Expression Pattern and Regulation 4739

Development 126, 4737-4748 (1999) 4737 Printed in Great Britain © The Company of Biologists Limited 1999 DEV5340

Patterning the ascidian nervous system: structure, expression and transgenic analysis of the CiHox3 gene

Annamaria Locascio1,*, Francesco Aniello1,*, Alessandro Amoroso1, Miguel Manzanares2, Robb Krumlauf2 and Margherita Branno1,‡ 1Department of Biochemistry and Molecular Biology, Stazione Zoologica ‘Anton Dohrn’, Villa Comunale, 80121 Naples, Italy 2Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 5 August; published on WWW 6 October 1999

SUMMARY

Hox genes play a fundamental role in the establishment of boundary and the absence of transcript in mesodermal chordate body plan, especially in the anteroposterior tissues are distinctive features of CiHox3 expression when patterning of the nervous system. Particularly interesting compared to the paralogy group 3 in other chordates. We are the anterior groups of Hox genes (Hox1-Hox4) since have investigated the regulatory elements underlying their expression is coupled to the control of regional CiHox3 neural-speciﬁc expression and, using transgenic identity in the anterior regions of the nervous system, analysis, we were able to isolate an 80 bp enhancer where the highest structural diversity is observed. responsible of CiHox3 activation in the central nervous Ascidians, among chordates, are considered a good model system (CNS). A comparative study between mouse and to investigate evolution of Hox gene, organisation, Ciona Hox3 promoters demonstrated that divergent regulation and function. We report here the cloning and the mechanisms are involved in the regulation of these genes in expression pattern of CiHox3, a Ciona intestinalis anterior vertebrates and ascidians. Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the Key words: Urochordate, Hox genes, Nervous system, central nervous system. The presence of a sharp posterior Transcriptional regulation, CiHox3

