ORIGINAL ARTICLE

Engrailed in cephalopods: a key gene relatedto the emergence of morphological novelties

S. Baratte & A. Andouche & L. Bonnaud

Received: 15 December 2006 /Accepted: 6 March 2007 / Published online: 30 March 2007# Springer-Verlag 2007

Abstract The engrailed gene is a transcription factorrequired in numerous species for major developmentalsteps (neurogenesis, limb development, boundary establish-ment), and its evolution is known to be closely related tothe evolution of the metazoan body plan. Cephalopodsexhibit numerous morphological peculiarities among mol-luscs, such as a direct development, a complex sensory andnervous system (eyes, brain, giant axons), a reduced shell, afunnel, and a brachial crown. We assessed a potentialrecruitment of engrailed in the development of thesederived traits and examined the expression pattern ofengrailed during the organogenesis of the cuttlefish Sepiaofficinalis, by immunostaining. Engrailed was detected atthe margin of the prospective internal shell, which isconsistent with studies on molluscs having an externalshell and confirms a conserved role of engrailed indelimitating the molluscan shell compartment. Interestingly,unexpected patterns were early detected in the emergingarms, funnel and optic vesicles and latter in tentacles andeye lids. We also identified an engrailed cognate in thesquid Loligo, which provides new evidence that engrailedin molluscs is not restricted to a ‘shell function’ and hasbeen recruited in the mollusc lineage for the emergence ofmorphological novelties in cephalopods.

Keywords Engrailed . Sepia . Shell differentiation .

Gene co-option . Derived structures

Introduction

A major issue in current biology is the evolution of themetazoan body plan. Widespread developmental processes,such as the establishment of the antero-posterior polarity orthe formation of the mesodermal layer, are conducted byhighly conserved groups of regulatory genes, whereasmorphological variations and novelties often result fromdivergences (duplication, mutation) in gene sequences and/or in regulatory networks (Force et al. 1999; Gibert 2002).These networks often involve many homeobox genes actingas transcription factors regulating gene expression duringdevelopmental patterning or cell differentiation. Studyinghow they act and which feature they control has become apowerful way of understanding organism complexity.Among those genes, engrailed is one of the most relevantexample demonstrating the evolvability and plasticity ofgene function during evolution. This transcription factorwas first shown in Drosophila to be a key gene in theestablishment of segment polarity (Kornberg 1981; Fjose etal. 1985), in neurogenesis (Patel et al. 1989), and inappendage development (Raftery et al. 1991). Highlyconserved in protostomes and deuterostomes, engrailedorthologues show similar roles in other arthropods (Patelet al. 1989; Abzhanov and Kaufman 2000), in annelids(Wedeen and Weisblat 1991; Seaver and Kaneshige 2006),in echinoderms (Lowe and Wray 1997; Byrne et al. 2005;Yaguchi et al. 2006), and in chordates (Joyner 1996;Holland et al. 1997). Extensive comparisons among taxasuggest that neurogenesis is likely the ancestral function of

Dev Genes Evol (2007) 217:353–362DOI 10.1007/s00427-007-0147-2

Communicated by D.A. Weisblat

S. Baratte (*) :A. Andouche : L. BonnaudBOME—Biologie des organismes marins et écosystèmes—CNRSUMR 5178, Muséum National d’Histoire Naturelle,55 rue Buffon,75005 Paris, Francee-mail: baratte@mnhn.fr

engrailed and that subsequent recruitments have increasedengrailed contributions (Patel et al. 1989; Gibert 2002). Inmolluscs, however, there is no strong data for theinvolvement of engrailed in neural development. Instead,engrailed is expressed in cells at the margin of the futureshell (protoconch) in a wide range of molluscs: in a bivalve(Transenella tantilla, Jacobs et al. 2000), in gastropods(Ilyanassa obsoleta, Moshel et al. 1998; Patella vulgata,Nederbragt et al. 2002), in a scaphopod (Antalis entalis,Wanninger and Haszprunar 2001), and in a polyplaco-phoran (Lepidochitona caverna, Jacobs et al. 2000). Fromengrailed role during shell development in molluscs,Nederbragt et al. (2002) proposed that its ancestral functionis the formation of a compartment boundary as analternative to the neurogenic hypothesis.

