Molecular Phylogenetics and Evolution 40 (2006) 872–879 www.elsevier.com/locate/ympev Short communication Unexpected variation of Hox genes’ homeodomains in

Mathieu Pernice b, Jean S. Deutsch a, Aude Andouche b, Renata Boucher-Rodoni b, Laure Bonnaud b,¤

a Développement et Evolution, UMR 7622, CNRS et Université P et M Curie (Paris 6), Case 24, 9 quai St Bernard, 75252 Paris Cedex 05, France b Muséum National d’Histoire Naturelle, Département Milieux et Peuplements Aquatiques, Biologie des Organismes Marins et Ecosystèmes (UMR 5178 CNRS), 55 rue BuVon, CP51, 75005 Paris, France

Received 2 September 2005; revised 24 February 2006; accepted 4 April 2006 Available online 26 April 2006

1. Introduction derived from the foot, a condensed nervous system associ- ated with several sensory structures and a speciWc direct Hox genes encode a family of transcription factors con- development. Among the eight recognised classes of extant taining a homeodomain-binding motif of 60 aminoacids molluscs six have been explored, few Hox genes have been (Gehring et al., 1994). They are extensively conserved identiWed and the number of genes in each cluster is far throughout Eu-metazoans (de Rosa et al., 1999). As they from being determined. The amount of available data have a role in the determination of the anterior-posterior recently increased with the characterisation of 11 Hox par- axis during development, they may give information about alogues in a bivalve, , the highest number the history of bilaterian lineages (de Rosa et al., 1999; Bal- found at present (Canapa et al., 2005). In gastropods, con- avoine et al., 2002). In bilaterians, they are organised in sidered as the sister group of cephalopods, six Hox genes clusters, the last common ancestor of protostomes and have been characterised in vulgata (de Rosa et al., deuterostomes having a cluster of 8–10 Hox genes (Cook 1999). In cephalopods, nine genes have been identiWed in et al., 2004). They appear to have undergone various inde- Euprymna scolopes, a sepiolid (Callaerts et al., 2002). The pendent duplications in the three clades Ecdysozoa, Lopho- aim of the present work was to compare Hox genes trochozoa and Deuterostomia. They present some between issued from the basal lineage conserved features that allowed determining lophotrocho- Nautiloidea ( D Tetrabranchiata) and those issued from the zoan signatures (de Rosa et al., 1999) but they have not derived lineage Coleoidea ( D Dibranchiata). In the present been used to distinguish between the main phyla within the study, we identiWed Hox genes in Sepia oYcinalis, a cuttle- Lophotrochozoa yet. Wsh belonging to the Decabrachia group like E. scolopes, To study Hox gene evolution it is crucial to include more and two nautiloids, macromphalus and Nautilus sequences from lophotrochozoan species. Among them, the pompilius. The latter have preserved a number of ancestral phylum is a Cambrian lineage showing a great morphological characters (e.g. external shell, numerous ten- diversity of body plans. It is considered as a monophyletic tacles without suckers) that were present in the Ammonites, group based on shared derived characters such as a foot, a a lineage close to extant nautiloids, which became extinct in , a mantle secreting the shell and a palleal cavity har- the Mesozoic era. We then compared our homeodomain bouring gills (Brusca and Brusca, 1990). sequences with those of available data from other molluscs Here we report the cloning of Hox genes from three and metazoans. cephalopod species. Cephalopods are distinct from other molluscs in numerous features. Cephalopoda is a mono- 2. Materials and methods phyletic group based on common derived characters (syna- pomorphies): a funnel and a brachial crown probably 2.1. Biological material and DNA extraction

Nautilus pompilius was collected on the east coast of * Corresponding author. Fax: +33 1 40 79 57 34. Cebu island (Philippines), N. macromphalus in New Cale- E-mail address: [email protected] (L. Bonnaud). donia and S. oYcinalis oV the coast of Banyuls (France).