INTRODUCTION by Holland, 1992; Burglin and Ruvkun, 1993). In chordates, only one cluster is present in the genome of the HOM/HOX genes are involved in the establishment of the Cephalochordate Amphioxus (Holland et al., 1994) and it has animal body plan by specifying the positional identity of been suggested that the Urochordate Ciona intestinalis also has different structures along the anteroposterior axis (reviewed in a single cluster (Di Gregorio et al., 1995). Four apparently McGinnis and Krumlauf, 1992; Krumlauf, 1994). It is also identical Hox clusters are present in all mammals so far believed that they contribute to the morphological diversity analyzed (Boncinelli et al., 1988; McGinnis and Krumlauf, throughout the animal kingdom. During evolution, Hox genes 1992; Duboule, 1994); however, variation in the number and have conserved both their organization in clusters and the organization of Hox clusters is observed in some lower ordered correlation between the position of the genes in the vertebrates (Aparicio et al., 1997; Amores et al., 1998). cluster and their expression pattern along the body axis (spatial Genetic analysis of vertebrate Hox genes, involving both colinearity) (Lewis, 1978; Graham et al., 1989; Duboule and loss- and gain-of-function mutations, has been carried out Dollè, 1989). From an evolutionary perspective, the primarily in the mouse through the use of gene targeting development of a more complex body organization seems to technology. Together, this large body of work has revealed correlate with the formation and amplification of Hox clusters, important functional roles for the overlapping and ordered probably achieved through successive tandem duplications of expression patterns of Hox genes in many different tissues of an ancestral homeobox-containing gene and subsequent cluster the embryo, such as the CNS, axial skeleton, limbs and gut duplication (Field et al., 1988; Schugart et al., 1988; Kappen (reviewed by McGinnis and Krumlauf, 1992; Maconochie et et al., 1989). al., 1996; Duboule, 1993). There are distinct individual roles In agreement with this hypothesis, a single cluster with a for most Hox genes, but there is also evidence for synergistic variable number of Hox genes has been found in most interactions and functional redundancy between members, metazoans, such as the Arthropods and Nematodes (reviewed which are most likely due to overlapping expression patterns 4738 A. Locascio and others and structural conservation between paralogous genes. Hence, patterns during C. intestinalis embryogenesis revealed that its Hox genes have multiple roles in patterning diverse axial mRNA is confined to the larval CNS and in particular to a well- structures. defined region of the visceral ganglion. In an effort to Organisms belonging to the Chordate phylum share some understand the mechanisms involved in the regulation of characteristics, such as the presence of an axial notochord CiHox3 expression during development, we have used flanked by muscles, a dorsal hollow nervous system and a electroporation (Corbo et al., 1997b) to introduce reporter ventral endodermal strand. Given the key role of Hox genes in constructs, containing different fragments from the 5′-flanking regulation of the body plan, study of their expression patterns region of the CiHox3 gene fused to the lacZ cistron, into and regulatory regions in different Chordates may aid in the fertilized Ciona eggs. With this approach, we identified an identification of both homologous structures and novel enhancer element responsible for the specific expression of the structures, thus shedding light on the evolution of reporter gene in the nervous system. morphological diversity. Therefore it is important to Furthermore, the CiHox3 promoter was tested in transgenic understand the regulation of restricted Hox expression in mouse embryos and mouse Hox3 regulatory elements were different species, as one suggested means of coupling this gene electroporated in Ciona in order to address whether regulatory family to a range of common or unique morphogenic processes elements of Hox3 genes have been conserved during chordate could be through the conservation, modification and/or evolution. acquisition of short regulatory elements (Gellon and McGinnis, 1998). The expression and regulation of the Hox genes has been MATERIALS AND METHODS studied in detail in the CNS (reviewed in Keynes and Krumlauf, 1994; Rubenstein and Puelles, 1994; Maconochie et Ascidians al., 1996; Lumsden and Krumlauf, 1996). In the vertebrate Adult C. intestinalis were collected in the Bay of Naples by the fishing CNS, Hox genes belonging to paralogy groups 1 to 4 are service of the Stazione Zoologica. Gametes were used for in vitro expressed with partially overlapping domains whose anterior fertilization and embryos were raised in filtered sea water at 16-18¡C. boundaries correlate with the subdivision of the hindbrain into Samples at appropriate stages of development were collected and used segmental units or rhombomeres (Hunt et al., 1991; Keynes for whole extract protein or RNA extraction, or fixed for whole-mount in situ hybridization. and Krumlauf, 1994). Mutational analysis in the mouse has shown that these rhombomere-restricted domains of Hox Isolation of cDNA and genomic clones expression underlie functional roles in multiple steps of A cDNA library made from embryos at larva stage (Gionti et al., segmental patterning, as illustrated extensively for the group 1 1998) was screened using as a probe a genomic fragment, named genes (Carpenter et al., 1993; Mark et al., 1993; Studer et al., CiHbox1, previously characterized by Di Gregorio et al. (1995) and 1996, 1998). The remaining cluster members (paralogy containing the homeobox sequence of CiHox3. A positive clone insert groups 5-13) are expressed along the entire spinal cord in was sequenced on both strands by the dideoxynucleotide termination progressively more posterior domains that are also functionally procedure (Sambrook et al., 1989). important (Tiret et al., 1998). A C. intestinalis cosmid library, constructed by Reference Library The use of a simpler model system than the higher Database (RLDB, MPI for Molecular Genetic, Berlin-Dahlem, Germany; Burgtorf et al., 1998), was screened at high stringency using vertebrates might contribute to the study of Hox genes and of as a probe the [32P]oligonucleotide 5′-TCTATCGGCAGCCATA- their role in the evolution of chordates. Ascidians have a basic AGAGTC-3′, complementary to the most 5′ coding region of the body plan and are considered prototypical chordates. Despite cDNA sequence. Three positive clones: MPMGc119L0224, their simple organization, recent evidences have pointed out MPMGc119B058 and MPMGc119D1338 were amplified and DNA a certain degree of conservation in the genetic pathways was purified with QIAGEN kit (Qiagen Inc., Chatsworth CA, USA). involved in the specification of different body structures The genomic inserts were subjected to digestion with the restriction between ascidians and vertebrates (Di Gregorio and Levine, endonucleases EcoRI or XbaI followed by Southern blot hybridization 1998). with the oligonucleotide described above. An XbaI- and an EcoRI- During ascidian embryogenesis, the nervous system positive fragments of about 4 kb and 1 kb in length, respectively, develops by the formation of a neural plate, which invaginates derived from the cosmidic clone MPMGc119B058 and containing the 5′ coding region of CiHox3, have been subcloned in the pBlueScript and seals dorsally leading to a hollow nerve cord in a process II KS vector and sequenced. reminiscent of vertebrate neurulation. At the larval stage, the The 4.2 kb promoter region was obtained by assembling three ascidian nervous system comprises an anterior vesicle in the partially overlapping EcoRI or XbaI genomic fragments obtained trunk, containing the pigmented sensory organs and thus called from the successive hybridization of Southern blot of the cosmidic sensory vesicle, followed, more posteriorly, by a visceral clone MPMGc119B058 with oligonucleotides at the 5′ end of each ganglion and a caudal nerve cord. This latter structure is devoid fragment. of neuronal cell bodies and lies above the notochord (Nicol and Introns position and length have been determined by PCR using the Meinertzhagen, 1991). same cosmid clone as template and useful primers complementary to Recent experiments have demonstrated the usefulness of C. the cDNA sequence. intestinalis as a model system for studying gene regulation RNA preparation and northern blotting (Corbo et al., 1997a,b). In the present report, we have focused Total RNA from various stage embryos was prepared according to our attention on a C. intestinalis Hox gene belonging to the Chomczynski and Sacchi (1987). Poly(A)+ RNA was purified by anterior group, sharing the highest degree of homology to the oligo(dT)-cellulose chromatography (Sambrook et al., 1989). class 3 Hox genes of vertebrates and thus referred to as CiHox3. Northern blot hybridization was carried out as described by Gionti et The analysis of CiHox3 spatial and temporal expression al. (1998) using 15 µg of poly(A)+ RNAs per lane and a 32P-labeled CiHox3: expression pattern and regulation 4739

