter Xmn1
Not found:
Aar1 Acc65 Age1 Apa1 Asc1 AsiS1 Avr2 Bbs1 BbvC1 Bcl1 BfuA1 Bgl2 BmgB1 Bpu10 BsaB1 BseR1 Bsg1 BsiW1 BsmB1 BspE1 BspM1 BssH2 BstAP BstE2 BstX1 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 I_Ceu Kas1 Kpn1 loxP Nar1 Nhe1 Nru1 Pac1 PflF1 PflM1 Pme1 PshA1 PspOM R4atB R4atL R4atP R4atR Rsr2 Sac2 Sal1 SanD1 Sbf1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 Sph1 Srf1 Stu1 Swa1 PISce Xcm1
238 2.3. NvAx2p(-3000)EGFP
Vector: EmptyEGFP x Hind3 (filling) Insert: Ax2-3127bp x Not1 (filling) (3127bp of the upstream sequences of Ax2 CDs) Plasmid: 12 Microsynth: 605776 (GFP-N-rev1), 743030 (Ax2NvR8) Comments: efficient by injection at the 1 cell stage of Nv (!20- 100ng/ul)
239 INSERT - Reverse complement:
XXX XXX: Hind3 + Not1 XXX: pGEM-T easy vector XXX: Ax2NvF8 XXX: Ax2p(3123bp) XXX: Ax2NvR7 XXX: HyactCDs XXX: EGFPCDs XXX: Hyact3’UTR aagctggccgcgaattcactagtgattgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtactttgatttt ctggtgagtcgttttgttttcggtcgccacgtctatgtcccgcagttctttttgtactaccgtcacaaggccctcaaaaatacctgtgaacataaaag ccaaacacaacagcctcaaagtgagcaaaaaacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaactgcagga tcacaaaaagtgtttcatgtttccttttctctcgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggcttgcga cgggtttgcggaggggcgaatacgctcctggttattgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaatttcagg ccaattaaattaattttccatatgtcagagaccttgccatcttactaggacttgatcaatcgttttttttttttctttttggtcaattgtgtttacgc tgttaactcgatcatgactttaattaagtcgcgttcgcaagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctattcgcgcgcgattc aaggctcattggtaatcttttgagctttgacttgcaagactaaatgagctccaactaggtactaaagctttagagatgtttcccgtggaattaaagag gcacaagtttgctttagtgttaccggggtaacttctaagaagaattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgcttgaa ttttttgtactgttgttattttttccatactaaaatcgtacatcttctattttacgccttgtttttagtactttgaaaacctttttcacaaataagtc agattgtttaagagaaatacattccagaagtttaaagataatgtatattgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttgatttt cgatcgtttgtgaggatgatttgtcttttcgttcataaccttcgttcgtttttacggttaatgttcttgacatttttgaataaatgttcttgtattca atctagttctcaaaacaccaggcttttatatttattgaagaggctgttcctatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgtta tgtccttaaactaattaactccgtgataaaattcagaattagatcatttgctatcggcatataaggaactaaatttaccaggcaatcttttgataaat attacccgatgagaattgtttattttgtctgcgaaagcaaatcaaagagctgacttctgtatgaagcctatttaccttcaacattcatttcatttcct tagtcaagtatttgagaggaagaaacttcggctagcgtattttgttcgagttcgttaaaattatgtttccggaaagattagagaaacaattattatag taaatacaggccgttacaatttgatatctttaattgttatctagccgttgtcgtccttttcatttagattagtaggcccaggttgttgaaccaaatcg tgtagagtacaatcattcggaacgaaaaatacttgagaatccttttttatatatattctttgagatactaagagattagatcaatcagaagagttaat gaactggtaggttaagaagtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaatagttgattcttgtttttgttaatgaacgattcatt cgtatctttgacgggttaaagttccccactgggcggcacctaatcttgtgtaactaacaagatagagagcctacgtggtgtaaagatttacctgctac gcacaaactaagcgcatgtaatgtgttcaccaagacgagaggcattcaatattttatcgctcagttgacgtacaatcgcttcaattgccccccagtgt tcactaactctactgttttcttcacgcaaaacgcaaaacgatcttttaatattttcctagaaaataattgcaataagtttggaagcgtgtgcgatatt tgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaatacctattctccttcggtctagtcgccaggc tatttatttttcttgttatatgattaaaacatatttcacattttggaattggggaacaattacccatgacgcgtgcagaacggccattctaacgtcct ggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaacaaataagttccgaaagaaaacttgc aggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaagaattatggtctacatctggaagtt tggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgagaagacgtcgattgcttacaca ggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgacaattacggacgaaaaaagg ccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttatactcaagggggaaaata attctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaatctccttaaaacatg tcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttgtgttgcgaacaat aggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcgcagctcccagacggc acacatctcctgataatcgaattcccgcggccagcttcagctttgcttgatactcagctcttctctttttaaacaaaacacttaatcaaaATGGCCGA TGATGAAGTTGCCGCCCTCGCTGCAGCCCCGGTAGAAAAAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATG GTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAA CTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAA GAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATA CCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTA TACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCA ACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAG ACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGcattcgtagaattcacaattcgattat atttatactggactatttttacatctgttcggttattttcacatttatttttctatatatatcttataaacgttttaaaacccatgtaatttttgtta agctgtaatataaaagacgtcctaacaaacttcttttattactgaatttcctttaattataataaataacaagttttaaaataaattcaggcaattaa ggcgctcctgaggtactaaaattaatgtaaacatttaaaattaacttggatggtcttaagtactgtactcgtgattttgttatactttattattagaa aagtcgtctattaactttttgttccttaatttacttgattaaattgtcgcttaatttatcaaatcaggttttgcgcgttattttagagaaaaacttat tagaaaaatgaataagcaaagtttaggctaacatgtttttttattattttaaatagttcaagtcaatgacgtataaaatgcatttgcaaaaaatttta agtaaccctataaacttagcaatagtagatactggatgcaagcattcagtagcagcattgcatatctgctgtctttacgtacaaataacagcaaaaat ggacctttattggcttcacatcgtcgtaaaacatgtgttattggacttgtcacaaatgtgttaagtatacagagcttagctcttgatgttgatcacta gt
Retriction sites without the backbone:
Unique:
Aar1 Afl2 Age1 Ale1 AlwN1 Apa1 Bae1a Bae1b Bcg1a Bcg1b BciV1 BfrB1 BmgB1 Bpm1 BsaXa BsaXb Bsg1 BspE1 BsrD1 BsrG1 BssS1 BstB1 Bsu36 BtgZ1 Drd1 Ecl2 Mlu1 Msc1 Nae1 Nco1 NgoM4 Nhe1 Nsi1 Pac1 PspOM Pvu1 Pvu2 Sac1 Sac2 Sap1 SnaB1 T7Ter Xho1
Not found:
Acc65 Afe1 Ahd1 ApaL1 Asc1 AsiS1 Avr2 BamH1 BbvC1 Bcl1 Bgl1 Bgl2 Bpu10 BsaB1 BseR1 BsiW1 BsmB1 BspH1 BsrB1 BstAP BstE2 BstX1 Bts1 BxatB BxatL BxatR BxatP _Chi Cla1 Dra3 Eag1 Eci1 EcoK EcoN1 FCatB FCatL
240 FCatR FCatP ScFRT Fse1 FspA1 Fsp1 I_Ceu Kas1 Kpn1 loxP Nar1 Not1 Nru1 PflF1 PflM1 PshA1 R4atB R4atL R4atP R4atR Rsr2 Sal1 SanD1 Sbf1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 Sph1 Srf1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1 Xmn1
241 242 2.4. EmptyEGFP
Vector: HyactEGFP-EcoR1 x Hind3 Self-ligation Plasmid: ?? Made by Virginie Voeffray Fasteris: ?? Comments: Control construct. Less fault-positif when the transgene is excised from the vector than when the construct is injected circular
243 INSERT - Reverse complement:
XXX: HyactCDs XXX: EGFPCDs XXX: Hyact3’UTR aagcttcagctttgcttgatactcagctcttctctttttaaacaaaacacttaatcaaaATGGCCGATGATGAAGTTGCCGCCCTCGCTGCAGCCCCG GTAGAAAAAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAG TGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTA CTTTCTGTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAA AGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTAT TGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAA TCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTC CTTTTACCAGACAACCaTTACCTGTCCaCACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGC TGCTGGGATTACACATGGCATGGATGAACTATACAAATAGcattcgtagaattcacaattcgattatatttatactggactatttttacatctgttcg gttattttcacatttatttttctatatatatcttataaacgttttaaaacccatgtaatttttgttaagctgtaatataaaagacgtcctaacaaact tcttttattactgaatttcctttaattataataaataacaagttttaaaataaattcaggcaattaaggcgctcctgaggtactaaaattaatgtaaa catttaaaattaacttggatggtcttaagtactgtactcgtgattttgttatactttattattagaaaagtcgtctattaactttttgttccttaatt tacttgattaaattgtcgcttaatttatcaaatcaggttttgcgcgttattttagagaaaaacttattagaaaaatgaataagcaaagtttaggctaa catgtttttttattattttaaatagttcaagtcaatgacgtataaaatgcatttgcaaaaaattttaagtaaccctataaacttagcaatagtagata ctggatgcaagcattcagtagcagcattgcatatctgctgtctttacgtacaaataacagcaaaaatggacctttattggcttcacatcgtcgtaaaa catgtgttattggacttgtcacaaatgtgttaagtatacagagcttagctcttgatgttgatcactagt
Retriction sites without the backbone:
Unique:
Aat2 Acl1 Afl2 Ale1 AlwN1 Ase1 Bae1a Bae1b BfrB1 Blp1 Bpm1 BpuE1 Bsa1 BseY1 BsrD1 BsrG1 BssS1 BstB1 Bsu36 BtgZ1 Drd1 Ear1 EcoR1 Hind3 Hpa1 Mfe1 Msc1 Nco1 Nde1 Nsi1 Pml1 Pst1 Pvu2 Sap1 Sca1 SnaB1 Spe1 T7Ter
Not found:
Aar1 Acc65 Afe1 Age1 Ahd1 Apa1 ApaL1 Asc1 AsiS1 Avr2 BamH1 Bbs1 BbvC1 Bcg1a Bcg1b BciV1 Bcl1 BfuA1 Bgl1 Bgl2 BmgB1 Bmr1 Bpu10 BsaB1 BsaXa BsaXb BseR1 Bsg1 BsiW1 BsmB1 BspE1 BspH1 BspM1 BsrB1 BssH2 BstAP BstE2 BstX1 Bts1 BxatB BxatL BxatR BxatP _Chi Cla1 Dra3 Eag1 Eci1 Ecl2 EcoK EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Fsp1 I_Ceu Kas1 Kpn1 loxP Mlu1 Nae1 Nar1 NgoM4 Nhe1 Not1 Nru1 Pac1 PflF1 PflM1 Pme1 PshA1 PspOM Pvu1 R4atB R4atL R4atP R4atR Rsr2 Sac1 Sac2 Sal1 SanD1 Sbf1 SexA1 Sfi1 Sgf1 SgrA1 Sma1 SpAcc Sph1 Srf1 Ssp1 Stu1 Swa1 T3RNA T7RNA PISce Xba1 Xcm1 Xho1 Xmn1
244 Reporter constructs: CMVDsRed2 derived
XXX: bglobI XXX: Kozak XXX: bglobpA XXX: BGH polyadenylation seq XXX: F1 ori & pUC origin XXX: SV40 early promoter and origin & SV40 early polyadenylation signal XXX: Neomycin & ampicillin
245 246 3.1. CMVDsRed2 p317 (from Ivan Rodriguez’s laboratory) Vector: pcDNA3.1 x Kpn1 Insert: p310 x Kpn1 (rabbit bglobinI – DsRed2 – bglobinpA)
247 Full sequence:
XXX: CMV propmoter XXX: DsRed2CDs gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcc catatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgcc aagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccac cccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtag gcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactataggg agacccaagctggctagcgtttaaacttaagcttggtacctcgagataagcggccgcagatcctgagaacttcagggtgagtttggggacccttgatt gttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccat gcatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaactttttcgt taaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaat cagggtatattatattgtacttcagcacagttttaggaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgt ggaaatattcttattggtagaaacaactacaccctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggataaa atactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctctttcctacagctcctgggcaacgtgctggttgttgtgctgtctc atcattttggcaaagaattcccaccatgggcgcggatccaccggtcgccaccATGGCCTCCTCCGAGAACGTCATCACCGAGTTCATGCGCTTCAAGG TGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCCACAACACCGTGAAGCTGAAGGTG ACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGA CTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGCGACCGTGACCCAGGACTCCTCCCTGC AGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCTCC ACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGACCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTC CATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACGCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGG AGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGTAGcggatcgataattcactcctcaggtgcaggctgcctatcagaaggtggtggctg gtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttct ggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatatgggagggcaaatcatttaaaaca tcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctgccatgaacaaaggttggctataaagaggtcatcagtatatgaaac agccccctgctgtccattccttattccatagaaaagccttgacttgaggttagattttttttatattttgttttgtgttatttttttctttaacatcc ctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactcccagtcatagctgtccctcttctcttatggagatccctcga tcgagggggggcccggtaccgagctcggatccactagtccagtgtggtggaattggccgctcgagtctagagggcccgtttaaacccgctgatcagcc tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaata aaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaata gcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgca ttaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgc cacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatt agggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaa actggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca aaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatct caattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgc ccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggcc gcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcgg atctgatcaagagacaggatgaggatcgtttcgcATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCT ATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC GGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAA TGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTT GTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGT CGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCT ATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGAT TCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatca cgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcat gctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttttt cactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtca tagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgag ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagag gcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcg gtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgct ggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccagg cgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctt tctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgc cttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatg taggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttc ggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatc tcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatct tcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagTTACCAATGCTTAATCAGTGAG GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCC CAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCG GCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAA GGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTAT TGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
248 Retriction sites full clone:
Unique:
Afl2 Age1 Ale1 Avr2 Bcg1a Bcg1b Bpu10 Bsm1 BssH2 BstB1 BstE2 BstZ1 Bsu36 Dra3 EcoK EcoR1 FspA1 Hind3 Kas1 Mlu1 Nar1 Nhe1 Not1 Nru1 PflF1 Rsr2 Sal1 SanD1 Sbf1 Sma1 SnaB1 T7RNA T7Ter Xba1
Not found:
Aar1 Afe1 Asc1 AsiS1 Bae1a Bae1b BbvC1 Bcl1 Blp1 BmgB1 BsaB1 BsiW1 BsmB1 BspE1 BsrG1 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 Hpa1 I_Ceu loxP Pac1 Pml1 PshA1 R4atB R4atL R4atP R4atR Sac2 SexA1 Sfi1 Sgf1 SgrA1 Srf1 Swa1 T3RNA PISce Xcm1
249 250 3.2. CMVEGFP
Vector: CMVDsRed2 x Age1 (filling) + Cla1 (filling) (-DsRed2) Insert: HyactEGFP-EcoR1 x Pst1 (filling) + EcoR1 (filling) (EGFP) Plasmid: 20 Fasteris: OG004 (seq with TAC2079) Comments: construct to check more precisely by sequencing
251 Full sequence:
XXX: CMV propmoter XXX XXX: Age1+ Pst1 XXX: EGFPCDs XXX XXX: Ecor1 + Cla1 gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcc catatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgcc aagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccac cccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtag gcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactataggg agacccaagctggctagcgtttaaacttaagcttggtacctcgagataagcggccgcagatcctgagaacttcagggtgagtttggggacccttgatt gttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccat gcatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaactttttcgt taaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaat cagggtatattatattgtacttcagcacagttttaggaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgt ggaaatattcttattggtagaaacaactacaccctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggataaa atactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctctttcctacagctcctgggcaacgtgctggttgttgtgctgtctc atcattttggcaaagaattcccaccatgggcgcggatccaccggtgcagccccggtagaaaaaATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGT CCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTA AATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCAT ATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGC TGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAAT ACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGC GTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTC GAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGcatt cgtagaattcgataattcactcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatct ttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgtt ggaattttttgtgtctctcactcggaaggacatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcc catatgctggctgccatgaacaaaggttggctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagcctt gacttgaggttagattttttttatattttgttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcc tcctctcctgactactcccagtcatagctgtccctcttctcttatggagatccctcgatcgagggggggcccggtaccgagctcggatccactagtcc agtgtggtggaattggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcc cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcat tctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctga ggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccg ctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggggg ctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagac ggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttg atttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagt tagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctcc ccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttc cgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggct tttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcATGATT GAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGC CGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTAT CGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAG GATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATT CGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCG CGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGA GCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCT TCTGAgcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgg gcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagct tataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgt atcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattc cacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttc cagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcac tgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaa agaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcac aaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttcc gaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtagg tcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca ccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgct cagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatc aatctaaagtatatatgagtaaacttggtctgacagTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
252 Retriction sites full clone:
Unique:
Afl2 Age1 Ahd1 Avr2 Bae1a Bae1b Bbs1 Bcg1a Bcg1b Bpu10 BsrG1 BssH2 Bsu36 Dra3 EcoK Hind3 Hpa1 Kas1 Mlu1 Nar1 Nhe1 Not1 Nru1 PflF1 PflM1 Pml1 Pst1 Rsr2 Sal1 SanD1 SgrA1 Sma1 SnaB1 Stu1 T7RNA T7Ter Xba1
Not found:
Aar1 Afe1 Ale1 Asc1 AsiS1 BbvC1 Bcl1 Blp1 BmgB1 BsaB1 BsiW1 BsmB1 BspE1 BstE2 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 EcoRV FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 I_Ceu loxP Pac1 PshA1 R4atB R4atL R4atP R4atR Sac2 Sbf1 SexA1 Sfi1 Sgf1 Srf1 Swa1 T3RNA PISce Xcm1
253 254 3.3. NvAx2p(-3000)DsRed2
Vector: CMVDsRed2 x Mlu1 (filling) + Nhe1 (filling) (-CMV) Insert: Nv-Ax2gDNApromF8/R7 – Ax2-3127bp x Not1 (filling) + Xmn1 (filling) (NvAx2p(3127)F8/R7) Plasmid: 18 Fasteris: OG003 (TAC2078), OG005 (Ax2F5), OG006 (Ax2F7), OG007 (Ax2R6), OG041 (DsRed2-1) Comments: efficient by injection at the 1 cell stage of Nv (!20- 100ng/ul), longer expression than NvAx2p(-3000)EGFP
255 Full sequence: XXX XXX: Mlu1 + Not1 XXX: pGEM-T easy vector XXX: Ax2NvF8 XXX: Ax2NvR7 XXX: Ax2p(3123bp) XXX XXX: Not1 + Nhe1 XXX: DsRed2CDs gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgatgtacgggccagatatacgcgggccgcgaattcactagtgattgctttgtttaaacgattttgcttggttggggttacttttt aaagggccttgagtaccggtattttgattttctggtgagtcgttttgttttcggtcgccacgtctatgtcccgcagttcttttgtactaccgtcacaa ggccctcaaaaatacctgtgaacataaaagccaaacacaacagcctcaaagtgagcaaaaaacgtgctcagccgagcgaaatcttgatatttttatta gttaggacattaaacatagtaactgcaggatcacaaaaagtgcttcatgtttccttttctctcgtttttgaaaggaggtattctaaactgggatggta attatgtcagttaattggaatggcttgcgacgggtttgcggaggggcgaatacgctcctggttattgcattcatgtgtcaaacgacatctgggttcag ggaatgtctcggaggaaccgaaatttcaggccaattaaattaattttccatatgtcagagaccttgccatcttactaggacttgatcaatcgtttttt tttttctttttggtcaattgtgtttacgctgttaactcgatcatgactttaattaagtcgcgttcgcaagaccaattaatatttgaacaccgcgctcg gctcgttgtgtcctatttgcgcgcgattcaaggctcattggtaatcttttgagctttgacttgcaagactaaatgagctccaactaggtactaaagct ttagagatgtttcccgtggaattaaagaggcataagtttgctttagtgttaccggggtaacttctaagaagaattctggaagtgcaaaaagaaaagaa aaaaaagaagacaaaaacacgtgcttgaattttttgtactgttgttattttttccatactaaaatcgtacatcttctattttacgccttgtttttagt actttgaaaacctttttcacaaataagtcagattgtttaagagaaatacattccagaagtttaaagataatgtatattgatcgtaaaatgaaacgata tcaaaacgaatcgaaataactttgattttcgatcgtttgtgaggatgatttgtcttttcgttcataaccttcgttcgtttttacggttaatgttcttg acatttttgaataaatgttcttgtattcaatctagttctcaaaacaccaggcttttatatttattgaagaggctgttcctatagtataaataaaaggt ttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaactccgtgataagattcagaatcagatcatttgctatcggcatataaggaac taaatttaccaggcaatcttttgataaatattacccgatgagaattgtttattttgtctgcgaaagcaaatcaaagggctgacttctgtatgaagcct atttaccttcaacattcatttcatttccttagtcaagtatttgagaggaagaaacttcggctagcgtattttgttcgagttcgttaaaattatgtttc cggaaagattagagaaacaattattatagtaagtacaggccgttacaatttgatatctttaattgttatctagccgttgtagtccttttcatttagat tagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattcggaacgaaaaatacttgagaatccttttttatatatattctttgagatact aagagattagatcaatcagaagagttaatgaactggtaggttaagaagtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaatagttga ttcttgtttttgttaatgaacgattcattcgtatctttgacgggttaaagttccccactgggcggcacctaatcttgtgtaactaacaagatagtgaa cctacgtggtgtcatgatttacctgctacgcacaaactaagcgcatgtaatgtgttcaccaagacgagaggcattcaatattttatcgctcagttgac gtacaatcgcttcaattgccccccagtattcactaactctactgttttcttcacgcaaaacgcaaacacgatcttttaatattttcctagaaaataat tgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaat acctattctccttcggtctagtcgccaggctatttatttttcttgttatatgattaaaacatatttcacattttggaattgggaaacaattacccatg acgcgtgcagaacggccattctaacgtcctggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcc taaacaaataagttccgaaagaaaacttgcaggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaa acaaagaattatggtctacatctggaagtttggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgc ggagttgagaagacgtcgattgcttacacaggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatgga aacttaatgacaattacggacgaaaaaaggccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaatta tcccgaatgttatactcaagggggaaaataattctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgc ccaaaatctagccaatctccttaaaacatgtcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattcctcatta tgtatgctgtgcttttgtgttgcgaacaataggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatctgaaccca gcagtagaacactcgcagctcccagacggcacacatctcctgataatcgaattcccgcggccctagcgtttaaacttaagcttggtacctcgagataa gcggccgcagatcctgagaacttcagggtgagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaa agttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccatgcatggaccctcatgataattttgtttctttcactttctactctgttga caaccattgtctcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgttta tttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttaggaacaattgttat aattaaatgataaggtagaatatttctgcatataaattctggctggcgtggaaatattcttattggtagaaacaactacaccctggtcatcatcctgc ctttctctttatggttacaatgatatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttc ttctctttcctacagctcctgggcaacgtgctggttgttgtgctgtctcatcattttggcaaagaattcccaccatgggcgcggatccaccggtcgcc accATGGCCTCCTCCGAGAACGTCATCACCGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGA GGGCGAGGGCCGCCCCTACGAGGGCCACAACACCGTGAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGT TCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATG AACTTCGAGGACGGCGGCGTGGCGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCC CTCCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGACCCACA AGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTG GACGCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGTAGcggat cgataattcactcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctc tgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttt tgtgtctctcactcggaaggacatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctg gctgccatgaacaaaggttggctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttgacttgagg ttagattttttttatattttgttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcct gactactcccagtcatagctgtccctcttctcttatggagatccctcgatcgagggggggcccggtaccgagctcggatccactagtccagtgtggtg gaattggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccg tgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctg gggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaag aaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttg ccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctcccttta gggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcg ccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataag ggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtg gaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggc agaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattc tccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggag gcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcATGATTGAACAAGAT GGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCG GCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGG CCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTG TCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCA AGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCG AACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGC CGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGG CGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcgg gactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaa tcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggt
256 tacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatca tgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaaca tacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggga aacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcga cgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgcc gcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgct ccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgactta tcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacac tagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggta gcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaac gaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaag tatatatgagtaaacttggtctgacagTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGC TCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAAT ACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
Retriction sites full clone:
Unique:
Aar1 Afl2 Ale1 Avr2 Blp1 BmgB1 Bpu10 BspE1 BstB1 BstE2 BstZ1 Bsu36 Dra3 EcoK FspA1 Kas1 Mlu1 Nar1 Nhe1 Not1 Nru1 Pac1 PflF1 Rsr2 Sac2 Sal1 SanD1 Sbf1 Sma1 T7Ter Xba1
Not found:
Afe1 Asc1 AsiS1 Bae1a Bae1b BbvC1 Bcl1 BsaB1 BsiW1 BsmB1 BsrG1 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 I_Ceu loxP PshA1 R4atB R4atL R4atP R4atR SexA1 Sfi1 Sgf1 SgrA1 SnaB1 Srf1 Swa1 T3RNA T7RNA PISce Xcm1
257 258 3.4. NvAx2p(-2000)DsRed2
Vector: CMVDsRed2 x Nru1 + Afl2 (-CMV) Insert: PCR281 6-9 x Nru1 + Afl2: Template: Nv-Ax2gDNApromF8/R1 – Ax2-4300bp Primers: Nru1-NvAx2p2000F/Afl2-NvAx2ATGR
Plasmid: 1 Fasteris: OG56 (Ax2NvF10), OG57 (Ax2NvR5) Comments:
259 Full sequence: XXX: Nru1-NvAx2p2000F XXX: Afl2-NvAx2ATGR XXX: Ax2p(1999bp) XXX: DsRed2CDs gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgagaataaatgttcttgtattcaatctagttctcaaaacaccaggcttttatatttattgaagaggctgttcctatagtataaat aaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaactccgtgataaaattcagaattagatcatttgctatcggcatat aaggaactaaatttaccaggcaatcttttgataaatattacccgatgagaattgtttattttgtctgcgaaagcaaatcaaagagctgacttctgtat gaagcctatttaccttcaacattcatttcatttccttagtcaagtatttgagaggaagaaacttcggctagcgtattttgttcgagttcgttaaaatt atgtttccggaaagattagagaaacaattattatagtaaatacaggccgttacaatttgatatctttaattgttatctagccgttgtagtccttttca tttagattagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattcggaacgaaaaatacttgagaatccttttttatatatattctttg agatactaagagattagatcaatcagaagagttaatgaactggtaggttaagaagtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaa tagttgattcttgtttttgttaatgaacgattcattcgtatctttgacgggttaaagttccccactgggcggcacctaatcttgtgtaactaacaaga tagtgaacctacgtggtgtcatgatttacctgctacgcacaaactaagcgcatgtaatgtgttcaccaagacgagaggcattcaatattttatcgctc agttgacgtacaatcgcttcaattgccccccagcgttcactaactctactgttttcttcacgcaaaacgcaaacacgatcttttaatattttcctaga aaataattgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaa aaagaatacctattctccttcggtctagtcgccaggctatttatttttcttgttatatgattaaaacatatttcacattttggaattgggaaacaatt acccatgacgcgtgcagaacggccattctaacgtcctggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattaca tgaatcctaaacaaataagttccgaaagaaaacttgcaggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttaccc ttgccaaacaaagaattatggtctacatctggaagtttggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaact cgagtgcggagttgagaagacgtcgattgcttacacaggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcga ggatggaaacttaatgacaattacggacgaaaaaaggccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagta caaattatcccgaatgttatactcaagggggaaaataattctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagg gccccgcccaaaatctagccaatctccttaaaacatgtcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattc ctcattatgtatgctgtgcttttgtgttgcgaacaataggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatct gaacccagcagtagaacactcgcagctcccagacggcacacatctcctgatatccttaagcttggtacctcgagataagcggccgcagatcctgagaa cttcagggtgagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgttta gaatgggaagatgtcccttgtatcaccatgcatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttat tttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtact ttctctaatcacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttaggaacaattgttataattaaatgataaggtagaa tatttctgcatataaattctggctggcgtggaaatattcttattggtagaaacaactacaccctggtcatcatcctgcctttctctttatggttacaa tgatatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctctttcctacagctcct gggcaacgtgctggttgttgtgctgtctcatcattttggcaaagaattcccaccatgggcgcggatccaccggtcgccaccATGGCCTCCTCCGAGAA CGTCATCACCGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACG AGGGCCACAACACCGTGAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTG TACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGT GGCGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTGATGC AGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGACCCACAAGGCCCTGAAGCTGAAGGAC GGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACGCCAAGCTGGACATCAC CTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGTAGcggatcgataattcactcctcaggt gcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggaca tcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaagg acatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctgccatgaacaaaggttg gctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttgacttgaggttagattttttttatatttt gttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactcccagtcatagct gtccctcttctcttatggagatccctcgatcgagggggggcccggtaccgagctcggatccactagtccagtgtggtggaattggccgctcgagtcta gagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagga cagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctaggg ggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgct cctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtcca cgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcc tattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctcccca gcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgca tctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaa ttttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaag ctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcATGATTGAACAAGATGGATTGCACGCAGGTTCTCC GGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCC CGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGC GCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGC CGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGC GAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG GCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGA CTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCC TCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcgggactctggggttcgaaatga ccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggc tggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcat cacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacct ctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaag tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgca ttaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgc ggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaag gccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccg cctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcac gaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccac tggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggta tctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaag cagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGC CGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA
260 ATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGG CGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCAT GGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGT GTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCAC CAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT CCCCGAAAAGTGCCACCTGACGTC
Retriction sites full clone:
Unique:
Aar1 Afl2 Age1 Ale1 Avr2 Bcg1a Bcg1b Bpu10 BspE1 BstB1 BstE2 BstZ1 Bsu36 Dra3 Ecl2 EcoK EcoR1 FspA1 Kas1 Mlu1 Nar1 Nhe1 Not1 Nru1 PflF1 Pml1 Rsr2 Sac1 Sal1 SanD1 Sbf1 Sma1 Spe1 T7Ter Xba1
Not found:
Afe1 Asc1 AsiS1 Bae1a Bae1b BbvC1 Bcl1 Blp1 BmgB1 BsaB1 BsiW1 BsmB1 BsrG1 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 I_Ceu loxP Pac1 PshA1 R4atB R4atL R4atP R4atR Sac2 SexA1 Sfi1 Sgf1 SgrA1 SnaB1 Srf1 Swa1 T3RNA T7RNA PISce Xcm1
261 262 3.5. NvAx2p(-1000)DsRed2