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.04.004 M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879 873 ) EEN534450 AF118832 ¤ continued on nextcontinued page on ( X79371 Hox9 Post1 Hox10 AbdB/Post2 Cdx cad Xlox X17566 AY439322 AF151669 AJ876627 AJ937217 AF144671 AF144678 Lox2 abdA/Lox4 AF400177 AF400175 7 AY052758 AY052759 AY052760 AY052761 AF127341 AY439325 AY439324 AY439326 AY439328 AY439329 in Swissprot database) ¤ 7 U26629 U26628 U26635 U26633 AF00168 AF400163 AF400170 AY497424 S76354 AY439316AY439317 AY439320 AY439327 AY439330 AY736274 L41848 L41851 L41846 Lsa Y16570 Y16571 Y16572 Y16573 Y16574 P81193 HroHtrHme AF004386 AF017253 LO7298 AF004387 AF006638 Y10888 AF137385 S79240 Sla S76355 S76357 S76361 Pex AY439315 AY439318 AY439319 AY439321 AY439323 CseCva U26539 AF163856 AF163857 U26625Ean AF163858 AF400169 U26626 U2662 AF400165 CVU68284 AF400167 AF400164 AF163860 AF163859 AF400162 AF400166 AF400180 AF400181 Nvi AF151663 AF151664 AF151665 AF151666 AF151667 AF151671 AF151668 AF151672 AF151673 AY117546 Has AF327746Hru X79374 X79372 AF327747 AF328863 X79377 AF275310 RphMgaPma AJ876619 AJ876620 AJ876621 AJ629073 AJ876622 MGA534452 AJ629072 AJ876623 AJ629071 PMA534457 AJ876625 AJ876626 AJ876628 AJ876629 AJ575213 AJ876631 TPH534460 TPH534459 TPH534461 YeiEen YEI534462 EEN534448 EEN534449 YEI534464 YEI534463 NmaPvuIob AF118831 AY736276 AF144666 AF118833 AF144667 AM050632 AM050631 AM050634 AM050633 AM050630 AF144669 AF144668 AF144670 AJ518062 Sof AJ937220Npo AJ937214 AJ937221 AJ937215 AJ937222 AJ937223 AJ937216 AM050636 AJ937224 Pni L41847 AJ937219 L41850 L41852 L41854 L41849 Btu AY497421 AY497422 AY497423 AY497425 AY497426 Esc AY052753 AY052755 AF127335 AY052756 AY05275 Lan AF144672 AF144673 AF144674 AF144676 AF144675 AF144677 AF144679 AF144680 cinalis Y Lineus sanguineus Hellobdella robusta triserialis Hellobdella Hirudo medicinalis Stylaria lacustris Perionyx excavatus Ctenodrilus serratus Chaetopterus variopedatus Eisenia andrei Nereis virens Haliotis asinina rufescens Haliotis Ruditapes philippinarum Mytilus galloprovincialis maximus Pecten Yoldia eightsi Ensis Nautilus macromphalus Patella vulgata Ilyanassa obsoleta Sepia o pompilius Nautilus nigra Polycelis Bugula turrita Euprymna scolopes Lingula anatina Anopla Hirudinida Oligochaeta Polychaeta Cephalopoda Nemerta Annelida Plathyhelmintha Table 1 Table Species taxonomicanalysedHox genesandwith name, database position their accessionin numbers EMBL (except Ectoprocta PhylumMollusca Species Abr. Hox1 lab Hox2 pbzen Hox3 Hox4Dfd Hox5 Scr Hox6 ftz/Lox5Antp Hox7 Ubx/ Hox8 Brachiopoda 874 M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879

Tissue samples (mantle and brain) were either frozen or preserved in alcohol and stored at 4 °C. DNA was extracted using the Dneasy Tissue Kit (Qiagen) and stored in aliquots at ¡20 °C.