Among these studies on molluscs, a major group islacking that could bring a larger and more completeoverview of the molluscan taxa and of the lophotrocho-zoans in general. The cephalopods have not yet beeninvestigated for engrailed, although they exhibit numerousderived traits among molluscs. As cephalopods undergo adirect development without a trochophore larval stage,investigating engrailed in a cephalopod provides a directinsight at the role of this gene in definitive organogenesis.In cephalopods, the typical molluscan foot is modified intoeight to ten prehensile appendages (arms and tentacles) anda funnel. Both this funnel and a muscular mantle areimplicated in jet propulsion. High capacities of cognitionand reaction are permitted by a compact brain, by aperipheral nervous system allowing rapid nervous trans-mission (giant axons, stellate ganglia), and by sensorystructures, such as complex camerular eyes (Boletzky1988). Whereas one of the molluscan characteristic is anexternal shell secreted by the mantle, coleoid cephalopods,comprising all extant cephalopods except nautiluses, pos-sess an internal shell, which may be regressed andsometimes absent.

Two paralogues of engrailed have been found inNautilus pompilius, a cephalopod with an external shell(Wray et al. 1995), whereas no en cognate was identifiedyet in the squid Loligo, a coleoid whose shell is internal andnot calcified (chitinous gladius). As a consequence,Wanninger and Haszprunar (2001) have correlated thepresence of engrailed with that of an external shell. In thispaper, we present the first report of an engrailed geneexpression pattern in a coleoid cephalopod, the cuttlefishSepia officinalis. We show that it is expressed in the shell-forming cells in early stages of organogenesis. Thissupports the role of engrailed in molluscan shell formationto organisms with an internal shell. Moreover, we show thatengrailed is also expressed in cephalopod-specific organs:the funnel and arms, the optic vesicle, and eye lids. We,here, identified an engrailed homologous in a Loligo

species that adds further lines of evidence for a widespreadrecruitment of engrailed in morphological novelties anddiversification of metazoan body plan.

Materials and methods

Collection of Sepia embryos

During spring and summer (April to September), fertilizedeggs were laid by captive S. officinalis females in thebiological stations of Luc-sur-mer (France) and Banyuls-sur-mer (France). From egg batches, individual eggs weredetached and embryos were taken out by removing thenumerous surrounding envelopes using forceps in seawater. Then, embryos were visually staged using Lemaire’s(1970) system for S. officinalis. As we focused onorganogenesis, embryos at stages 16 to 25 were selectedfor immunochemistry. After chorion removal, embryoswere fixed for 1 h in 4% paraformaldehyde (PFA) at roomtemperature, washed in phosphate-buffered saline (PBS),gradually dehydrated in methanol, and stored at −20°C.Until stage 16, the chorion and embryo are in intimatecontact, and an additional fixation for 1 h in PFA 4% wasperformed to strengthen the chorion and reduce rupturehazard.

Cloning of engrailed gene homeodomain

Genomic DNA of S. officinalis was extracted from adultbrain using the DNeasy Tissue kit (Qiagen, Valencia, CA,USA). mRNAs of Loligo vulgaris (collected in Caen,France) were extracted from stage 30 embryos using TriReagent (MRC, Cincinnati, OH, USA), then converted intocDNA by the Omniscript reverse-transcriptase (Qiagen).Initial amplification primers for polymerase chain reaction(PCR; Eng-2: 5′-GACAAGCGRCCDMGVACVGCNTT-3′: KPPRTAF; Eng-3: 5′-ATCAAGCTTWTTYTKRAACCANAYYTTNAYYTG-3′: QIKIWFQN) were used toamplify a 106-bp homeodomain fragment in S. officinalis.Primers Eng-2 and Eng-4 (5′-TGRTTRRTANARNCCYTGNGCCAT-3′:MAQGLYN) were used to amplify a 189-bphomeodomain fragment in L. vulgaris. PCR conditionswere: 95°C for 5 min+(95°C for 1 min; 45°C for 1 min;72°C for 1 min) for five cycles+(95°C for 1 min; 50°C for1 min; 72°C for 1.5 min) for 30 cycles+72°C for 10 min.PCR products were cloned into TOPO vector (Invitrogen,Carlsbad, CA, USA) sequenced by Genome Express(Meylan, France) and analyzed with the BioEdit software(Ibis Therapeutics, Carlsbad, CA, USA) and GenBankBLASTn (BLAST, basic local alignment search tool).