Vector: CMVDsRed2 x Nru1 + Nhe1 (-CMV) Insert: PCR281 1-4 x Nru1 + Nhe1: Template: Nv-Ax2gDNApromF8/R1 – Ax2-4300bp Primers: Nru1-NvAx2p1000F/Nhe1-NvAx2ATGR
Plasmid: 39 Fasteris: OG55 (Ax2NvF10) Comments:
263 Full sequence: XXX: Nru1-NvAx2p1000F XXX: Nhe1-NvAx2ATGR XXX: Ax2p(1999bp) XXX: DsRed2CDs gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgatgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaatacctattctccttcg gtctagtcgccaggctatttatttttcttgttatatgattaaaacatatttcacattttggaattgggaaacaattacccatgacgcgtgcagaacgg ccattctaacgtcctggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaacaaataagttc cgaaagaaaacttgcaggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaagaattatggt ctacatctggaagtttggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgagaagacg tcgattgcttacacaggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgacaatt acggacgaaaaaaggccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttatac tcaagggggaaaataattctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaa tctccttaaaacatgtcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttt tgtgttgcgaacaataggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcg cagctcccagacggcacacatctcctgatatcgctagcgtttaaacttaagcttggtacctcgagataagcggccgcagatcctgagaacttcagggt gagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaa gatgtcccttgtatcaccatgcatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttc attttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaat cacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttaggaacaattgttataattaaatgataaggtagaatatttctgc atataaattctggctggcgtggaaatattcttattggtagaaacaactacaccctggtcatcatcctgcctttctctttatggttacaatgatataca ctgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctctttcctacagctcctgggcaacgt gctggttgttgtgctgtctcatcattttggcaaagaattcccaccatgggcgcggatccaccggtcgccaccATGGCCTCCTCCGAGAACGTCATCAC CGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCCACA ACACCGTGAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAG CACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGCGACCGT GACCCAGGACTCCTCCCTGCAGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTGATGCAGAAGAAGA CCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGACCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCAC TACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACGCCAAGCTGGACATCACCTCCCACAA CGAGGACTACACCATCGTGGAGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGTAGcggatcgataattcactcctcaggtgcaggctgc ctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggacatcatgaagc cccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatatggg agggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctgccatgaacaaaggttggctataaag aggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttgacttgaggttagattttttttatattttgttttgtgt tatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactcccagtcatagctgtccctctt ctcttatggagatccctcgatcgagggggggcccggtaccgagctcggatccactagtccagtgtggtggaattggccgctcgagtctagagggcccg tttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccact cccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggg ggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatcccc acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgct ttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacct cgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttta atagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggtta aaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcaga agtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaatta gtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaatttttttta tttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccggga gcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTG GGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTT TTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTG CTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGT ATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTA CTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATG CCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG GCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcgggactctggggttcgaaatgaccgaccaag cgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatc ctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaattt cacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctaga gcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcc tggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaat cggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcgg tatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaac cgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgac aggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcc cttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctc tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagatt acgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcat gagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagTT ACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGG GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGC CGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACA TGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGC AGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATT GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA GTGCCACCTGACGTC
264 Retriction sites full clone:
Unique:
Aar1 Afl2 Age1 Ale1 Avr2 Bcg1a Bcg1b Bpu10 Bsm1 BstB1 BstE2 BstZ1 Bsu36 Dra3 Ecl2 EcoK EcoR1 EcoRV FspA1 Kas1 Mlu1 Nar1 Nhe1 Not1 Nru1 PflF1 Pml1 Rsr2
Not found:
Afe1 Asc1 AsiS1 Bae1a Bae1b BbvC1 Bcl1 Blp1 BmgB1 BsaB1 BsiW1 BsmB1 BspE1 BsrG1 BxatB BxatL BxatR BxatP _Chi Cla1 EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 I_Ceu loxP Pac1 PshA1 R4atB R4atL R4atP R4atR Sac2 SexA1 Sfi1 Sgf1 SgrA1 SnaB1 Srf1 Swa1 T3RNA T7RNA PISce Xcm1 Sac1 Sal1 SanD1 Sbf1 Sma1 Spe1 T7Ter Xba1
265 266 3.6. CMVAx2CDsFLAG
Vector: CMVDsRed2 x Age1 (filling) + Cla1 (filling) Insert: p-Xma1-Ax2CDs-FLAG-Nar1 X Sma1 + Nar1 Plasmid: 9 Fasteris: Fasteris OG39 (TAC2079), OG40 (NvAx2F4) Comments: Never worked, high toxicity
267 Full sequence: XXX: CMV propmoter XXX XXX: Age1+Sma1 XXX: NvAx2CDs + NvAx23’UTR XXX XXX: Nar1 + Cla1 + XXX: FLAG gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcc catatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgcc aagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccac cccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtag gcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactataggg agacccaagctggctagcgtttaaacttaagcttggtacctcgagataagcggccgcagatcctgagaacttcagggtgagtttggggacccttgatt gttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccat gcatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaactttttcgt taaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaat cagggtatattatattgtacttcagcacagttttaggaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgt ggaaatattcttattggtagaaacaactacaccctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggataaa atactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctctttcctacagctcctgggcaacgtgctggttgttgtgctgtctc atcattttggcaaagaattcccaccatgggcgcggatccaccgggggccaccATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGT CGGTACCAACGAGTACTTCAGAGCCGCGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTA TGCCAGCTCTGCATTCCTACTAGTGCTAGCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGA ACTACAGAAAGATCATATTTTGCTGCAACAACACTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTTATGCATCATCACGAGATCCTGACA GTCCATCAAGGGGAGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAGCATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGA TATCTTTCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAGCAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAA GAAGGACAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCACCGGTAGAATGGATGGTGTAGACTACA AGGACGACGATGACAAGTGAacactaaaattgaaccataattgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcataat ttgtatagaatctatagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgagggata aattgtaaaacaaaaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaacttttctgaga attttaccatgcatttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaggcgat aattcactcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgcc aaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtg tctctcactcggaaggacatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctg ccatgaacaaaggttggctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttgacttgaggttag attttttttatattttgttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgact actcccagtcatagctgtccctcttctcttatggagatccctcgatcgagggggggcccggtaccgagctcggatccactagtccagtgtggtggaat tggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcc ttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggg gtggggtggggcaggacagcaagggggaggattgggaagacaatagcangcatgctggggatgcggtgggctctatggcttctgaggcggaaagaacc agctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccag cgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggt tccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccct ttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggat tttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaa gtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaa gtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccg ccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcct aggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcATGATTGAACAAGATGGAT TGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTG TCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCAC GACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCAT CTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCG AAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACT GTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCT TTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAA TGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcgggact ctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgt tttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttaca aataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtc tgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacg agccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacc tgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgc tcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagc aaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgct caagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctt accggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactaga agaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcgg tttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaa actcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtata tatgagtaaacttggtctgacagTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAG CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGG TTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAG CATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
268 Retriction sites full clone:
Unique:
Afl2 Age1 Ahd1 Avr2 Bpu10 Bsg1 BsrG1 BssH2 BstZ1 Cla1 Dra3 EcoK EcoR1 EcoRV Kas1 Nar1 Not1 Nru1 PflF1 PflM1 Pst1 Rsr2 Sal1 SanD1 Sma1 SnaB1 Stu1 T7RNA Xba1
Not found:
Aar1 Afe1 Ale1 Asc1 AsiS1 Bae1a Bae1b BbvC1 Bcl1 Blp1 BmgB1 BsaB1 BsiW1 BsmB1 BspE1 BstE2 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT Fse1 FspA1 Hpa1 I_Ceu loxP Pac1 Pml1 PshA1 R4atB R4atL R4atP R4atR Sac2 Sbf1 SexA1 Sfi1 Sgf1 SgrA1 Srf1 Swa1 T3RNA PISce Xcm1