2.2. PCR ampliWcation

The central part of the homeodomain Hox gene was ampliWed by PCR from Nautilus and Sepia DNA using degenerate primers HoxA and HoxB (5Ј-GARY TNGARAARGARTT-3Ј corresponding to amino acids sequence ELEKEF and 3Ј-CKNCKRTTYTGRAACCA- 5Ј corresponding to WFQNRR, respectively). Lox2 was ampliWed in N. pompilius using HoxB and 5Ј-CCNAAYT CNTMNCARAGRAGRAAR-3Ј corresponding to amino Hox9 Post1 Hox10 AbdB/Post2 Cdx cad Xlox acids sequence PNS(S/Y)QRRK. Post2 homeodomain gene was ampliWed in S. oYcinalis only using 5Ј-MGNTAYCA RACNATGGTNYTNGARAAYGARTTY-3Ј corre- AJ007434 AJ007436 AF241657 X54453 AF017415 AJ005422 Lox2 abdA/Lox4 sponding to amino acids sequence RYQTMVLENEF and 3Ј-YTTYTTNCKYTTCATNCKNCKRTTYTGRAACC A-5Ј corresponding to amino acids sequence WFQNRR MKRKK. The reaction mixture contained 0.6 mol of each primer, 0.4 mmol of each deoxynucleoside triphosphate, 1X SuperTaq buVer and 2.5 U of SuperTaq polymerase

4 M20704 X76210 X51663 X03062 (ATGC) in a total volume of 50 l. PCR were conducted with an initial denaturing step (94 °C for 5 min) followed by 40 cycles at 94°C for 1min, 42°C for 2min and 72°C for 1 min and a Wnal elongation step at 72 °C for 7 min. PCR products of the expected size (150 bp) were puriWed with a Qiaquick gel extraction Kit (Qiagen).

2.3. Cloning and sequencing

PuriWed PCR products were then cloned by insertion into the plasmid vector PCR 2.1 TOPO TA Cloning (Invitrogen) following the manufacturer’s instructions. Plasmids were pre- pared from pelleted cells with a QIAprep Miniprep kit (Qiagen). A total of 30 clones for each species, i.e. S. oYcinalis, N. pompilius and N. macromphalus, were sequenced.

AF362086 AF3620892.4. AF362092 AF362094 Alignments and sequence comparison

The central part of the homeodomain (aminoacids) Z35142 Z35143 X68045 AB028208sequences Z35145 Z35146 of Z35147lophotrochozoans, Z35148 Z35150 ecdysozoans AF052465 AF052464 and deuter-

X ostomes were aligned using the Bioedit program. Species Alo AF071407 Csa AJ007431B Pma AJ005643Omy AJ007432 AJ007433 AJ007435 AAM19473 AY567802 CSA131397 Z35149 ScaSla U79468 AF148935 U79469 AF148936 AF148937 U79476 AF1011431 AF393441 AF393444 AF393443 AF393442 AF241662 AF241661 AF481736 AY779183 U79471 Tdo AF104008Tca AF104009 AF231104 AY456922 AF187068Dme X97819 X13103 AF104005 AF104010 X63728 AAB393556 AAK16422 AF321227 X05136 AF104003 AF104001 X14475 AY074761 X0085 AF104002 AF227923 AJ005421 Lfo AF362084 AF362085 AF362087 AF362088 AF362090 AF362091 AF362093name, AF362095 taxonomic status and accession number of each gene included in the alignments are given in Table 1. Alignments are described in supporting material (Fig. S1). Based on oridae X

catus current literature and our present analysis, we grouped the W genes into paralogy groups from Hox1 to Hox9/10. Lox2, Lox4, Ubx and abdA were included in the same Hox8 group, Post1/Post2/AbdB in the same Hox9/10 group. Archegozetes longisetosus Branchiostoma Cupiennius salei Petromyzon marinus Oncorhynchus mykiss Lithobius for Sacculina carcini Porcellio scaber Thermobia domestica Tribolium castaneum Drosophila melanogaster )