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Whole-mount immunochemistry

The engrailed protein (En) was detected by using amonoclonal antibody Mab4D9 (Developmental StudiesHybridoma Bank, University of Iowa, USA) raised againsta portion of the Drosophila En as primary antibody (Patelet al. 1989). It has been shown to selectively bind anengrailed protein in some molluscs: a gastropod and achiton (Moshel et al. 1998; Jacobs et al. 2000), and we,therefore, assumed that it detected an engrailed-like proteinin Sepia. Embryos were incubated in hydrogen peroxide(3% in pure methanol) for 1 h to inactivate endogenousperoxidases; they were preincubated in blocking solution(PBS 1×+bovine serum albumin 1%+Triton X-100 0.1%)for 1 h at room temperature and incubated with theMab4D9 (1:200) in blocking solution overnight at 4°C(without Ab in controls). After five washes of 30 min inpolybutylene terephthalate (PBT; PBS 1×+Triton X-1000.1%), embryos were preincubated again for 1 h at roomtemperature in blocking solution and incubated with the1:500 biotin-conjugated universal secondary antibody(ABC Kit, Vector Laboratories, Burlingame, CA, USA)overnight at 4°C. After four washes of 30 min each in PBT,embryos were incubated with streptavidin-conjugatedhorseradish peroxidase (ABC kit) for 30 min, rinsed threetimes for 10 min in PBT, and MAb4D9-binding sites werefinally revealed using 3,3-diaminobenzedine (DAB-nickelkit, SK-4100, Vector Laboratories Burlingame, CA, USA),a colored substrate of peroxidase (dark brown). After 10–15 min, the coloration process was stopped in PBT, andembryos were fixed with PFA 4%–PBT.

Results

By PCRs on genomic DNA of S. officinalis, we detected asingle engrailed gene (Fig. 1, accession number: AM114934), and we were able to similarly confirm the presenceof two en paralogues in N. pompilius (accession numbers:AM114935, AM114936). Using cDNA of L. vulgaris, weidentified the first engrailed cognate in a squid (accessionnumber: AM422130). As expected, the En homeodomain ishighly conserved among cephalopods, coleoid sequencesbeing more similar between each other than with those ofNautilus (nautiloid; Fig. 1). For immunostaining, we used the4D9 antibody, raised against the Drosophila Engrailed proteinbut able to recognize many other Engrailed homologuesdepending on the residue present at position 40 of thehomeodomain (Patel et al. 1989). En proteins with G or S aredetected, whereas some residues, such as R, T or Q, areknown to prevent 4D9 binding (Patel et al. 1989; Wedeen andWeisblat 1991). The S. officinalis and L. vulgaris homeo-domains are alone to exhibit a cysteine at position 40 (Fig. 1,arrow). It cannot be excluded that Mab4d9 recognizes anengrailed-like protein or a cognate of engrailed, not yetsequenced in Sepia. Recent molecular studies, however,showed that this antibody recognizes an engrailed protein inmolluscs (Moshel et al. 1998; Jacobs et al. 2000). Thestaining we obtained in Sepia, especially in the shell sac (seebelow), corresponds to that found in other molluscs, whichsuggests that 4D9 is able to detect the cysteine at position 40or, at least, an engrailed-like protein.

S. officinalis develops directly with no larval intermedi-ate and no metamorphosis. Organogenesis proceeds during

10 20 30 40 50 60 70 ..|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Sepia officinalis KRPRTAFTSDQLQRLKQEFDDCRYLTEERRKKLARDLCLSEAQVKVWFQNLoligo vulgaris ............................E.......I.I....KRAKIKKSSGVKNPLAIHLM AQGLYNEuprymna scolopes .....L..............D.......E.S.....I.I..............M.....L.. Nautilus pompilius A ......Y..K..EE......D..R....E.S.... Nautilus pompilius B ..E.....RR..EAGK....D..QT..KE.G.N.S

Lepidochiton caverna SDS..E...K...SS...S.AK.QD...E.R.N.S.I.I........L...TSGRTG..L... Patella vulgata .N.......H...EN.....T..QH..NE.G.H.S...I........L..AT.TT....LK.. Illyanassa obsoleta A ..E..S...R...E......T..RH..AE.G.T.S.I.I............S....E..MQ.. Illyanassa obsoleta B ..E..S...R..EE......T..RH..AE.G.T.S.I.I............S....E..MQ.. Cadulus fusiformis SNE..D..QH..ETS.....Q..RN.SME.D...T.I.I........L..TG.GSSD..Q... Dentalium eboreum SNE..D..QV..EA......T...D.SDE.K...T.I.I........L..TGSNSPS..L... Placopecten magellanicus .N.......R..EE......Q..LD..QE.N.T...I.I........Ö...VAPR.T..LS.. Transennella tantilla .TE..RK..D..EANK..S.N..QQ..HE.N.N.S.I.I...........CN.D..G..ME.. Crassostrea virginica .T.......S..EENH....K..LE.SEE.K...S.I.I............T.G..T..LK..