CiHox3 cDNA insert as a probe. In addition, a Ci-CaM cDNA (Di 0.9% agarose-coated dishes, until the required stage, fixed in 1% Gregorio et al., 1998) probe was used as a control. glutaraldehyde in sea water for 30 minutes at room temperature, washed twice in PBS 1× and stained at 30¡C in a solution containing In situ hybridization 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1 mM MgCl2, 0.1% Tween20 Two Dig-11-UTP-labeled RNA probes were used for whole-mount in and 200 µg/ml X-gal in PBS. situ hybridization experiments: the CiHox3 cDNA insert and a DNA fragment, amplified by PCR, corresponding to the 5′ coding region of Band-shift assay CiHox3 (extending from position −70 to +500). Conditions for in vitro Band-shift assay was carried out essentially as described by Yuh et al. transcription were as described by the manufacturers in the (1994) with 8 µg of protein extracts from embryos at larva stage, 50 Digoxigenin RNA Labeling Kit (Boehringer-Mannheim). Whole- ng of poly(dIdC) and 4 fmol of 32P-labeled AL2 oligonucleotide. The mount in situ hybridizations on Ciona embryos and larvae were sequence of the random oligonucleotide was: 5′-CTGCTTTGA- carried out as described in Caracciolo et al. (1997). Semithin sections TGGATGGAGCTG-3′. Protein extracts were prepared according to were performed as described by Di Gregorio et al. (1998). Tomlinson et al. (1990). RNase protection analysis The transcriptional initiation sites were determined by RNase RESULTS protection assay basically according to the RNase protection kit instructions (Boehringer Mannheim). Isolation CiHox3 gene A 353 bp fragment (spanning −179 bp to +174 bp), amplified by PCR, was cloned in the EcoRV site of the pBluescript II KS vector. CiHox3 was isolated from a cDNA library prepared with + The plasmid was digested with XbaI and used as template for in vitro poly(A) mRNA from C. intestinalis larvae using a genomic transcription of [32P]UTP-labeled antisense riboprobe. 5 µg of fragment, containing the homeobox sequence (previously poly(A)+ RNA from embryos at larva stage were hybridized overnight named CiHbox1 in Di Gregorio et al., 1995) as a probe. at 45¡C with the probe. After RNase A and T1 digestion, the protected Sequence analysis of the isolated cDNA clone, 1994 bp in fragments were run on 8% denaturing polyacrylamide gel along with length, showed that the translation start codon was missing. To a reference sequence for size determination. obtain the remaining 5′ coding sequence, a cosmid DNA Preparation of constructs library, prepared by the RLDB (Burgtorf et al., 1998), was screened with an oligonucleotide complementary to the The basic electroporation vector was pBlueScript II KS containing the lacZ and SV40 polyadenylation sequences (pBS+LacZ). Genomic anteriormost region of the cDNA. Three positive clones were fragments of 0.2, 0.45, 1.0 and 1.5 kb were amplified by PCR from isolated and analyzed, and an EcoRI fragment from the cosmid the cosmid clone MPMGc119B058. The amplified fragments were clone MPMGc119B058, partially overlapping with the cDNA, ligated with the pBS+LacZ vector in the 5′ to 3′ orientation. An XbaI- was subcloned and sequenced. This fragment was found to KpnI CiHox3 genomic fragment, obtained from the cosmid clone contain the remaining 5′ coding region, preceded by a series MPMGc119B058, was subcloned in the 0.45 construct, XbaI and of stop codons, and part of the promoter sequence of CiHox3. KpnI digested, to obtain the construct 3.0. The analysis of the genomic organization of CiHox3 has been The 4.2 construct was obtained by cloning an XbaI (end-filled)- performed by PCR amplification on the cosmid clones using XbaI genomic fragment, from the same cosmid clone, in the KpnI oligonucleotides complementary to the CiHox3-coding region. (end-filled)-XbaI-digested 0.45 construct. The complete genomic structure of CiHox3 thus obtained is To generate the internal deletion mutants ∆4-3 and ∆3-25 the regions from −3 kb to −0.5 and from −2.5 kb to −0.5 kb of the 4.2 schematically reported in Fig. 1A. It includes two introns of and 3.0 constructs, respectively, were removed by KpnI and SnaBI or 2700 and 2173 bp respectively, and three exons including the only SnaBI digestions and the resulting linear DNA fragments were 5′ (90-167 bp) and the 3′ UTR (280 bp) regions, and a coding self-ligated. The deletion mutant ∆3-2 was generated by exonuclease region of 2199 bp, encoding for a putative protein of 733 amino III digestion of the region from −2 kb to −0.45 kb of the 3.0 construct acids. according to manufacturer’s instructions (Promega). All the other It is interesting to note that the first intron, positioned constructs, P-1 to P-6, were obtained by cloning the corresponding between the sequences encoding the hexapeptide and the genomic fragments, amplified by PCR, in the end-filled HindIII site homeodomain, is conserved among all Hox3 genes of of the 0.45 construct. chordates, while the presence of an intron in the homeobox For transgenic mice, a 2.3 kb KpnI-HindIII fragment (construct 1, sequence seems to be a peculiar feature of C. intestinalis Fig. 7A) or a 500 bp PCR product (construct P1, Fig. 7A) from the Ciona Hox3 genomic region were cloned downstream of a lacZ homeobox-containing genes (Di Gregorio et al., 1995). reporter expression construct (pBGZ40) containing the human β- The comparison of the deduced amino acid sequence of globine promoter (Yee and Rigby, 1993). DNA purified from vector CiHox3 with those deposited in the GenBank and EMBL sequences was microinjected in mouse oocytes, and lacZ expression databases revealed the highest degree of homology with the in embryos detected as described by Whiting et al. (1991). homeodomains of the paralogy group 3 HOX proteins. A The mouse Hoxb3 and Hoxa3 constructs used for electroporation comparison of the homeodomain, the hexapeptide and the in Ciona embryos were the constructs N. 5 and 6 by Manzanares et interposed sequence of CiHox3, AmphiHox3, the HOX3 al. (1997) and #1.4, #3.3 (Manzanares et al., 1999). proteins of mouse and the Drosophila Proboscipedia is shown Electroporation in Fig. 1B. The highest percentage of identity was found in the homeodomain of AmphiHox3 (81%) and the lowest in the Electroporation of fertilized and dechorionated C. intestinalis eggs was as described in Corbo et al. (1997b) with a few modifications: homeodomain of Proboscipedia (72%). Interestingly, the capacitance setting was between 700 and 850 µF so that the pulse degree of homology (78%) is the same among the three HOX range was 15-18 mseconds and the final volume in the 0.4 cm cuvettes group 3 proteins of mammals (HOXa3, HOXb3 and HOXd3). was 700 µl. A lower degree of conservation can be identified in the Embryos were allowed to develop at 16¡C, in fresh sea water in region interposed between the homeodomain and the 4740 A. Locascio and others