3. Results and discussion continued The most conserved motives of all Hox and paraHox Chelicerata Myriapoda Crustacea Hexapoda Chordata Table 1 ( Table Phylum Species Abr. Hox1 lab Hox2 pbzen Hox3 Hox4Dfd Hox5 Scr Hox6 ftz/Lox5Antp Hox7 Ubx/ Hox8 included. are species Mollusca the All Arthropoda genes are situated within helix 1 and helix 3 of the homeo- M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879 875 domain. The sequence located in between contains typical Characterisation of a unique fragment per gene in each aminoacids (“signatures”) speciWc to each so-called paral- species suggests that a single set of Hox genes is present in ogy group of Hox genes. We used classical degenerate prim- the genome of all three cephalopod species here studied. A ers designed from the conserved peptides of helices 1 and 3 chromosomal duplication has been suspected to have to isolate by PCR ampliWcation (see Materials and meth- occurred during the evolution of the Decabrachia lineage ods) Hox genes’ fragments from genomic DNA of the (Bonnaud et al., 2004). Nevertheless, chromosomal duplica- cephalopod species S. oYcinalis (sepiid), N. pompilius and tions in “invertebrates” do not generally lead to duplication N. macromphalus (nautiloids). We were able to recover six of the Hox cluster, contrary to what is known in vertebrates diVerent gene fragments from N. pompilius, eight from N. (Wagner et al., 2003). macromphalus and six from S. oYcinalis. Among the 11 Hox paralogues described in other lopho- ClassiWcation of our isolated gene fragments into Hox trochozoan species (de Rosa et al., 1999), we have isolated paralogy groups was determined by comparison of their all but the proboscipedia/Hox2 and Post1 genes. Eight para- translated sequence, using diagnostic aminoacids, to the logues were characterised summing up the two Nautilus homeodomain of Hox genes from a panel of species. These species: one anterior group gene labial/Hox1, one ortho- included all available Hox genes from lophotrochozoan logue of Hox3, six median group genes, i.e. Dfd/Hox4, Scr/ species, a number of arthropod species and the amphioxus Hox5, Lox5/Hox6, Antp/Hox7, Lox2 and Lox4. Five para- Branchiostoma Xoridae as a chordate representative (see logues were characterised in S. oYcinalis: one anterior supplementary Fig. S1). group gene labial/Hox1, one orthologue of Hox3, two cen-

Fig. 1. (A) Mollusc-speciWc signatures identiWed in the Hox5 (Scr) paralogy group. (B) Mollusc-speciWc signatures identiWed in the Post paralogy group. 876 M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879

Fig. 1 (Continued) tral group genes Scr/Hox5, Lox4/Hox8 and one posterior been lost in parallel with morphological innovations of this gene, Post2/Hox9-10. Sequences of paraHox genes (Brooke group. et al., 1998) were also identiWed: Xlox in N. pompilius and Both Lox2 and Lox4 have been identiWed in Nautilus caudal/Cdx in S. oYcinalis. (Detailed results and discussion species. These two median Hox genes, Wrst found in a of each group are reported in supporting material and leech, are thought to derive by duplication from a single Fig. S1). ancestor (Wong et al., 1995; Balavoine et al., 2002). They Adding to the sequences already known in E. scolopes were also found in P. vulgata (de Rosa et al., 1999) and P. (Callaerts et al., 2002), all Hox genes identiWed in other lop- maximus (Canapa et al., 2005) among the Mollusca and hotrochozoans are present in the cephalopod lineage, with in the brachiopod Lingula anatina (de Rosa et al., 1999) the possible exception of proboscipedia. showing that this duplication preceded the divergence In Drosophila, pb is required for the speciWcation of between the Lophotrochoza and the other protostomes. mouthparts in adults (Percival-Smith et al., 1997). We have We grouped both of them into a Hox8 group, together not retrieved a proboscipedia/Hox2 (pb) gene in any of the with Ultrabithorax (Ubx) and abdominal-A (abdA) of three cephalopod species here studied. This is consistent arthropods and Hox8 of the amphioxus. Our data show with the results obtained by Callaerts et al. (2002) on the that Lox4 sequences are more similar to Ubx/abdA than sepiolid E. scolopes. Thus, at present, there is no evidence to Lox2. It means that the Lox group is paraphyletic, that pb is present in Cephalopoda. Homologues of this gene with the “UbdA” group (Balavoine, 1997) included have been reported in Bivalvia (Barucca et al., 2003; within. This could suggest a diVerent scenario for the Carpintero et al., 2004) and Gastropoda (Degnan and evolution of these Hox genes, in that the Lox2/Lox4 and Morse, 1993). Our data are not exhaustive, so that it could Ubx/abdA duplications may not be independent from have escaped our search. Alternatively, the gene may have each other. M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879 877