Drosophila melanogaster S.E..A...R..NEN.....R..QQ.SSE.G.N...I.I........I...T.S..PLALQ..

Fig. 1 Predicted amino-acid sequence deduced from the fragment ofengrailed homeodomain in S. officinalis (PCR primers are underlined).Additional partial sequences of engrailed homeodomain are shown inother cephalopods, other molluscs, and in Drosophila. The residue atposition 40 (arrow head) is crucial for the 4D9 antibody-specificrecognition of Engrailed. Accession numbers: S. officinalis

AM114934; L. vulgaris: AM422130; E. scolopes AF181095; N.pompilius A AM114935; N. pompilius B AM114936 ; Lepidochitonacaverna U21675; P. vulgata AF440097; I. obsoleta A :U23432 B :U23433; Cadulus fusiformis CFU23153; Dentalium eboreum U23154;Placopecten magellanicus U23213; Transennella tantilla U23212;Crassostrea virginica U23214; D. melanogaster M10017

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2–3 weeks, from stage 16 to hatching at stage 30,eventually resulting in adult anatomy (Boletzky et al.2006). Zygote cleavage gives rise to a disk-shaped embryoat the animal pole of the egg, whereas the vegetal pole ismade of a thin layer of ‘extra-embryonic’ ectoderm cellsthat covers the yolk. After a disk-shaped phase whereprospective organs start delineating (Fig. 2, first column),the embryo expands as all organs gain volume (Fig. 2,second column). Yet, the oral (or anterior) pole of the futureadult lies at the periphery of the embryo (arm crown andmouth), whereas the future aboral (or posterior end of theadult) pole is central (mantle, gills, funnel). The final adultarrangement is reached at stage 21, where the wholeembryo straightens: Eyes, mouth, and the arm crown are

then located at the yolk side and the visceropallium(visceral mass, palleal cavity, and surrounding mantle) atthe opposite side (Fig. 2, third column).

In all molluscs, the shell is an ectodermal product of themantle. It holds true for the internal shell of cephalopods, inwhich the embryonic mantle invaginates and delineates acircular inner ‘shell sac’ (Boletzky et al. 2006). In S.officinalis, as this cavity grows in size during stages 17 to19, its aperture at the mantle surface decreases in size andbecomes a small pore at stage 20 (Fig. 2h). Near the closurepoint, fins develop from stage 20. The enclosed ‘shell sac’starts producing both the organic matrix of the shell and aperiostracum at stage 21 and the calcite mineralizationstarts at stage 24 (Spiess 1972). In this central area of the

Fig. 2 S. officinalis organogenesis summed up into three majorphases visually based on the embryo shape. a–c Apical views of theanimal pole. d–f Lateral views of the animal pole (d right side, e and fprospective ventral side). First column (a, d, g): following cleavageand gastrulation, the embryo remains disk-shaped until stage 19.Second column (b, e, h): from stage 20 to 22, the body parts expand,yet in a two-dimensional pattern. Third column (c, f, i): from stage 21,

the embryo straightens up and acquires the adult orientation, mostvisible organs being in place and almost achieved. a1, a2, a3, a4, anda5: arms 1 to 5; ssa shell sac aperture; e: eye; f fin; fp funnel pouch; ftfunnel tube; g gill; ma mantle; me mantle edge; mo mouth; sse shellsac edge; ssa shell sac aperture; st statocyste; y yolk. Scale bar: 500 μm.Orientation (relating to adult animal in the so-called physiologicalorientation): L left; R right; V ventral; D dorsal; AO aboral; O oral

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embryo (i.e., the aboral pole), the En protein was firstdetected at stage 16 in both the mantle and the whole shellsac area, at the same time, as these two structures appeared(Fig. 3a). At stage 17, En immunostaining was restricted toboth the shell sac and mantle edges (Fig. 3b). At stage 18,4D9-positive cells were only found at the posterior,prospective ventral edge of the mantle (Fig. 3c). Stainingthen vanished and was no more detected at stage 19.