Fig. 1. Structure of the CiHox3 gene, amino acid comparison of the homeodomain and transcription initiation sites. (A) The coding sequence of 2199 bp in length is boxed and includes the homeobox (white box). Interrupted lines indicate the two introns of about 2700 and 2173 bp in length. 5′ and 3′ UTR are shown in striped boxes. The 5′ genomic region is presented in bold line and is reproduced in magniﬁed view to show the alternative transcription start site, indicated by arrows, and the putative TATA and CAAT boxes (black and white dots, respectively). The bar −179 +174 indicates the 353 bp fragment used for the RNase protection assay. (B) The sequences shown in the comparison are as follows: CiHox3 (C.i., Ciona intestinalis; accession no. AJ132778), AmphiHox3 (B.f., Branchiostoma ﬂoridae; accession no. A49127), Hoxd3, Hoxb3 and Hoxa3 (M.m., Mus musculus; accession no. U03485; P09026; Q61197) and Proboscipedia (D.m., Drosophila melanogaster; accession no. P31264). The identical residues are indicated by a dash. To optimize continuity of conserved regions gaps, indicated by dots, are introduced. Number of glycine residues in the interposed region between the hexapeptide and the homeodomain characteristic of mouse HOXb3 are indicated by numbers in brackets. Per cent of identity to the ascidian homeodomain are given in the right column. (C) CiHox3 mRNA start sites were determined using poly(A)+ RNA from embryos at larva stage. Lanes A, C, G, T are sequencing reactions of unrelated DNA used for size determination. Lane 1, undigested probe corresponding to the 353 bp antisense probe; lane 2, protected fragments obtained after RNase T1 and RNase A digestion are indicated by arrows and correspond to alternative transcription start sites 167, 101 and 90 bp upstream from the translation start codon.

hexapeptide where 10-12 identical amino acids are conserved. To establish the site of transcript initiation, RNase protection In this domain, CiHox3 does not include the stretch of glycine assay was carried out on poly(A)+ RNA from the larval-stage residues, characteristic only of mouse and human HOXb3, embryos using a [32P]UTP-labeled riboprobe which extends suggested to have a hinge function (Beachy et al., 1985; Sham from position −179 to +174 and includes both part of the 5′ et al., 1992). Therefore this may represent characteristic coding region and of the adjacent promoter sequence. Three sequence acquired only by the HOXb3 proteins later in alternative transcription start sites were found (Fig. 1C): evolution. two giving a stronger signal corresponding to position −90 and CiHox3: expression pattern and regulation 4741