269 270 3.7. Ax2(-3000)Ax2CDsFLAG
Vector: CMVAx2CDsFLAG pl9 x Nru1 (filling) + Not1 (filling) (-CMV) Insert: Nv-Ax2gDNApromF8/R7 – Ax2-3127bp x Not1 (filling) + Xmn1 (filling) Plasmid: 15 Fasteris: OG51 (Ax2NvF11) Comments: Never worked, high toxicity
271 Full sequence: XXX XXX: Nru1 + Not1 XXX: pGEM-T easy vector XXX: Ax2NvF8 XXX: Ax2p(3123bp) XXX: Ax2NvR7 XXX XXX: Not1 + Not1 XXX: FLAG gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtg ttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgggccgcgaattcactagtgattgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtattt tgattttctggtgagtcgttttgttttcggtcgccacgtctatgtcccgcagttcttttgtactaccgtcacaaggccctcaaaaatacctgtgaaca taaaagccaaacacaacagcctcaaagtgagcaaaaaacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaact gcaggatcacaaaaagtgcttcatgtttccttttctctcgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggc ttgcgacgggtttgcggaggggcgaatacgctcctggttattgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaat ttcaggccaattaaattaattttccatatgtcagagaccttgccatcttactaggacttgatcaatcgtttttttttttctttttggtcaattgtgtt tacgctgttaactcgatcatgactttaattaagtcgcgttcgcaagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctatttgcgcgc gattcaaggctcattggtaatcttttgagctttgacttgcaagactaaatgagctccaactaggtactaaagctttagagatgtttcccgtggaatta aagaggcataagtttgctttagtgttaccggggtaacttctaagaagaattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgc ttgaattttttgtactgttgttattttttccatactaaaatcgtacatcttctattttacgccttgtttttagtactttgaaaacctttttcacaaat aagtcagattgtttaagagaaatacattccagaagtttaaagataatgtatattgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttg attttcgatcgtttgtgaggatgatttgtcttttcgttcataaccttcgttcgtttttacggttaatgttcttgacatttttgaataaatgttcttgt attcaatctagttctcaaaacaccaggcttttatatttattgaagaggctgttcctatagtataaataaaaggtttaaacggtgtcccgtaataaagt cgttatgtccttaaactaattaactccgtgataagattcagaatcagatcatttgctatcggcatataaggaactaaatttaccaggcaatcttttga taaatattacccgatgagaattgtttattttgtctgcgaaagcaaatcaaagggctgacttctgtatgaagcctatttaccttcaacattcatttcat ttccttagtcaagtatttgagaggaagaaacttcggctagcgtattttgttcgagttcgttaaaattatgtttccggaaagattagagaaacaattat tatagtaagtacaggccgttacaatttgatatctttaattgttatctagccgttgtagtccttttcatttagattagtaggcccaggttgttgaacca aatcgtgtagagtacaatcattcggaacgaaaaatacttgagaatccttttttatatatattctttgagatactaagagattagatcaatcagaagag ttaatgaactggtaggttaagaagtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaatagttgattcttgtttttgttaatgaacgat tcattcgtatctttgacgggttaaagttccccactgggcggcacctaatcttgtgtaactaacaagatagtgaacctacgtggtgtcatgatttacct gctacgcacaaactaagcgcatgtaatgtgttcaccaagacgagaggcattcaatattttatcgctcagttgacgtacaatcgcttcaattgcccccc agtattcactaactctactgttttcttcacgcaaaacgcaaacacgatcttttaatattttcctagaaaataattgcaataagtttggaagcgtgtgc gatatttgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaatacctattctccttcggtctagtcg ccaggctatttatttttcttgttatatgattaaaacatatttcacattttggaattgggaaacaattacccatgacgcgtgcagaacggccattctaa cgtcctggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaacaaataagttccgaaagaaa acttgcaggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaagaattatggtctacatctg gaagtttggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgagaagacgtcgattgct tacacaggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgacaattacggacgaa aaaaggccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttatactcaaggggg aaaataattctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaatctccttaa aacatgtcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttgtgttgcg aacaataggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcgcagctccca gacggcacacatctcctgataatcgaattcccgcggccggccgcagatcctgagaacttcagggtgagtttggggacccttgattgttctttcttttt cgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccatgcatggaccctca tgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaactttttcgttaaactttagctt gcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattat attgtacttcagcacagttttaggaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgtggaaatattctta ttggtagaaacaactacaccctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggataaaatactctgagtcc aaaccgggcccctctgctaaccatgttcatgccttcttctctttcctacagctcctgggcaacgtgctggttgttgtgctgtctcatcattttggcaa agaattcccaccatgggcgcggatccaccgggggccaccATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGTCGGTACCAACGAG TACTTCAGAGCCGCGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTATGCCAGCTCTGCA TTCCTACTAGTGCTAGCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGAACTACAGAAAGAT CATATTTTGCTGCAACAACACTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTTATGCATCATCACGAGATCCTGACAGTCCATCAAGGGG AGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAGCATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCC TTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAGCAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAA GCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGTCGCCAGCAAGCACCGGTAGAATGGATGGTGTAGACTACAAGGACGACGATGA CAAGTGAacactaaaattgaaccataattgtacagtttgtatatagtttaatgtactatattcggggcaaccttgttttcataatttgtatagaatct atagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtttgtgagggataaattgtaaaacaa aaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaacttttctgagaattttaccatgca tttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtcaggcgataattcactcctca ggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatgggg acatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcgga aggacatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctgccatgaacaaagg ttggctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttgacttgaggttagattttttttatat tttgttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactcccagtcata gctgtccctcttctcttatggagatccctcgatcgagggggggcccggtaccgagctcggatccactagtccagtgtggtggaattggccgctcgagt ctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctg gaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggca ggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctcta gggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgccc gctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgc tttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagt ccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctcc ccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcat gcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgac taattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaa aagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcATGATTGAACAAGATGGATTGCACGCAGGTTC TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGC GCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCT TGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCC TGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCG AGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTC AAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCAT CGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCT
272 TCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcgggactctggggttcgaaa tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatag catcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcga cctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcata aagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagct gcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggc tgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaa aaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg gcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgt ccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtg cacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagc cactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttg gtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgc aagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagg gattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaactt ggtctgacagTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATA ACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCC AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAG TTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACT CATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTGACGTC
Retriction sites full clone:
Unique:
Aar1 Ahd1 Avr2 Bgl1 Blp1 BmgB1 Bpu10 BspE1 BsrG1 BstZ1 Cla1 Dra3 EcoK Fse1 Kas1 Nar1 Pac1 PflF1 PflM1 Rsr2 Sac2 Sal1 SanD1 Sma1 Stu1 Xba1
Not found:
Afe1 Afl2 Ale1 Asc1 AsiS1 Bae1a Bae1b BbvC1 Bcl1 BsaB1 BsiW1 BsmB1 BstE2 BxatB BxatL BxatR BxatP _Chi EcoN1 FCatB FCatL FCatR FCatP ScFRT FspA1 I_Ceu loxP Not1 Nru1 PshA1 R4atB R4atL R4atP R4atR Sbf1 SexA1 Sfi1 Sgf1 SgrA1 SnaB1 Srf1 Swa1 T3RNA T7RNA PISce Xcm1
273
274 Séquences des gènes et amorces / Gene sequences and primers
Nematostella vectensis sequences 276 1.1. Nematostella vectensis Anthox2 genomic sequence 276 1.2. Nematostella vectensis Actin 277 1.3. Nematostella vectensis SoxB2 277 1.4. Nematostella vectensis Sox2 277 1.5. Nematostella vectensis Forkhead 278
Clytia hemisphaerica sequences 279 2.1. Clytia hemisphaerica Gsx 279 2.2. Clytia hemisphaerica Pdx 279 2.3. Clytia hemisphaerica CnoxC 280
Turritopsis dohrnii sequences 281 3.1. Turritopsis dohrnii Pdx 281 3.2. Turritopsis dohrnii CnoxA 281
Hydra vulgaris sequence 282 4.1. Hydra vulgaris cnox2 genomic sequence 282
Hydra actinEGFP EcoR1 283
CMV-bglobinintron-DsRed2- bglobinpolyA 284
275 Nematostella vectensis sequences
1.1. Nematostella vectensis Anthox2 genomic sequence Ax2F8-> <-Ax2R8 cgctttgtttaaacgattttgcttggttggggttactttttaaagggccttgagtaccggtattttgattttctggtgagtcgttttgttttcggtcgcc 100 <-Ax2R9 acgtctatgtcccgcagttctttttgtactaccgtcacaaggccctcaaaaatacctgtgaacataaaagccaaacacaacagcctcaaagtgagcaaaa 200 aacgtgctcagccgagcgaaatcttgatatttttattagttaggacattaaacatagtaactgcaggatcacaaaaagtgtttcatgtttccttttctct 300 cgtttttgaaaggaggtattctaaactgggatggtaattatgtcagttaattggaatggcttgcgacgggtttgcggaggggcgaatacgctcctggtta 400 ttgcattcatgtgtcaaacgacatctgggttcagggaatgtctcggaggaaccgaaatttcaggccaattaaattaattttccatatgtcagagaccttg 500 ccatcttactaggacttgatcaatcgttttttttttttctttttggtcaattgtgtttacgctgttaactcgatcatgactttaattaagtcgcgttcgc 600 aagaccaattaatatttgaacaccgcgctcggctcgttgtgtcctatttgcgcgcgattcaaggctcattggtaatcttttgagctttgacttgcaagac 700 Ax2F9-> taaatgagctccaactaggtactaaagctttagagatgtttcccgtggaattaaagaggcataagtttgctttagtgttaccggggtaacttctaagaag 800 aattctggaagtgcaaaaagaaaagaaaaaaaagaagacaaaaacacgtgcttgaattttttgtactgttgttattttttccatactaaaatcgtacatc 900 ttctgttttacgccttgtttttagtactttgaaaacctttttcacaaataagtcagattgtttaagagaaatacattccagaagtttaaagataatgtat 1000 EcoRV <-Ax2R6 Ax2F7-> attgatcgtaaaatgaaacgatatcaaaacgaatcgaaataactttgattttcgatcgtttgtgaggatgatttgtcttttcgttcataaccttcgttcg 1100 Nru1-Ax2pr-2000F-> tttttacggttaatgttcttgacatttttgaataaatgttcttgtattcaatctagttctcaaaacaccaggcttttatatttattgaagaggctgttcc 1200 tatagtataaataaaaggtttaaacggtgtcccgtaataaagtcgttatgtccttaaactaattaactccgtgataaaattcagaattagatcatttgct 1300 atcggcatataaggaactaaatttaccaggcaatcttttgataaatattacccgatgagaattgtttattttgtctgcgaaagcaaatcaaagagctgac 1400 Nhe1 ttctgtatgaagcctatttaccttcaacattcatttcatttccttagtcaagtatttgagaggaagaaacttcggctagcgtattttgttcgagttcgtt 1500 <-Ax2R5 Ax2F6 EcoRV aaaattatgtttccggaaagattagagaaacaattattatagtaaatacaggccgttacaatttgatatctttaattgttatctagccgttgtagtcctt 1600 ttcatttagattagtaggcccaggttgttgaaccaaatcgtgtagagtacaatcattcggaacgaaaaatacttgagaatccttttttatatatattctt 