Lox2 has not been identiWed up to now neither in (K) at positions 22 and 24, respectively, whereas all other S. oYcinalis nor in the other decabrachian species, E. sco- bilaterians exhibit a phenylalanine (F) and an arginine (R) lopes. This gene is very conserved among mollusc clades (Fig. 1A). The Post genes of molluscs show two residues and must have been present in the mollusc ancestor. It is that may represent mollusc-speciWc signatures : a serine (S) conserved in the Nautilus lineage but it may have been lost at position 23 and a hydrophobic leucine (L) at position 37 in the Decabrachia lineage. in Post1 from molluscs, whereas a basic residue is found at We thus propose a number of 10–11 Hox genes as primi- this position in other Post proteins, i.e. R or Q in Post1, K, tive in the Cephalopoda group, possibly one less than sug- R or H in Post2 (Fig. 1B). gested for other molluscs such as the bivalve Pecten In several instances, coleoids are distinct from nauti- (Canapa et al., 2005). loids by presenting derived features. In Lox4, a Hox pro- Our data on cephalopod sequences conWrm that Hox tein speciWc to lophotrochozoans, phenylalanine (F), genes show speciWc features at the “supra-phylum” level histidine (H) and cysteine (C) at positions 22, 24 and 39 such as lophotrochozoan-speciWc genes (Lox2 and Lox4; of the homeodomain, respectively, are clearly primitive. Post1 and Post2) and signatures (de Rosa et al., 1999). These residues, present in all other lophotrochozoans More unexpectedly, Hox genes can be informative at lower (with the unique exception of Y22 in the bivalve Rudi- taxonomic levels. At the phylum level, in spite of the diver- tapes) are also present in Nautilus. They are replaced by a sity of body plans within the Mollusca, Hox homeodo- tyrosine (Y) at position 22 and an asparagine (N) at posi- mains show mollusc-speciWc signatures. This is exempliWed tions 24 and 39 in both Euprymna and Sepia (Fig. 2A). by the Hox5 group, where all mollusc sequences are identi- Similarly, in the Hox3 protein, an arginine (R) is found at cal in the central part of the homeodomain, with the single position 24 in all bilaterians, except in some insects where exception of the Mytilus galloprovincialis (Mga- the Hox3 homologues zen and bicoid genes are known to Hox1). Very interestingly, all seven mollusc sequences, be extremely derived in both sequence and function (Fal- including that of Mytilus, show a tyrosine (Y) and a lysine ciani et al., 1996; Stauber et al., 1999). A glutamine (Q) is

A

Fig. 2. (A) Decabrachian-speciWc signatures- Lox4 protein. (B) Decabrachian-speciWc signatures- Hox3 (zen) paralogy group. 878 M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879

B

Fig. 2 (Continued) found at this position in both Euprymna and Sepia signatures speciWc to a certain clade. This opens the per- (Fig. 2B). These two cephalopod lineages, nautiloids and spective to explore Hox genes’ full-length sequences in a decabrachian coleoids, have diverged since the Ordovi- variety of mollusc species. Phylogenetic analysis of com- cian (ca. 400 MY). These changes in the otherwise so con- plete Hox genes sequences may allow determining more served central part of the Hox homeodomain among precisely evolutionary relationships within the phylum bilaterians are noteworthy. Derived residues in decabra- Mollusca. chians might be correlated to derived morphological characters. In that respect, expression patterns of Hox Acknowledgments genes in both nautiloid and coleoid species would be worth studying and comparing. Of particular interest is We are grateful to all people who participated in the Wnding that the Hox3 and Lox4 genes have been collecting biological material: Pascale Joannot for shown to be expressed in the stellate ganglia of providing Nautilus macromphalus specimens from New- Euprymna, a neuro-anatomical novelty speciWc to cole- Caledonia, Aquascapes Philippines for collecting oids (Lee et al., 2003). Nautilus pompilius specimens, and the Observatoire We have shown that the central part of Hox homeodo- Océanologique of Banyuls (Université Pierre et Marie mains present aminoacid residues that can be viewed as Curie, Paris 6) for providing Sepia oYcinalis. We thank M. Pernice et al. / Molecular Phylogenetics and Evolution 40 (2006) 872–879 879

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