fragment starting immediately upstream from CiHox3 translation start site and linked to a lacZ reporter gene (Fig. 4A). The electroporated Ciona embryos were allowed to develop until the stage of interest, they were then fixed and assayed for β-galactosidase activity by X-gal staining. As shown in Fig. 4C, this construct drove the expression of the reporter gene in the nervous system of the larvae. In particular, staining was visible in the visceral ganglion showing virtually the same localization as the endogenous transcript. However, this construct also showed ectopic expression in the anteriormost region of the nervous system, at the level of the sensory vesicle and around the otolith. In about 5% of the electroporated embryos, the trunk mesenchyme was also ectopically stained (not shown). The onset of expression was Fig. 2. Northern blot analysis of CiHox3. Each lane contains 15 µg at early tailbud stage (Fig. 4B). In these embryos, staining of poly(A)+ RNA from Ciona eggs and embryos at the indicated became visible in the anterior region of the developing nervous stages of development. Blot was hybridized with a 32P-labeled system after 10 days of staining. Three internal deletion CiHox3 cDNA (A) and with the Ci-CaM cDNA probe (B) as transgenes and a series of deletion constructs progressively control. truncated at the 5′ end were then prepared and their effects was analyzed (Fig. 4A). The same pattern of expression found with −101, and a third one giving a weaker signal at position −167 the 4.2 kb transgene was obtained only with the construct 3.0, from the translation start codon (Fig. 1A). The same result was while the progressively shorter constructs of 1.5, 1.0, 0.45 and obtained using different riboprobes or poly(A)+ RNAs from the three deletion mutants ∆4-3, ∆3-25 and ∆3-2 were unable tailbud-stage embryos (data not shown). to give expression in the nervous system. These constructs, however, showed strong ectopic labeling in the trunk CiHox3 expression in the CNS of the Ciona embryo mesenchyme at the larval stage (Fig. 4E). The embryos The timing of CiHox3 expression during C. intestinalis electroporated with the 0.45 construct were also analyzed at development was determined by northern blot analysis. Using earlier stages of development and after 2 weeks of staining the cDNA clone as a probe, no signal was detected in stages mesenchymal cells of the neurula stage embryos were weakly prior to the early tailbud in which a single positive band of labeled (not shown). During the subsequent stages of about 2.6 kb appears (Fig. 2A). The signal remained faint in development, the signal became stronger and was clearly the middle tailbud and gradually increased in the following recognizable after one week of staining in the mesenchymal stages of development, peaking in the swimming larva. The pockets of the early tailbud stage embryo (Fig. 4D). The same northern, hybridized with the Ci-CaM cDNA (Di smallest deletion construct 0.2 (shown in Fig. 4A), containing Gregorio et al., 1998), served as a control for RNA loading only the putative TATA and CAAT boxes was unable to drive (Fig. 2B). the expression of the reporter gene (data not shown). In situ hybridization assays were then carried out to determine the localization of CiHox3 message during Ciona Identification of a neural-specific CiHox3 enhancer embryogenesis. No signal could be detected even after long and comparative study of Ciona and mouse Hox3 staining times in early tailbud embryos probably due to the low promoters abundance of the message (not shown). CiHox3 expression The analysis of the results obtained with the constructs shown became visible at larval stage and was restricted to the nervous in Fig. 4A suggested that the region from −2 kb to −1.5 kb system (Fig. 3A,B). In particular, the transcript was localized (indicated as red box) of the promoter contained elements in the anteriormost region of the visceral ganglion showing responsible for the restricted expression of the lacZ transgene well-defined both anterior and posterior limits. Transverse in the larval nervous system. To confirm this hypothesis a sections, cut at different levels of the hybridized larva, construct (P1, shown in Fig. 5A) was prepared by fusing this confirmed this conclusion. In particular, staining seemed to be region upstream from the 0.45 construct of Fig. 4A. The β- confined in the lateral and most superficial region of the galactosidase activity in the embryos electroporated with this visceral ganglion (Fig. 3C) which, according to the studies by construct was assayed at the larval stage. The reporter gene was Nicol and Meinertzhagen (1991), contains the cellular bodies expressed both in the visceral ganglion and the sensory vesicle of ganglion cells. No signal was detected outside the CNS. of the CNS (Fig. 5C) thus reproducing the result obtained with the 4.2 and 3.0 constructs of Fig. 4C. Also in this case about CiHox3 promoter analysis in electroporated 5% of the embryos showed ectopic staining in the trunk embryos mesenchyme (not shown). These results indicate that the As indicated in Fig. 1A, the CiHox3 promoter region contains sequence contains all the element(s) necessary to activate several putative TATA and CAAT boxes in the region adjacent transcription in the CNS. to the three alternative transcription start sites. In order to In an attempt to isolate the minimal sequence responsible for identify the elements required for neural-specific expression of the neural-specific expression pattern, the P1 sequence was CiHox3, the 5′ genomic sequence of this gene was further subdivided in a series of smaller and partially overlapping examined using an electroporation method (Corbo et al., fragments obtained by PCR, which were subcloned upstream 1997b). Initially, we assayed the 4.2 kb genomic DNA from the 0.45 construct as shown in Fig. 5A. 4742 A. Locascio and others

Fig. 3. In situ localization of CiHox3 transcript at the larva stage. Whole-mount Ciona larvae were hybridized with digoxigenin-labeled CiHox3 antisense RNA probe and stained with alkaline phosphatase. (A,B) Lateral and dorsal view of a larva showing CiHox3 expression in the visceral ganglion (arrow). Line s indicates level of transverse section. (C) Transverse section at the (s) level of the visceral ganglion of a hybridized larva. CiHox3 mRNA expression is observed in the most external lateral cells of the ganglion. cg, ganglion cells; En, endoderm; oc, ocellus; ot, otolith; sv, sensory vesicle; vg, visceral ganglion.

Fig. 4. Summary of CiHox3 transgene constructs and their expression in electroporated Ciona embryos. (A) Diagram of different 5′ CiHox3 promoter sequences analyzed in the electroporated embryos. At the top, genomic sequence of CiHox3 gene with the coding region represented as a wide rectangle. The restriction sites (H, HindIII; E, EcoRI; K, KpnI; S, SnaBI; X, XbaI) used for the isolation of the promoter region and the construction of the transgenes are also indicated. BamHI (B) was introduced by PCR. Right side, construct names and tissues where the reporter gene is expressed. The red box indicates the minimal element necessary for the expression in the Ciona CNS. All fragments start immediately upstream from the translation start codon of CiHox3 and extend in the 5′ direction for the indicated length. Constructs ∆4-3, ∆3-25 and ∆3-2 are internal deletion mutants where the dot line represents the deleted region. (B) Embryo at tailbud stage electroporated with construct 4.2. X-gal staining is visible after 12 days in the anterior region of the developing nervous system. (C) Larva electroporated as in B, where the signal is present, after 8-9 days, in the visceral ganglion and ectopically in the sensory vesicle. (D,E) Embryos at tailbud and larva stages electroporated with 0.45 construct. The staining is lost from the nervous system and is found, after 3-6 days, in ectopic localization in the mesenchyme. ans, anterior nervous system; mp, mesenchymal pocket; tm, trunk mesenchyme; sv, sensory vesicle; vg, visceral ganglion. CiHox3: expression pattern and regulation 4743

Fig. 5. Analysis of the 500 bp genomic sequence containing the CiHox3 enhancer element(s) and of the mouse kreisler binding elements. (A) On the left, diagram of the 500 bp region and of derived fragments studied in the electroporation experiments. The red box indicates the 80 bp containing the minimal element necessary for the CNS expression. Constructs names and their expression territories are shown on the right. (B) On the left, diagram of the mouse kreisler binding element for the HOXa3 and HOXb3 promoters. The green boxes indicate the localization of the kreisler elements. Constructs names and their expression territories are shown on the right. Larvae electroporated with the P1 construct (C) and with the P6 construct (D). X-gal staining is visible in the visceral ganglion and in the sensory vesicle. sv, sensory vesicle; vg, visceral ganglion.