1700 tgagatactaagagattagatcaatcagaagagttaatgaactggtaggttaagaagtttattttgctcaagttcctgtgatcaagaaatgccgcaaaaa 1800 tagttgattcttgtttttgttaatgaacgattcattcgtatctttgacgggttaaagttccccactgggcggcacctaatcttgtgtaactaacaagata 1900 gtgaacctacgtggtgtcatgatttacctgctacgcacaaactaagcgcatgtaatgtgttcaccaagacgagaggcattcaatattttatcgctcagtt 2000 Ax2F5 gacgtacaatcgcttcaattgccccccagcgttcactaactctactgttttcttcacgcaaaacgcaaacacgatcttttaatattttcctagaaaataa 2100 Nru1-Ax2p-1000F -> ttgcaataagtttggaagcgtgtgcgatatttgaatgaagtcttgtcgccggcaaattaaaggtgtttattccatttttgaacagggggtaaaaagaata 2200 cctattctccttcggtctagtcgccaggctatttatttttcttgttatatgattaaaacatatttcacattttggaattgggaaacaattacccatgacg 2300 cgtgcagaacggccattctaacgtcctggaaagcctaattaacgctctcaatgccataaagctcaatttgaaaaattagtttattacatgaatcctaaac 2400 aaataagttccgaaagaaaacttgcaggtttccaaaaacagtttacattgattaaacgttttatagcgtctagcttgcgtttacccttgccaaacaaaga 2500 attatggtctacatctggaagtttggaattattggggagagaattggggagtttgtttcaagtggttgtagaaaaccggaaactcgagtgcggagttgag 2600 aagacgtcgattgcttacacaggaagcgtggcgcgcattgtagcaaaagttaactcggatacaatagctttggcgagtgcgaggatggaaacttaatgac 2700 aattacggacgaaaaaaggccgagggattgttaacaattcaaatcggaatgtcactttcagtagcaggtgacagcaagtacaaattatcccgaatgttat 2800 actcaagggggaaaataattctgtgaaacaggaccaatgaaatttaaaaggcttcgttattttagtttagttctgagggccccgcccaaaatctagccaa 2900 tctccttaaaacatgtcaactacctgtatcagtaattaaccttgctttaaagggcttttctaaagctttaaaattcctcattatgtatgctgtgcttttg 3000 Ax2F11 tgttgcgaacaataggcgacgtcttagacacgtgtgctctatccagggtggtcaatatatagagccctgcatctgaacccagcagtagaacactcgcagc 3100 Ax2MO Ax2F10 / Ax2R7 / Afl2/Nhe1-Ax2ATGR Ax2R4 / Ax2R3 Ax2F1 TCCCAGACGGCACACATCTCCTGATATCATGTCTTCGTCCTTCTACATTGACTCGCTTATTTCAAAAGCCAAGTCGGTACCAACGAGTACTTCAGAGCCG 3200 M S S S F Y I D S L I S K A K S V P T S T S E P Ax2F2 -> Ax2F3 Nhe1 CGACACACTTACGAATCTCCTGTTCCTTGTTCTTGTTGTTGGACTCCGACGCAACCTGACCCAAGTAGCCTATGCCAGCTCTGCATTCCTACTAGTGCTA 3300 R H T Y E S P V P C S C C W T P T Q P D P S S L C Q L C I P T S A S GCGTGCACCCGTATATGCATCATGTGAGAGGCGCATCGATACCCTCAGGGGCCGGACTCTACTCTAGGGAACTACAGAAAGATCATATTTTGCTGCAACA 3400 V H P Y M H H V R G A S I P S G A G L Y S R E L Q K D H I L L Q Q ACACTACGCTGCGACAGAGGAGGAGAGACTTCATCTTGCGAGTTATGgtgagttgcgctttccacgccaaaacaaggcttcaaggaccctaacaggtgtc 3500 H Y A A T E E E R L H L A S Y A Cgacactaaagctctgtcaaaaatatccatgatgtcctttgaaaaaatccattgaacttaatggtgaaatataattattatttccttaattctgtgttta 3600 atagaattttttgtctttttttagCATCATCACGAGATCCTGACAGTCCATCAAGGGGAGGAAATTCACGGTCAAAGCGGATCAGAACGGCATACACCAG 3700 S S R D P D S P S R G G N S R S K R I R T A Y T S CATGCAACTACTCGAGCTTGAGAAAGAGTTCAGTCAAAACAGATATCTTTCTCGCCTTCGCCGCATTCAAATCGCCGCTTTGCTAGATCTTTCCGAGAAG 3800 M Q L L E L E K E F S Q N R Y L S R L R R I Q I A A L L D L S E K CAAGTGAAAATCTGGTTCCAAAACCGACGCGTTAAATGGAAGAAGGACAAGAAAGCAGCGCAACATGGCACAACAACCGAGACTTCTTCTTGTCCTTCGT 3900 Q V K I W F Q N R R V K W K K D K K A A Q H G T T T E T S S C P S S CGCCAGCAAGCACCGGTAGAATGGATGGTGTATGAACACTAAAATTGAACCATAATTGTACAGTTTGTATATAGTTTAATGTACTATATTCGGGGCAACC 4000 P A S T G R M D G V * ttgttttcataatttgtatagaatctatagtttggcgaacgaactgtgatcgcccaatttatttcgacttctaatttggttttaacaccatttcgaagtt 4100 Ax2R2 tgtgagggataaattgtaaaacaaaaacaatttaaaagccttaaatggaaaggcggggggatatacacaaaaaaattgcatgtaaattttcgtataaact 4200 Ax2F4 Ax2R1 tttctgagaattttaccatgcatttgtataaaacggcaagagatttgccggcctgtaacaataattagttaatgaagttggaagtgaataaaagcttgtc 4300 acttat 4306
276 1.2. Nematostella vectensis Actin Aau-actinF-> <-Nv-actin-R2 <-Nv-actin-R1 AGGTCATCACCATAGGAAACGAGCGCTTCAGGTGCCCCGAGGCTATGTTCCAGCCTTCCTTCCTGGGTATGGAATCCGCTGGTATCCATGAGACCACCTA 100 V I T I G N E R F R C P E A M F Q P S F L G M E S A G I H E T T Y Ch,Nv-actin-F1-> CAACTCCATCATGAAGTGCGACGTCGACATCCGTAAAGATTTGTATGCTAACACTGTCCTGTCTGGAGGCTCAACCATGTACCCAGGCATCGCCGACCGC 200 N S I M K C D V D I R K D L Y A N T V L S G G S T M Y P G I A D R ATGCAAAAAGAAATTACTTCCCTCGCTCCCCCGACCATGAAAATCAAGATCATCGCTCCACCAGAGAGGAAATACTCCGTCTGGATCGGAGGCTCCATCC 300 M Q K E I T S L A P P T M K I K I I A P P E R K Y S V W I G G S I L Nv-actin-R3-> Aau-actinR-> TCGCTTCCCTGTCCACCTTCCAACAGATGTGGATCTTCAAA 341 A S L S T F Q Q M W I F K
1.3. Nematostella vectensis SoxB2 Nv-SoxB2-F1-> CGCGCCTACCACTGGTACCATAatactgtgatgttgctcctttggtaacgtcagttcactagagcagagctcgcgatcacagctcagcgatcacatagcg 100 SoxB2MO Nv-SoxB2-F1-> acacagcggagagtatacacacagttcctacaacATGGGCAAGCAAGAAGACGGGCATGTGAAGAGACCGATGAATGCGTTCATGGTTTGGTCACGAGGC 200 M G K Q E D G H V K R P M N A F M V W S R G AAGAGAAAGCATTACGCCAGTATTAATCCACGAATGCACAACTCTGAGATCAGCAAGAGGTTAGGAGCGGAGTGGAAAATGTTGACGGCTGAAGAGAAGG 300 K R K H Y A S I N P R M H N S E I S K R L G A E W K M L T A E E K E AGCCATTTATAGCGGAGGCAAAGCGCCTGCAAGCACTTCACATCCAGGAGCACCCGGACTATAAGTACAAACCCAAACGGCGCAAACCCAAATCAGTGCA 400 P F I A E A K R L Q A L H I Q E H P D Y K Y K P K R R K P K S V Q GAAGAAAGACCTCGCTTCCCCTGTCTTCTCCCCGTACGCTGCATCTATGATGGCGGTTGACAAATTCTCGACGAATCAATTGCCACAAACAATAGCTCAT 500 K K D L A S P V F S P Y A A S M M A V D K F S T N Q L P Q T I A H TCAGTTGCTTTGTCCCAGGATCCAATGTATTCGAAAATAAATGGTGGACCGCCGTTTCATCACTCCGTGTCGCCGGGTTACCCCGTGATATACCCGAGTG 600 S V A L S Q D P M Y S K I N G G P P F H H S V S P G Y P V I Y P S V TCAGTAATGGAGGGAATGTGCACTCAGGCTCGCCTTCTTCCCGCCAAATCTTCGCCGGCGCCATGGACAGTACTCACTCGTTTCGCGCGAGTGATATGAT 700 S N G G N V H S G S P S S R Q I F A G A M D S T H S F R A S D M M GGCGGGTCGACCAGTGTATAGCAGTCAGGGCTACCAGGGGGCTCTCCACTCGCAGGTCCAGCAGCGACTATCGCAAGTGGAGGATAGCAGAGGTATGAAG 800 A G R P V Y S S Q G Y Q G A L H S Q V Q Q R L S Q V E D S R G M K TCCATGAACGCGACTCCGAGTCCACCCGTGTCAAGTCCCGACCCCATGTCTAAAGCGTACTCGAGCTCCGAGCTGAGTAACCAACGAGTGTGGCAGCCAC 900 S M N A T P S P P V S S P D P M S K A Y S S S E L S N Q R V W Q P Q <-Nv-SoxB2-R1 AGCAGGACCTAACCAGAACCGTCGCTTACGTACCGGTATTGCTCTAGtgtatataaaaacaccagtactcacatataggacagttaaaacctttgtctac 1000 Q D L T R T V A Y V P V L L * cttgaatagtcaatgtgcaggggtttagattcggccatgaccttgtttacgcaggttcggtatatttaagttcgaaaagttttaatttgaaaccggagtc 1100 gagggagagagagcgtagattattaggtataagtacgtgctaatttgtcagtgacaaagaggccggtctcgatgacagtatttaggggtctatattttct 1200 ctagtggtgaaatttccgttcgggctgcgctaaatgatgtttaatgagtcgaaatttcttatttgccaaaaaggttaatggcacagaccacacaataact 1300 tggttaacacgcacaactataccactgttataaattcgtcttgatcgctaatttatcttggccacggggcccaaaatcgaaagctgatacggcttcaacg 1400 cgcaccaactcgaaagtgcaattggatttaatatatgcgcattttggttgcgaacgggcagggtaatcaacaagaaacggttctcttccaggcggaattt 1500 cacactggagaaagtgattagaaaatccgaatccagactacgagctctatgctttccttttgggctcatcggccgtcccttggaacaatactctaatctt 1600 tggccccaaaccaacacaaatcaggcagtacttttgttatgtaaagtcggtaattcagtttatgtgataacatgagagagaaacgtcaacgaagaaacaa 1700 aacaatttctaaatacatgacttagatttcctctaaacacaagccaaatgatactcatgtttgcgtctttttcgtacaagtttctgacaatgcgcgcgac 1800 actttgtttgcggaactcgaccaaggacattacgaaaccatcctatttttgcatattggcgacgagcgcaatttaaggtgccatttagtttcggggtgaa 1900 ttgacgctgagtactgcgttactaagtatcaatctaggtggataataaggatgaacctttctaaatcggcaatatgctctcttctgaacacggtttattt 2000 ggagttatttgttgttcagattctagcacatgtggaacgcgtgtacgcgctccggtgaccatgctttcgatctttctcattataacaaagaaaaaaaata 2100 cggctccacttcgaaggttttttaagcaacatttaaactaattctcgttatagagccataggattacgaggatttcgaaaaggtttaagctagcgcacgt 2200 ggacttctcaaggtgactcggcaacccgaattaatttattgaagattcttgacacgttacaaatttatgatctaagcgctgttgacagagagattgttag 2300 tctaaaaagtgtacatatctatgctagctcaacacatgccgatatcaagatgccgataagggtttatttagtaaatcatgacactcgcgaaagcttctgc 2400 ctcagtaaacgctatggaaatcaatagattatcccaccgcgcgctgcatatttctctacttaagagtctttttttgctaagtatgaagaacgagcgtaga 2500 actagaagtacctctcgggtgaacaagcatacctactgcatcaaataagttcagagttgatgaaagtgttaacctttttaataataaagcataaatcttg 2600 gttcaataatactatttactctatcaaatgaaatgttttctctctccacatttattccataataggcatttatataggtgtttatttatttttgtagaat 2700 aaacctaaagtaacatatgtatgtatatatattggccctatgctacgtttctgcgatgtttgaaaaaaaataggaaaggcttatatcatatgtaatttaa 2800 gatataggaaaagcattttatgatgaaaaaaataaacgaaattgatttcccaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2880
1.4. Nematostella vectensis Sox2 Nv-Sox2-F1-> ATGACCAAACCAGGAGATCATATTAAACGGCCCATGAACGCGTATATGGTATGGTCGAGAAAAGAGCGAAGGAGGATAGCAGAGGAATGCCCGCGTATGT 100 M T K P G D H I K R P M N A Y M V W S R K E R R R I A E E C P R M L TGAACTCGGAGATAAGCAAAAGGCTAGGACTCGAGTGGAATTCGCTCACTTTGGACGAAAAACAGCCTTACGTAGAAGAAGCCAAAAGACTGCGCGAACT 200 N S E I S K R L G L E W N S L T L D E K Q P Y V E E A K R L R E L GCATAAAAAGGACCATCCGGATTACAAATATCAACCGAAGCGCAAACCCAAAACAAGTCCAAAACTTAAAACACCGGGACTAAACCCGTTTATGCATGGC 300 H K K D H P D Y K Y Q P K R K P K T S P K L K T P G L N P F M H G TATGGGGAGATGCCAGGGATAGGTATGCCTCCTCCAACGAACCTATGCCAACCAATGGCTTCCCATATGGGTCCTATGGTTAACATGGCAAGCTGCCCTG 400 Y G E M P G I G M P P P T N L C Q P M A S H M G P M V N M A S C P G GATCGTGCACACTGCCAGAGCCTCCACCGCCCTACCATTTCTCCCCGCACTACTCCTTCGTGCAGAATATCTCGGACTATAAGAATCAATGTGGTTCGCA 500 S C T L P E P P P P Y H F S P H Y S F V Q N I S D Y K N Q C G S H TCTATCACTGATGAGCAGGGATCTGCCGTACCCGTCACCCATCGGATATCCATCGCACGGCGCATCGCACCCGGTGCAGTTTGTACATCGCAGTCTTATT 600 L S L M S R D L P Y P S P I G Y P S H G A S H P V Q F V H R S L I <-Nv-Sox2-R1 CCAGACTCTGGGTCTGTCGTACACGCGACTTCACCCATAGACGCACGAAGTGGGCTAGCGAGACATCCTTTAGACTGTGTTGTCATGCGGCCGGAGGGTT 700 P D S G S V V H A T S P I D A R S G L A R H P L D C V V M R P E G Y ACTGA 705 *
277 1.5. Nematostella vectensis Forkhead Nv-FH-F1-> ATGATGGAGCACACGGGGGTGCCTCCAGCGGCCATGCAAGACCCATCACAAAACCCGCACGAGCTCAAGAAATCCAAGGACAAGGAGAAAGCGTATCGCC 100 M M E H T G V P P A A M Q D P S Q N P H E L K K S K D K E K A Y R R GGAGCTACACGCACGCCAAGCCGCCATATTCATATATCTCACTCATCACGATGGCCATTCAACAGAGCCCAAACAAGATGCTCACACTGAGCGAGATCTA 200 S Y T H A K P P Y S Y I S L I T M A I Q Q S P N K M L T L S E I Y CCAATTCATCATGGACTTGTTTCCCTACTACAGGCAAAACCAACAGCGCTGGCAGAACTCTATCCGGCACAGTTTATCATTCAATGATTGCTTCGTGAAG 300 Q F I M D L F P Y Y R Q N Q Q R W Q N S I R H S L S F N D C F V K GTGCCGCGCTCTCCTGACCGCCCCGGGAAAGGCAGTTACTGGACTCTCCACCCGGAATGCGGTAATATGTTCGAGAACGGGTGCTACCTTCGCAGGCAGA 400 V P R S P D R P G K G S Y W T L H P E C G N M F E N G C Y L R R Q K AGCGCTTCAAAGCCGAGAAAAAACCGGACCTGAGTCACCTTAGCAAGGTGAGCAGTATGACACACAACCCGGTCACAGTACAAAGCATGGCGAAGAGCAT 500 R F K A E K K P D L S H L S K V S S M T H N P V T V Q S M A K S M GGCGGCACAACCTCGCTCAATGGGAACCCCTAGCTTCCTTGCACCGTCTCCGTACGGGACGGCTATGGGCATGGGACACGTTGGGGGCATGACCGCCATG 600 A A Q P R S M G T P S F L A P S P Y G T A M G M G H V G G M T A M GGTATGGCAGGTATGCCCATGAATAAGTCGTTTAATCACCCATTCGCCATCAAGAATATCATCGCGCAAGATCACGAAGCTGAGCTTCGAGGCTACGACC 700 G M A G M P M N K S F N H P F A I K N I I A Q D H E A E L R G Y D P CCATGCACTTCAGTCCTTATCATCCATCACTCCAATCCATGGGTTCCCTAGGACTCCCTAAATCCGCCTACGAATCGCAACCTATCACGACGGATACGAG 800 M H F S P Y H P S L Q S M G S L G L P K S A Y E S Q P I T T D T S <- Nv-FH-F1 TCCGTACTACCAGGGTTGCGTCTTCACGCCGTCGAGTTGTGGTATATCAAATCTTTCGTGA 861 P Y Y Q G C V F T P S S C G I S N L S *
278 Clytia hemisphaerica sequences