Embryonic arms, supposed to be foot-derived structures,emerge as a peripheral crown at stage 15 (Fig. 2a,g). As thewhole embryo expands and straightens upward above theyolk, arms become regularly arranged around the mouth atthe oral end of the animal (Fig. 2f,i). They first appear assmall buds and then grow as cylinders with suckers locatedon their oral surface starting from stage 22. Suckers arepresent all along the arms, except for tentacles (derivedarms 4) showing suckers at their extremities only (club).From stages 17 to 19, En immunostaining sequentiallyappeared in arm buds (Fig. 4e). At stage 17, the prospectivedorsal arms (1 and 2) started producing Engrailed (Fig. 4a,b),soon followed by arms 4 from stage 18 (Fig. 4c). At latestages 18 and 19, only arms 2 and 4 expressed En withhigh intensity, whereas arms 3 and 5 were less stronglycolored (Fig. 4d). From stages 19 to 23, no expression wasdetected; then, a thin and linear En staining briefly

appeared along the aboral side of arms 4 at stage 24(Fig. 4f), which get restricted to the distal future ‘club’ atstage 25 (Fig. 4g).

Also deriving from the molluscan foot, the adult funnelis an unpaired organ made of two structures, a tube and apouch, developing from distinct areas in the embryo. Thefunnel tube arises from two ventral bands that earlyseparates from the arm crown at stage 16, close to arms 4and 5 (Fig. 2, first column). At stage 20, they join as acylinder that eventually closes at stage 22 (Fig. 2, secondcolumn). The funnel pouch, essentially providing theretracting muscles, develops from two narrow bands alongthe lateral mantle edges (Fig. 2, first column) that laterconnect the funnel tube (Fig. 2, second column). At stages16 and 17, both tube and pouch areas show engrailed-expressing cells (Fig. 3a,b). The most noticeable changeoccurred at stage 18 when suddenly two intensely stainedparallel lines included within a larger band of 4D9-positivecells appeared in the pouch area (Fig. 3c,c’). Subsequently,the funnel pouch differentiates and appears as two ‘walls’running along the mantle edge (Fig. 2g,h) and engrailedstopped being expressed.

In cephalopods, successive ectodermal folds lead to theformation of the eye. At stage 16, the invagination of anectodermic thickening in the cephalic area yields the optic

Fig. 3 Results of 4D9 immu-nostaining in the aboral pole ofthe embryo, showing both thedeveloping mantle and shell. aAt stage 16, En is located in theshell area (before invagination),in the mantle and in both funneltube and pouch; b at stage 17,En is located at the edge of theshell, at the edge of the mantleand in both funnel tube andpouch; c at stage 18, engrailed islocated at the edge of the mantlein both funnel tube and pouch;c’ a focus on the funnel pouchshows two parallel lines of 4D9-positive cells (black arrows)included in a larger band of4D9-positive cells (double ar-row). Scale bar: 100 μm.Orientation and abbreviations:cf. Fig. 2

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Fig. 4 Arm development in S.officinalis from stages 17 to 25and En location revealed by 4D9immunostaining. a, b Stage 17,Engrailed is located in arm 1(not shown) and arm 4 (squaredin a and enlarged in b); c earlystage 18 where arms 1, 2, and 4are stained ; d late stage 18,arms start growing as cylinders;e synopsis table of engrailedlocation in arms 1 to 5 fromstage 16 to stage 25. Staining inthe whole arm bud is indicatedby a circle filled in black or gray(depending on the intensity),whereas discrete staining is in-dicated by a line; f stage 24,arms 4 exhibit En within anaboral line along the proximo-distal axis; g stage 25, theengrailed expression becomesrestricted to the distal part ofarm 4, where suckers are present(dotted line). Scale bar: 100 μm.Orientation and abbreviations:cf. Fig. 2

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vesicle, the edge of the remaining pore is sutured at stage19 (Fig. 5a). The inner hemisphere of this vesicle, whicheventually differentiates as the retina (Lemaire and Richard1978), showed 4D9-reactive cells from stage 20. The Enprotein was first detected as one spot of contiguous cells(stage 20), then as two anterior and posterior spots (stage21, Fig. 5c), and later as an equatorial ring parallel to thehead surface (Fig. 5d,e). At stage 21, the outer hemispheredevelops the lens, soon encircled by the iris, a secondectodermal fold. A third and last fold arises at stages 25–26yielding the lid. At stage 24, the head mass exhibits ridges,

called ‘arm pillars’, somewhat prolonging arms 2, 4, and 5.From stages 25 to 26, the ‘arm pillars’ 2 (prospective dorsalface) and 4 (prospective ventral face) expand laterallytoward the eyes, enclose them, and form the upper and thelower elements of the lids, respectively. During thisprocess, 4D9-reactive cells were detected as rows at thelateral margin of arm pillars 2, 4 (Fig. 5d,e), and 5 (Fig. 5f),and staining persisted until they joined and formed the twolids at late stage 25 (Fig. 5g,h). Finally, the ventral lidproduces a thin secondary cornea that fuses with the dorsallid and isolates the lens from external environment. This