Embryos electroporated with the construct P2 presented a 200-fold excess of cold AL2 was able to decrease complex only ectopic staining in the trunk mesenchyme of the larva. formation while an unrelated oligonucleotide had no effect. Constructs P3 and P4 were able to drive expression of lacZ in Two oligonucleotides, partially overlapping with AL2, named the larval nervous system, both in the visceral ganglion and AL1 and AL3 (Fig. 6A), were tested for the ability to compete ectopically in the sensory vesicle (not shown). The P4 with AL2 for complex formation. As shown in Fig. 6B, only sequence was further subdivided into two fragments and AL1 was able to decrease the binding, suggesting that the embryos electroporated with the construct corresponding to P5 common region between the AL1 and AL2 oligonucleotides did not show expression in the nervous system while P6 was involved in the binding. The analysis of the AL2 sequence fragment reproduced the result obtained with P4 (Fig. 5D). with the MATINSPECTOR 2.2 and TRANSFAC databases did Therefore, the element(s) necessary for the expression in the not reveal any likely recognition sequence for known CNS are present in the 80 bp of the 5′ flanking region from transcription factors. position −1943 to −1864 (Fig. 6A). To date, only the CNS-specific regulatory elements of mouse In order to study the ability of the 80 bp sequence to bind and chicken Hoxb3 or mouse Hoxa3 (Manzanares et al., 1997, nuclear proteins, different overlapping oligonucleotides of 20- 1999) promoters have been studied and several kreisler-like 24 bp each were used in gel shift assays on whole-cell extracts elements have been found to be involved in their activation in of Ciona larvae. Only the oligonucleotide AL2, indicated in rhombomeres (r) 5 and 6. Therefore, we decided to test the Fig. 6A, was able to form a specific complex (Fig. 6B). In fact, transcriptional activity of CiHox3 regulatory elements 4744 A. Locascio and others in mouse embryos. For this purpose, reporter constructs with lacZ for microinjection in mouse oocytes were prepared. We first tested a 2.3 KpnI- HindIII fragment (Fig. 7A), which includes the region involved in the neural-specific expression of CiHox3 in Ciona embryos. Transgenic embryos for this construct consistently gave reporter expression in a Fig. 6. Gel-shift assay with pattern reminiscent of mouse Hox regulatory AL2 double-stranded elements. Expression was detected in the ventral oligonucleotide and protein portions of rhombomere (r) 4 and also in a domain extracts from Ciona larvae. with an anterior boundary at the r6/r7 boundary that (A) Nucleotide sequence of the extended posterior into the spinal cord (Fig. 7B,C). We regulatory region contained in then tested in mouse embryos the CiHox3 P1 promoter the P6 construct. The fragment that was responsible for specific lacZ underlined sequences AL1, expression in the Ciona nervous system (Fig. 7A). In AL2 and AL3 correspond to contrast to the first case, this construct did not generate the oligonucleotides used in any specific pattern of expression in transgenic mouse bandshift assays. (B) A single- shifted band is observed in embryos, but showed only ectopic expression lane 2 where no cold presumably due to effects of the integration site (data competitor was added. not shown). Therefore, these results suggest that the Incubation with 200-fold molar CNS-specific regulatory elements from CiHox3 gene excess of oligonucleotides AL2 that direct reporter expression in that species are or AL1, but not AL3 or not the ones recognized by the mouse embryo random oligonucleotide (R), transcriptional machinery. Hence, other elements must substantially reduced the be present in Ciona genomic fragments that are able complex formation. to mediate a segmental expression in the mouse hindbrain. These elements might correspond to auto- or cross- 1B). The temporal and spatial expression pattern of CiHox3 regulatory regions that are capable of responding to were analyzed by northern blot and whole-mount in situ endogenous mouse Hox genes (see Discussion). hybridization. The earlier stage at which a clear signal could Conversely, when the mouse kreisler-dependent enhancer be detected in the northern blot was the early tailbud. CiHox3 regions, involved in the expression of Hoxa3 and Hoxb3 in the mRNA localization was restricted to the larval nervous system hindbrain (schematized in Fig. 5B), were electroporated in and, in particular, to the visceral ganglion. Ciona embryos, no nervous-system-specific expression was The larval nervous system in C. intestinalis can be observed. Only ectopic expression was detected, such as subdivided into three regions, which have been attributed staining in mesenchymal cell using Hoxa3 and in the muscle distinct functions (Nicol and Meinertzhagen, 1991): in the of the tail using Hoxb3 (data not shown). Hence the mouse trunk, an anterior sensory vesicle, containing the two sensory kreisler-like response elements, conserved in higher organs, followed by the visceral ganglion and, in the tail, the vertebrates, seem unable to regulate any part of a CiHox3-type spinal cord. The visceral ganglion has been considered the expression pattern in Ciona. It is possible that other CNS integrating center of the ascidian nervous system and its regulatory regions recently identified in the mouse Hoxa3 locus architecture has been studied in detail and was found to (Manzanares et al., 1999) might function in Ciona. However, contain, in its outer region, neuronal cell bodies (ganglion our results reveal that the regulation of Hox3 gene expression cells), while in the inner part are present fibrous neuropil in the mouse and Ciona CNS does not appear to be mediated (Barnes, 1971). Transverse sections of hybridized larvae (Fig. by the same highly conserved elements, as either of the 3C) showed that CiHox3 message was localized in the external enhancers shown to direct specific expression in one species area of the visceral ganglion suggesting it could be expressed fails to direct reporter expression in the other. This might in the neuronal cell bodies. The comparison of Hox3 indicate that different regulatory elements and components are expression territories between ascidians, cephalochordates and utilized by these species and/or that similar components are vertebrates might shed light on the evolution of these genes involved but they have slightly diverged and are unable to along this phylogenetic line and could permit the establishment function across such an evolutionary distance. of structural homologies among such different taxa. Studies on Amphioxus AmphiHox3 expression revealed that it is restricted to a specific domain of the dorsal nerve cord with a sharp DISCUSSION boundary only in the anterior part, in particular, at the level of somite pairs 4 and 5 and it is also expressed in the posterior We have isolated a homeobox-containing gene called CiHox3 mesoderm (Holland et al., 1992). In vertebrates, the encoding for a protein with 81 and 78% of identity in its localization of paralogy group 3 genes transcripts is not homeodomain and hexapeptide sequences with AmphiHox3 confined to the nervous system, where they are expressed in and HOX paralogy group 3 of vertebrates, respectively (Fig. the hindbrain with anterior limit at the level of rhombomere 5, CiHox3: expression pattern and regulation 4745