2.1. Clytia hemisphaerica Gsx <-Ch-Gsx-R4 Ch-Gsx-F5-> gatatctaatggaacaccttgaatggtaagttattcttcctgaatgcttgttatttgttaaagtttgaaggaacccctggcccttcactttatttcccct 100 Ch-Gsx-F4-> atttcatgattattgagctttgaaacagtccgcataaaaaaacaaaatataaagttcaatttcattaattcacattaattcttaatgattgatttattct 200 tctgtttatagtattaaatttgaatggaaaaaaatcgatatattcatgggagtaaagaaatcaaaagaatttaaatggacgggagagaaaatgaagatga 300 aaaagaaaaagaacattcttttttaatagcataaatagagagatagaaatgtcaaatagtttaaaatctgtgtcgcaagtgcgcatgcccaaagtaagac 400 Ch-Gsx-F1-> aacatctataaagaggntgttaaaaaattctgatatatcattaacagaaacgtcgcgtcacagtacactgcacacgcagaaagtgaagaagaagaagaag 500 <-Ch-Gsx-R3 aaagcagaaaatagatttaaaaggaattttatttgtgaagaaaaatttattaaaaaaATGTCGCGATCATTTTTCATCGATACGATCATTCACGAGAAAG 600 M S R S F F I D T I I H E K E <-Ch-Gsx-R2 Ch-Gsx-F2-> Ch-Gsx-F3-> AGAAGTTGTTACGACAGACCTCGCCGATCAAACAACAACCGATCGCATCGTCGTCGCCATCATCACGCGAACCATCGCCGATCTCCGAACACGGAAATCA 700 K L L R Q T S P I K Q Q P I A S S S P S S R E P S P I S E H G N Q ATATCAACTTTCACCGATATCCCGATACAATCAATCGCCATCACCTCGCTCACCTCCCTCACCGACTGGACATCAAGATTATTCAAGATCATCGCAAAAT 800 Y Q L S P I S R Y N Q S P S P R S P P S P T G H Q D Y S R S S Q N ATCTCTTCCACATCGTTACACCATGGCTCCGCATTGTGCGGATGTTGTCCACCACAGCCACACTCACGCTTATGCATGTGCTCCTCGTGTGAAAGCGGAC 900 I S S T S L H H G S A L C G C C P P Q P H S R L C M C S S C E S G P CCAGAGAAGGGGAAGCGGCGCCGTTCGGATCACCAAGAGAGGTAACTCATCACACAACAAGATATTTATATGGTGGAAGCGAAAGAGGGCGTATCTTTTC 1000 R E G E A A P F G S P R E V T H H T T R Y L Y G G S E R G R I F S TCTTGCATCTCCGATCAATTCAAGAGCTCGACCACAATTTCCCGCGCTTTATGgtaaatttcattatctcattattctatcatttttcttgatctctttt 1100 L A S P I N S R A R P Q F P A L Y V cttgactcccaaacaaattttacattggaagggtaattttcttgactttctgcatttttaaaaggacacaaatgcaattttaagaaatttggcgcgcatt 1200 gaggtaaaagcgcgcaaattgtctttgcatgatattgtaaacatatttatgctgaagaatattgcgcctgaaataatgatattttcgcgccaaaaacaca 1300 tattacgtgttgcgataattgcaaacaaagtagatattctgaattgtttgataattatgttttaaacgttcgatcgtttatgaagacgcaaacattaaaa 1400 acattcttgttttgacaaaacaatcgcgtgctacagagaagagtactatcaagatgacccctccctttctgtaaagctacaatgaatgaaacttaacatt 1500 cttcttctttttctttcaacagTACGAGACTTGGACAGCCGTCGCTTACAACTTCAACAACAGGTACAACACCAACGACAACAACAACTTGAAGAACAAC 1600 R D L D S R R L Q L Q Q Q V Q H Q R Q Q Q L E E Q Q AAGGAGGAGGTAGCAAAAGCAAACGAGTCCGTACAGCCTATACTAGTATCCAACTTCTTGAACTTGAAAAAGAATTCCAAAATAATCGATATCCTTCACG 1700 G G G S K S K R V R T A Y T S I Q L L E L E K E F Q N N R Y P S R <-Ch-Gsx-R1 ATTAAGGCGTATTCAAATTGCAGCTATGTTAGATCTGACGGAAAAACAAGTGAAGATTTGGTTTCAGAACCGACGTGTGAAATGGAAGAAGGATAAGAAA 1800 L R R I Q I A A M L D L T E K Q V K I W F Q N R R V K W K K D K K AGCGGTTT 1808 S G
2.2. Clytia hemisphaerica Pdx Stefano sequence:
Ch-Pdx-F1 / Ch-Pdx-F3-> Ch-Pdx-F2-> CGGAAGCGAACGGCTTATACGCGAGCACAGCAACTTGAACTCGAAAAAGAATATCGTTACAACCGTTACATCTCGAGAGCGAGGAGGATCGAGTTGGCTA 100 R K R T A Y T R A Q Q L E L E K E Y R Y N R Y I S R A R R I E L A K <-Ch-Pdx-R4 <-Ch-Pdx-R3 <-Ch-Pdx-R2 AGAATTTGACTTTAACAGAAAAACATATCAAGATTTGGTATCAGAATCGAAGAATGAAGGAGAAAAGAGACGAGGAAGATATCATGAGAGGTACAACTGT 200 N L T L T E K H I K I W Y Q N R R M K E K R D E E D I M R G T T V <-Ch-Pdx-R1 TTTGGATCCTCGATCTTCTTATTATTAACTTATTTTTTAATTTATTTTTCAAACTTTTTGCCTCGCTTCGTTTTTAGTTATTTCTTGGACATATTCTTGT 300 L D P R S S Y Y * L I F * F I F Q T F C L A S F L V I S W T Y S C TGTTTTTTTT 310 C F F
BG’s lab sequence:
Ch-Pdx-F2 / F3 / F6 -> Ch-Pdx-F4-> GCTTATACGCGAGCACAGCAACTAGAACTGGAAAAAGAATATCGATATAATCGATATATATCGAGAGCACGTCGAATCGAATTAGCTAAAAATTTGACGC ------+------+------+------+------+------+------+------+------+------+ 100 CGAATATGCGCTCGTGTCGTTGATCTTGACCTTTTTCTTATAGCTATATTAGCTATATATAGCTCTCGTGCAGCTTAGCTTAATCGATTTTTAAACTGCG <-Ch-Pdx-R5 / R6 A Y T R A Q Q L E L E K E Y R Y N R Y I S R A R R I E L A K N L T L Ch-Pdx-F5-> TAACAGAAAAACATATCAAAATATGGTATCAGAATCGAAGAATGAAGGAGAAAAGAGACGAGGAAGATATTATGAGAGGTACAACTGTTTTGGATCC ------+------+------+------+------+------+------+------+------+------197 ATTGTCTTTTTGTATAGTTTTATACCATAGTCTTAGCTTCTTACTTCCTCTTTTCTCTGCTCCTTCTATAATACTCTCCATGTTGACAAAACCTAGG <-Ch-Pdx-R3 / R4 <-Ch-Pdx-R2 / R7 T E K H I K I W Y Q N R R M K E K R D E E D I M R G T T V L D
279
2.3. Clytia hemisphaerica CnoxC TCTGCTATTTTGGAACCAAATTTTCCACAAGGATTAAATAACAACAATACACCACCACCCCTTTCATCACCATCTTACCTTCGAAATGATGCTAACAGAT 100 S A I L E P N F P Q G L N N N N T P P P L S S P S Y L R N D A N R Y Ch-CnoxC-F ACGGTGGCACGGGAACCCCAACAACAGACTCTCTCGAACAATCTGCATTCAGTCACATCTCATCGCCAGATACGACAGCTAATTCACCACCAATGACAGA 200 G G T G T P T T D S L E Q S A F S H I S S P D T T A N S P P M T E ACCAACTTACAGCACAACAGCAGGAAGCACGTTCGACACCTCCGTGCAATCCTACCTTGCCAACACGTCTTTAGCTAACCCACCATCAACCTCAGCAATG 300 P T Y S T T A G S T F D T S V Q S Y L A N T S L A N P P S T S A M TCATTTTACAACACTCCCACCTCTCTACTATCCAACCAACACTTATCCACAGATTACTCACAATTTGGATATGCTTCTCCTTCAAACTATTTCTACAGCT 400 S F Y N T P T S L L S N Q H L S T D Y S Q F G Y A S P S N Y F Y S S Ch-CnoxC-R CTGGTTACCCATCCATCGGATCTGGGTACAATACTTATCCAGGAATGACCAACACTGGGCCTTTACCAGCTGGTCCATGGATATGCCGAGATATCGATAC 500 G Y P S I G S G Y N T Y P G M T N T G P L P A G P W I C R D I D T CAAACGAAAACGAATGACTTATTCACGAAAACAACTTTTAGAATTGGAAAAAGAATTTCATTTCAGTCAATTCTTAAAGAAAGAACGAAGATCAGACTTG 600 K R K R M T Y S R K Q L L E L E K E F H F S Q F L K K E R R S D L GCAAAACAACTCAGTTTAACCGAACGACAAATCAAAATTTGGTTTCAAAACAGCCGA 657 A K Q L S L T E R Q I K I W F Q N S R
280
Turritopsis dohrnii sequences
3.1. Turritopsis dohrnii Pdx TuxF4-> AGCAGTGGTAACAACGCAGAGTACGCGGGATTTCTGAAGTTATTGACACAATATCATCTTTAACAAGTAAAGGGAAGTTGAAGACAACCAGCACAAACGA 100 Q W * Q R R V R G I S E V I D T I S S L T S K G K L K T T S T N E TuxF5-> ACAGAACCAAACTGAAAAGGAGAAAACGAAAGATGACAAACCACCAAAAGCTTGGCAGTCAGCTAAAGTGAAGAAGAGGAATCGCACGACCTACACACGA 200 Q N Q T E K E K T K D D K P P K A W Q S A K V K K R N R T T Y T R TuxF1-> <-TuxR2 / F2-> <-TuxR1 GTACAGCAGCTGGAGCTGGAGAAAGAATATCGGTACAGTAAGTACATATCAAGAGCCAGGAGAATCGAGTTAGCTAAAAATTTAACTCTGACAGAAAAAC 300 V Q Q L E L E K E Y R Y S K Y I S R A R R I E L A K N L T L T E K H <-TuxR4 ATATTAAGATCTGGTATCAAAATCGCAGGATGAAAGAAAAGCGGGACGAAGAAGACGCTTTGAGAAATGAGAATAGTCTGGATGGGCAACTAAGGTTTTT 400 I K I W Y Q N R R M K E K R D E E D A L R N E N S L D G Q L R F F <-TuxR5 TTGATGTTAGAAATAACATCTGAAGGAATTTAAATATCATATCGAGAAAAAAAAAAAAAAAAAA 464 * C * K * H L K E F K Y H I E K K K K K K
3.2. Turritopsis dohrnii CnoxA gacattaacatactaatgtaaaaaaaaagaggggtttatttattcgtggctttggtttcttcaattcactagttgaccacctttcacaccATGTGGAGAA 100 TH5F3-> M W R M TGAACTGTTCAACATCGTCTTGTGAAACGTGCTCTTCGCGATGGTCACATGTTATTCCAGCACCCTACCAAAATCGATATTGTAATCCAAGAAGAACTGT 200 N C S T S S C E T C S S R W S H V I P A P Y Q N R Y C N P R R T V TGACGATAAATATGGTAAAGTAAGGCCATGGATAGTTGCTCGAAATTCCGTCGACAATAAACATCTGGTCTCAAGGGATTATCGTCCTCCCTGCCATGCC 300 D D K Y G K V R P W I V A R N S V D N K H L V S R D Y R P P C H A TCAAGAGTTAGTTTGTGTCACTGTCGAAGTTGTCACCTAGCCGCAGAAACGCAATATATCACGTATTTTGCTCGCCAATTTATCCCGAGAGAATGCAATT 400 S R V S L C H C R S C H L A A E T Q Y I T Y F A R Q F I P R E C N C GTATCGATTGTGATAGGGAAGGGATGAAAAGATTTCGAACAAGTTCCAACACGTCACAACTAACAGAACTGGAAAAAGAATTCCAACAGAACAAATACTT 500 I D C D R E G M K R F R T S S N T S Q L T E L E K E F Q Q N K Y L AACACGTCGTCGTCGAGTCGAACTTGCTGTCGGCTTAAAGTTGTCAGAAAAACAGGTCAAGGTGTGGTTTCAAAATAGAAGAATGAAGTGGAAAAAGCAG 600 T R R R R V E L A V G L K L S E K Q V K V W F Q N R R M K W K K Q <-TH5R6 ACAAAATTTGAAGAAGAAGAAGAAGAGGGGAGATTCACTTGAtagcgaatacgtatgaaagttatcgtcaattagatatcagatatatccaaaagcaagc 700 T K F E E E E E E G R F T * tggaaatacctgaaacctttttttgattcataatctgaaagggacaaaatatatagtcgaaactttcactcaaaatgtcgattaaaaagattttgttaaa 800 tatcaacattcctatgccc 819
281 Hydra vulgaris sequence
4.1. Hydra vulgaris cnox2 genomic sequence
cx2hvXmn5’-> <- cx2hvR3iPCR gaacctcttcttaaaacgagttagatttttttatggttgttaaggatatagaATGTCAACTTCGTTTTTAATAGATTCTCTAATACATGAAAAAGAAAAG 100 M S T S F L I D S L I H E K E K <- cx2hv-5ipm cx2hvF1iPCR-> cx2hvF4iPCR-> TATAAGATACGGCAGCAGCCTGGAACATCTTTTTTATTTCGTGAATCATCTCCTCCAGATCGATCGCCGAGTTATTCACCCGGTGCGTCAATGATTAGAT 200 Y K I R Q Q P G T S F L F R E S S P P D R S P S Y S P G A S M I R ATTCCAATTCTTCTTCTCCAAGAAGTTTAGATTCACCTATAAATCCATTGGATCGACACCCTCTTGAACGAGTACATCAAGTAGTTAGTTGTATGAGAGG 300 Y S N S S S P R S L D S P I N P L D R H P L E R V H Q V V S C M R G ACCTTCGATGTGTAATTGCTGTCGGCCTCCGACTGTTCAACCTATGTGTACAGTATGTGAACCTAGGGAACCAGGTGAAGGTACCTCTTCACAATATCCT 400 P S M C N C C R P P T V Q P M C T V C E P R E P G E G T S S Q Y P cx2hv1for-> cx2hvF5iPCR / cx2cvQfor-> TATACCCGCGAACCTCATGACCATGCAAGAGGCTTGTATGGAAATGATAGGTCAAGACTTTTTCCAATATTATCTCCTTTACACGGGCAAAGAGCGCAGT 500 TTTCCCCAAATTATggtaagcttaatttttttttttattcataaaaagtttttgttttaactttacaactttattatttgtgaaaaaaataggtagttaa 600 F S P N Y acgtatgtatagaataaaaaaacaaataaaattgctttgttagtaaattcggcattttagtgtataaagttctaactaaaagtttaaaaactgttcacaa 700 gttcggcataaatcgcgcactttttttatcactaaatttgagcgcccttttgcactaatggtcgcgtcaatttaagcggtgtataatgtcctgaatgaca 800 taagatgaccctagatgaaactctactaaatatattaacaacttaattttttcatttaGTTTACGATTTGGAACTTCGTCATTCCCGTCAACTTCAACTG 900 V Y D L E L R H S R Q L Q L CAACACCAAGAACACGAAACAGATCTCTACGGAAAATCTAAACGCATTCGAACCGCCTATACTAGCATTCAGTTACTTGAACTTGAAAAAGAGTTTCAAA 1000 Q H Q E H E T D L Y G K S K R I R T A Y T S I Q L L E L E K E F Q ATAATCGTTATTTATCGAGATTACGGAGAATCCAAATAGCTGCTATTCTTGACTTAACAGAAAAACAAGTTAAAATATGGTTTCAAAATCGACGTGTAAA 1100 N N R Y L S R L R R I Q I A A I L D L T E K Q V K I W F Q N R R V K <-cx2hv16HB3 ATGGAAAAAAGATAAGAAAGGATATAGCTATTCCCCTACTGGAAGTCCGCAATCTCCAGAATAActaccggTtttctttctcaaaagttcttctgcatct 1200 W K K D K K G Y S Y S P T G S P Q S P E * ttcaaaacgaagatatttttttataaaaacaaacgatattaaaatgatttcccatagtcattttataatatgtacataaaaattcatcaataatttattc 1300 atcttttacgacatttttttgtttatctttgtatactgaaactttattttcaggtgaagcgtaagttgttccgaa 1375
282 Hydra actinEGFP EcoR1 aagcttgcatgcctgcaggtcgactctagaggatcccccatcgatctgactaacctaaccagtgcaaaaaaatttaaaagatttgcattgtgaaagttag 100 aatattataaaaaatctaaaacgagtattactcgagtaaatgttatacgatctatagattaaatatattaaaaatgtatagcgaatgttaaactaaatat 200 ataatataaacttgaaaacttactaaattgcaaaaactcaaaaccgactgtatcatttttacaggaaaccgttattcaagatacttaagttgtttactac 300 attattataacatcttgcaattagcaagacaatcgttattttaacatcacggtatcgaaaggattttgagaaattttattgaaacattttaaacaaaaaa 400 tatcatatttagatgcattttaagccgagatgcaggattctgaatgaaaaagaaaaaaagaagtctcggtagagtaaaagtgatcggtttgcaactgtaa 500 aatttattgaagtaccaataattttatttaaaataaaactgaaatataaagttaaagttgctgttctataagtttactcgaattttaaaaccattgtaac 600 gctagagtaatatttgagtctactaagttagtccccgcactttttaatcaagcaataaatacccaaactttgcttattcaaatcaataaaccaatatatc 700 Hyact-promF2-> tcttaaaataaagtaaaaacttctgaaattctataaaaaaaaatttaatttcgaaatatcaaatgtaacttcaacaccgcactattttcttttaaacaac 800 tgatatagtaattacttctcaaaaacgttatctcaaggtttgtgatgtacttaaaaccactcctattttgttacgcgtttaaaaaagcaaacataagttg 900 gtttctattgatgaatgagaacatatttcatttaaagttaaaatcctaccagtggtttcactgtacgtaaacaccgtcaaaaaaacaggaacgtttttaa 1000 agattaataattgaagtaaaaaaaatttaataccgggggttaaaaaaatcttttaaaataattataaatatatatattaaaatttataaatttttaaaca 1100 catttaaaatatatattaagtataataaaagtaatattataaaaaaaaatttaattttataattatttttattaaatttataaataataggtaaaactta 1200 hyact-promf1-> catatccgttttattttttcttaataaaataacgcgtgcaaatttttgtccatataaagaccttttcgaacaataacttttttgcttagccgtttttttt 1300 cttatatggtcaaaaaagcgctcaagcgattcaccataaaaagcgcaattagttcagcgttcgttattcagaagcttcagctttgcttgatactcagctc 1400 ttctctttttaaacaaaacacttaatcaaaATGGCCGATGATGAAGTTGCCGCCCTCGCTGCAGCCCCGGTAGAAAAAATGAGTAAAGGAGAAGAACTTT 1500 M A D D E V A A L A A A P V E K M S K G E E L F <-GFP-N-rev1 TCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAA 1600 T G V V P I L V E L D G D V N G H K F S V S G E G E G D A T Y G K ACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTTTCAAGATAC 1700 L T L K F I C T T G K L P V P W P T L V T T F C Y G V Q C F S R Y GFP-C-for2-> CCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA 1800 P D H M K R H D F F K S A M P E G Y V Q E R T I F F K D D G N Y K T <-GFP-C-rev3 CACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATT 1900 R A E V K F E G D T L V N R I E L K G I D F K E D G N I L G H K L GGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGA 2000 E Y N Y N S H N V Y I M A D K Q K N G I K V N F K I R H N I E D G <-GFP-C-rev2 / GFP-C-for1-> AGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCaTTACCTGTCCaCACAATCTGCCCTTT 2100 S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q S A L S CGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGcattc 2200 K D P N E K R D H M V L L E F V T A A G I T H G M D E L Y K * <-HyactTermrev1 gtagaattcacaattcgattatatttatactggactatttttacatctgttcggttattttcacatttatttttctatatatatcttataaacgttttaa 2300 aacccatgtaatttttgttaagctgtaatataaaagacgtcctaacaaacttcttttattactgaatttcctttaattataataaataacaagttttaaa 2400 ataaattcaggcaattaaggcgctcctgaggtactaaaattaatgtaaacatttaaaattaacttggatggtcttaagtactgtactcgtgattttgtta 2500 tactttattattagaaaagtcgtctattaactttttgttccttaatttacttgattaaattgtcgcttaatttatcaaatcaggttttgcgcgttatttt 2600 agagaaaaacttattagaaaaatgaataagcaaagtttaggctaacatgtttttttattattttaaatagttcaagtcaatgacgtataaaatgcatttg 2700 caaaaaattttaagtaaccctataaacttagcaatagtagatactggatgcaagcattcagtagcagcattgcatatctgctgtctttacgtacaaataa 2800 cagcaaaaatggacctttattggcttcacatcgtcgtaaaacatgtgttattggacttgtcacaaatgtgttaagtatacagagcttagctcttgatgtt 2900 gatcactagt 2910