Fig. 7. Analysis of CiHox3 promoter regions in transgenic mouse embryos. (A) Diagram of CiHox3 promoter fragments fused to the reporter lacZ gene and summary of their territories of expression in transgenic mice. N, indicates the number of mouse embryos analyzed. (B,C) Lateral and dorsal view of a mouse embryo microinjected with construct 1. ov, otic vesicle; sc, spinal cord.

1997; Wada et al., 1997) and neural anteroposterior patterning are regulated by the same mechanisms described in vertebrate CNS. Wada et al. (1998) have proposed a subdivision of the ascidian nervous system based on the comparison of the territories of expression of genes involved in the patterning of anteroposterior structures of vertebrates and their ascidian counterparts. Our results indicated that CiHox3 is expressed in Ciona

but are also detectable in posterior and lateral mesoderm (Hunt et al., 1991). Therefore, our analysis on CiHox3 expression, which is restricted to the nervous system, demonstrates that the common feature of Chordates Hox3 genes is in the spatial patterning of the nervous system. Fig. 8 shows a schematic representation of the territories of expression of CiHox3 and Cihox5, the Ciona Hox genes so far analyzed in the larva, compared with the neural expression of mouse Hox counterparts. A previous study on the Ciona homologue of the paralogy group 5 genes, Cihox5 (Gionti at al., 1998), revealed that its expression is limited to the larva neural tube. This demonstrated that the spatial colinearity rule, i.e. the Fig. 8. Comparison of Hox3 and Hox5 expression in the nervous expression pattern of Hox genes follows their position in the system of a C. intestinalis larva and a mouse embryo. Hox3 cluster, is fulfilled in Ciona. As shown in the scheme (Fig. 8), territories of expression are shaded, Hox5 are striped. The CiHox5 is expressed in the most anterior region of the larval expression domain of Hox3 genes in the CNS of a murine embryo neural tube, therefore posteriorly to CiHox3 and without (right side) has an anterior boundary in the hindbrain at the r4/r5 overlapping domains. border and posteriorly extends along the spinal cord. The Moreover, their territory of expression has well-defined homologous CiHox3 gene is expressed, in the anterior region of the anterior and posterior limits, feature which, together with their visceral ganglion of the Ciona larva (left side) with well-defined restricted expression to the nervous system, seems to be unique anterior and posterior limits. Murine Hox5 genes are expressed to Ciona Hox genes. In cephalochordates and vertebrates, Hox exclusively in the spinal cord while CiHox5 mRNA was found in the neural tube of the larva. Note the presence of a posterior limit genes are present in different mesodermal regions and their only for the Ciona Hox5 gene. The expression domains of murine neural expression has only a defined anterior limit, while Hox3 and Hox5 genes in tissues other than the neural tube is not posteriorly extends and overlaps along the entire spinal cord. shown. The murine embryo was from Lumsden (1990). nt, Previous studies suggested that ascidians neural notochord; r, rhombomeres; sc, spinal cord; sv, sensory vescicle; vg, dorsoventral patterning (Corbo et al., 1997a; Glardon et al., visceral ganglion. 4746 A. Locascio and others visceral ganglion and are in agreement with the suggestion that might have been differentially distributed amongst the multiple the mechanisms involved in the anteroposterior regionalization mouse group 3 members. of the nervous system have been conserved between ascidians The converse experiment, testing two different CiHox3 and vertebrates. promoter fragments in transgenic mouse embryos for their In Halocynthia roretzi, the expression pattern of HrHox1 ability to drive reporter lacZ expression, showed that they did was analyzed by Katsuyama et al. (1995). In the nervous not function effectively in mice. The smallest fragment that system of this ascidian, which lacks a defined visceral drives nervous system expression in Ciona was negative in ganglion, its expression is limited in the larva to the sensory mice. This again suggests a divergence of control elements or vesicle and to the junction of trunk and tail. It has been mechanisms between these species. While this may arise due suggested that this latter localization corresponds to the region to totally different mechanisms, it is also possible that small of the visceral ganglion of other ascidian species (Wada et al., divergence of the binding site sequences, and/or associated 1998). Since HrHox1 territory of expression is equivalent to changes in the binding capabilities of the same trans-acting the visceral ganglion, there would be an overlapping of factors, would render Ciona sites unrecognizable by the mouse expression with CiHox3. Therefore, the study of C. intestinalis machinery or vice versa. Hox1 and Hox2 homologues would be necessary to compare Nevertheless, it is interesting that when a slightly larger the expression territories of Ciona and Halocynthia Hox genes. Ciona enhancer fragment was tested, a reproducible segmental To better understand the evolutionary relationships of Hox3 pattern of expression in the mouse hindbrain was obtained. genes between ascidians and vertebrates, we carried out the This consisted of expression in the ventral portions of r4 and analysis of CiHox3 promoter regulatory elements with the a second domain starting at the r6/r7 boundary, which spreads electroporation method. The results indicated that a sequence posteriorly into the spinal cord. This pattern is reminiscent of of 80 bp, from position −1943 to −1864 from the translation those seen using the mouse Hoxb1 r4 enhancer (Marshall et al., start site, contained the positive regulatory element(s) able to 1992; Popperl et al., 1995), and the Hoxb4 r6/7 region A activate transcription in both the visceral ganglion and the enhancer (Whiting et al., 1991). One possible explanation for sensory vesicle. the similarities in these patterns, comes from the observation Analysis of this sequence by band-shift assay indicated that that both the Hoxb1 r4 and the Hoxb4 r6/7 domains, are only the AL2 sequence (Fig. 6A) was able to form a complex dependant on auto-/cross-regulatory loops involving Hox/Pbx with protein extracts from Ciona larvae. This suggested the interactions (Popperl et al., 1995; Gould et al., 1997). presence of an unknown transcription factor binding site in the Therefore, the larger CiHox3 CNS enhancer that also functions AL2 sequence presumably involved in the neural-specific in mice, might contain both the minimal CNS element defined activation of CiHox3. in this study and an auto-/cross-regulatory element. The pattern It has been proposed that the conservation of a cluster obtained in mice would then reflect a read-out of mouse organization for Hox genes during evolution was also due to Hox/Pbx complexes acting through a CiHox3 Hox-responsive the presence of common regulatory elements for different Hox element. This may indicate that the Ciona Hox complex also genes (Lufkin, 1996). Therefore, we decided to see whether the uses auto-/cross-regulatory interactions for maintaining later regulatory elements identified from CiHox3 would be expression patterns. In conclusion, this comparative regulatory recognized by vertebrate transcriptional networks and, on the analysis between Ciona and mice demonstrates that, at least contrary, whether the mouse HOXa3 and HOXb3 promoters for the group 3 Hox genes, there is considerable divergence in were able to work in Ciona. In fact, although there is ample sequences implicated in mediating CNS expression. Further proof of the common evolutionary origin of Hox complexes analysis in other chordates and intermediate species will be throughout all animal phyla, this has been studied basically on required to elucidate the molecular basis of these differences. the coding regions for the Hox genes. Much less is known about the conservation and/or divergence of regulatory We are indebted to Dr Albertina Fanelli for her invaluable help in elements. The regulation of vertebrate group 3 Hox genes has the preparation of the manuscript. We thank Professor Roberto Di recently been analyzed, and it has been shown that the b-ZIP Lauro and Dr Rosaria De Santis for helpful discussion and Gennaro transcription factor encoded by the kreisler gene is responsible Iamunno for sectioning C. intestinalis larvae. We thank Amanda Hewett, Peter Mealyer and Rosemary Murphy for help in animal for the upregulation of Hoxb3 in r5 and Hoxa3 in r5 and r6 husbandry. M. M. was supported by HFSP and EEC Marie Curie (Manzanares et al., 1997, 1999). Therefore, direct comparisons postdoctoral fellowships and by an EEC Biotechnology Network between regulation of mouse and Ciona group 3 Hox genes is grant (#BIO4 CT-960378). This work was also funded in part by Core possible. MRC Programme support and an EEC Biotechnology Network grant Mouse Hoxa3 and Hoxb3 rhombomere-specific regulatory (#BIO4 CT-960378) to R. K. elements tested in Ciona embryos were unable to reproduce any neural-specific expression. This suggests that indeed the regulation of Hox3 genes between mouse and Ciona is REFERENCES somewhat different. However, the kreisler-dependent enhancers could represent a recent acquisition by the higher Amores, A., Force, A., Yan, Y. L., Joly, L., Amemiya, C., Fritz, A., Ho, R., vertebrates, which occurred during the early duplications Langeland, J., Prince, V., Wang, Y. L., Westerfield, M., Ekker, M. and leading to four Hox complexes. 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