283
CMV-bglobinintron-DsRed2- bglobinpolyA gttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaa 100 tggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaa 200 tgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccg 300 cctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggca 400 gtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg 500 gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaacta 600 gagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtacctcgagataa 700 gcggccgcagatcctgagaacttcagggtgagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaag 800 <-TAC2078 ttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccatgcatggaccctcatgataattttgtttctttcactttctactctgttgacaac 900 cattgtctcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtc 1000 agattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttaggaacaattgttataattaaat 1100 TAC2079-> gataaggtagaatatttctgcatataaattctggctggcgtggaaatattcttattggtagaaacaactacaccctggtcatcatcctgcctttctcttt 1200 atggttacaatgatatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctctttccta 1300 cagctcctgggcaacgtgctggttgttgtgctgtctcatcattttggcaaagaattcccaccatgggcgcggatccaccggtcgccaccATGGCCTCCTC 1400 M A S S CGAGAACGTCATCACCGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCC 1500 E N V I T E F M R F K V R M E G T V N G H E F E I E G E G E G R P TACGAGGGCCACAACACCGTGAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGG 1600 Y E G H N T V K L K V T K G G P L P F A W D I L S P Q F Q Y G S K V <-DsRed2-R1 TGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGT 1700 Y V K H P A D I P D Y K K L S F P E G F K W E R V M N F E D G G V GGCGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTGATGCAG 1800 A T V T Q D S S L Q D G C F I Y K V K F I G V N F P S D G P V M Q AAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGACCCACAAGGCCCTGAAGCTGAAGGACGGCG 1900 K K T M G W E A S T E R L Y P R D G V L K G E T H K A L K L K D G G GCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACGCCAAGCTGGACATCACCTCCCA 2000 H Y L V E F K S I Y M A K K P V Q L P G Y Y Y V D A K L D I T S H CAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGTAGcggatcgataattcactcctcaggtgcaggctg 2100 N E D Y T I V E Q Y E R T E G R H H L F L * cctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggacatcatgaagcc 2200 ccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatatgggagg 2300 gcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctgccatgaacaaaggttggctataaagaggtc 2400 atcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttgacttgaggttagattttttttatattttgttttgtgttattttt 2500 ttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactcccagtcatagctgtccctcttctcttatgg 2600 agatccctcgatcgagggggggcccggtacc 2631
284 Manon QUIQUAND, PhD CURRICULUM VITAE 06/2009
Name: Manon QUIQUAND Professional address: Private address: Department of Zoology and Animal Biology Av. du Devin du Village, 25 Birth: 27/09/79 (29 years old) Sciences III, Quai Ernest Ansermet, 30 CH-1203 Geneva - Switzerland CH-1211 Geneva 4 – Switzerland Citizenship: French Phone : 00 33- (0)6-18-56-39-23 Languages: French (mother tongue) Phone: 00 41-(0)22-379-67-65 E-mail : [email protected] English (spoken) E-mail : [email protected]
EDUCATION 2004-2009 PhD in developmental biology, University of Geneva, Switzerland: “Mention Très Bien” 2001-2003 Master in Biology, University Pierre et Marie Curie (UPMC), Paris, France: 2002-2003 “DEA” in "Biology of invertebrates", Rank: 4th/13, “Mention Bien” 2001-2002 “Maîtrise” in "Biology of populations and ecosystems", option "Marine ecology" 1998-2001 Bachelor in Biology, University Pierre et Marie Curie (UPMC), Paris, France: 2000-2001 “Licence” in "Biology of organisms", option "Animal biology and physiology" 1998-2000 “DEUG” in " Sciences of life and earth "
SCIENTIFIC TRAINING 2009 PhD thesis (5 years) Department of Zoology and Animal Biology in Dr B. Galliot laboratory, University of Geneva, Switzerland: “Tracing back the early evolution of ParaHox genes and the ancestral neurogenic function of Gsx/Anthox2 in the developing sea anemone, Nematostella vectensis” 2003 Master thesis Part II (6 months) Defense and Resistance in Marine Invertebrate laboratory of Dr. J.M. Escoubas, CNRS/IFREMER, Montpellier, France: "Expression and regulation of Cg-timp, an immune gene of the Pacific oyster Crassostrea gigas" 2002 Master thesis Part I (2 months) Developmental Biology laboratory of Dr. E. Houliston, CNRS, Villefranche-sur- Mer, France: "Research of cellular type markers in larvaes, polyps and jellyfishes of the Cnidarian Clytia hemisphaerica, by immunofluorescence" 1999-2002 Voluntary training at the National Natural History Museum, Paris, France: 2002 Laboratory of Cryptogamy, Pr. A. Couté: Isolation of phytoplanctonic organisms (37hrs) 2001 Laboratory of Ichthyology, Dr. D. Paugy: "Osteological study of sub-species Brycinus macrolepidotus (Teleosteen Characidae) in the Guinean zone" (40 days) 2000 Laboratory of Cryptogamy, Dr. S. Gombert: "Study on metallic fallouts of atmospheric origine by measurement on French moss", (160hrs, European Campaign, 2000) 1999 Laboratory of Cryptogamy, Pr. A. Couté: “Inventory and classification of the Algology herbarium” (180hrs)
SCIENTIFIC COURSES 2008 Statistical analysis applied to genome and proteome analyses, EMBnet course, Lausanne 2007 Basic Light Microscopy & Imaging Course National Center of Competence in Research (NCCR)-Frontiers in Genetics; Geneva, Switzerland 2006 Molecular Genetics of Development (selected chapters), University of Geneva 2005 Molecular Genetics of Development (advanced course), University of Geneva 2004 Experimental Developmental Biology Course in Marine Invertebrates, University of Geneva, Roscoff, France
CONFERENCES & ORAL PRESENTATIONS 2009 Thesis defense, University of Geneva, Switzerland PhD results, oral presentation 2008 Swiss Stem Cells Network meeting, Geneva, Switzerland Poster 2007 International Workshop, Hydra and the development of animal form, Tutzing, Germany Poster 2006 EMBO Workshop, Cellular and Molecular Basis of Regeneration and Tissue Repair, Ascona, Switzerland Poster First joint French/Swiss Developmental Biology meeting (SFBD/ZMG), Praz-sur-Arly, France Poster 38th annual meeting of the USGEB/USSBE swiss societies, Geneva, Switzerland Poster Department seminar, “Tracing back the early evolution and the ancestral function of ParaHox genes through the cnidarian phylum”, University of Geneva, Switzerland PhD work progress, oral presentation 2005 International Workshop, Hydra and the molecular logic of regeneration, Tutzing, Germany Poster Department seminar, “How to trace back the ancestral function of Gsx/ind ParaHox gene family in cnidarians ?”, University of Geneva, Switzerland PhD one year project, oral presentation
AWARDS 2006 USGEB (Union of the Swiss Societies for Experimental Biology) Fellowship travel award
EXPERTISES Molecular biology: consensus primers, gene cloning, RT-PCR, reporter gene construct assay, in situ hybridization Cellular biology: immunohistochemistry, confocal and classical microscopy, microinjection Zoology: culture and life cycle of cnidarian species (Nematostella, Clytia, Hydra) Bioinformatics and phylogeny: BLAST, Protein alignments, Mr Bayes, PhyML Computing softwares: Word, Excel, Power point, Photoshop, Illustrator, R-software (basic) Matlab (basic) Computer language: Pascal (basic) SCIENTIFIC PUBLICATIONS
1. Montagnani C., Avarre J.C., de Lorgeril J., Quiquand M., Boulo V., Escoubas J.M. (2007) First evidence of the activation of Cg-timp, an immune response component of Pacific oysters, through a damage-associated molecular pattern pathway. Dev. Comp. Immunol. 31, 1-11. Quiquand M., Yanze N., Schmich J., Schmid V., Galliot B* and Piraino S. (2009) More constraint on ParaHox than Hox gene families in early metazoan evolution. Dev. Biol. 328, 173-187 *corresp. author. 2. Galliot B., Quiquand M., Ghila L., de Rosa R., Miljkovic-Licina M., Chera S. (2009) Origins of neurogenesis, a cnidarian view. Dev. Biol. 332, 2-24. 3. Quiquand M. (2009) Tracing back the early evolution of ParaHox genes and the ancestral neurogenic function of Gsx/Anthox2 in the developing sea anemone, Nematostella vectensis. PhD dissertation (defense). 4. Quiquand M. and Galliot B. The ParaHox gene Gsx/Anthox2 regulates neurogenesis in developing Nematostella vectensis. (to be submitted). 5. Quiquand M., Montoya-Burgos J. and Galliot B. Potentials and limits of phylogenetic analyses limited to evolutionarily- conserved functional domains. (in preparation). 6. Quiquand M. and Galliot B. Strategies to express gene reporter constructs in developing Nematostella. (in preparation).
POSTER PRESENTATIONS 2009 Quiquand M. & al., Origin of Neurogenesis: What Were the Actors of this Invention? Swiss Stem Cells Network meeting (Geneva, Switzerland). 2007 Quiquand & al., The cnidarian Pdx/Xlox gene reshuffles the puzzle of Hox/ParaHox evolution. Poster presented at the International Workshop, Hydra and the development of animal form (Tutzing, Germany). 2006 Quiquand & al., The Gsx/cnox-2 ParaHox gene family, a key player in neurogenesis and apical patterning from cnidarians to vertebrates ? Poster presented at the EMBO Workshop on Regeneration and Tissue Repair (Ascona, Switzerland). 2006 Quiquand & al., The Gsx/cnox-2 ParaHox gene family, a key player in neurogenesis and apical patterning from cnidarians to vertebrates ? Poster presented at the First joint French/Swiss Developmental Biology meeting (SFBD/ZMG, Praz-Arly, France). 2006 Quiquand & al., The Gsx/cnox-2 ParaHox gene family, a key player in neurogenesis and apical patterning from cnidarians to vertebrates ? Poster presented at the 38th Annual Meeting of the USGEB/USSBE (Geneva, Switzerland). 2005 Quiquand & al., Comparative analysis of the regulation of the Gsx/cnox-2 gene in full-life cycle and polyp-restricted cnidarians. Poster presented at the International Workshop, Hydra and the molecular logic of regeneration, (Tutzing, Germany).