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Fig. 5 Eye development in S. officinalis from stages 17 to 25 andEngrailed location revealed by 4D9 immunostaining. a scheme of theeye placode invagination at stage 17; b stage 20, a large spot of 4D9-positive cells can be detected on the floor of the freshly closed opticvesicle (empty arrow head); c stage 21, Engrailed is located in a thickring within the optic vesicle (empty arrow heads); d, e stage 24, En-positive cells are present in a thin ring within the eye (empty arrowheads) and at the lateral margin of arm pillars 4 and 5 (black arrow

heads); f ventral view of the d individual showing En at the lateralmargin of the arm pillar 2 (black arrow head); g, h stage 25, armspillars 2 and 4, respectively, form the ventral and dorsal lid folds, atthe margin of which En-positive cells are present (black arrow heads);i diagram of the eye elements at stage 25. a1, a2, a3, a4, a5: arms 1 to5; ap arm pillar; dl dorsal lid fold; i iris; ov optic vesicle; r retina; lelens; vl ventral lid fold. Scale bar: 100 μm

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constitutes the last developmental process of the so-calledmyopsid eyes, present in some Decabrachia, including S.officinalis and L. vulgaris.

Discussion

Engrailed and compartment boundaries

The engrailed expression patterns that we detected in thedeveloping shell sac are consistent with observations madein previous studies on molluscs (Moshel et al. 1998; Jacobset al. 2000; Wanninger and Haszprunar 2001; Nederbragt etal. 2002). As En was no longer detected in the shell sacwhen shell formation started (at around stage 21), weprovide additional evidence that En in molluscs is notrequired for the shell formation (i.e., the skeletogenesis,Jacobs et al. 2000) but rather for delimiting the shellcompartment boundary, as proposed by Nederbragt et al.(2002). This engrailed role, molecularly based on cellsignaling together with the secreted Dpp protein, is similarto that observed in the formation of the antero-posteriorboundary in arthropod parasegments (Gritzan et al. 1999)and of the dorso-ventral boundary in vertebrate limbsdevelopment (Hidalgo 1998). Our study contrasts withpreviously studied molluscs in that S. officinalis has notrochophore larval stage and no external shell. Therefore,we show that the role of engrailed also concerns adult shelldevelopment and can be extended to those molluscs whoseshell sac invaginates leading to an internal shell.

The detection of En at the mantle edge in the first stagesof organogenesis is a novel data among molluscs. Giventhat the mantle edge and the shell edge are two distinctboundaries in cephalopods, engrailed could have been co-opted in S. officinalis to set up the future mantle edgeboundary in addition to the shell edge boundary. Thiswould add a novel example of boundary delimitationtriggered by the action of En. However, as the mantle edgesecretes the shell edge in molluscs with an external shell,these two boundaries overlap, and it cannot be excludedthat a ‘mantle delimitation’ function also exists in previ-ously studied molluscs, not distinguishable from the ‘shelldelimitation’ function.

Engrailed and cephalopod-derived structures

Another striking result is that engrailed turned out to beexpressed in structures specific to cephalopods, such as eyelids and the funnel and arms, derived from the foot,suggesting a recruitment of engrailed for the developmentof major morphological novelties representing cephalopodsynapomorphies.

The linear expression patterns observed in both thefunnel and lid suggest that engrailed has been recruited forthe delineation of novel compartment boundaries, similar tothat found in mantle and shell. The delineation of twoparallel lines of 4D9-positive cells predates the emergenceof the funnel pouch that stands from stage 19 as a ‘wall’ atthe surface of the embryo. Each line could correspond to anectodermal boundary that later separates the funnel pouchfrom the mantle at one side and from the cephalic region atthe other side. This engrailed expression stopped as thefunnel pouch thickening visually appeared (stage 19). Thissuggests that engrailed is required for the establishment ofthese boundaries, but not for their maintenance. Along thearm pillars 4 and 2, lines of 4D9-positive cells predate themigration and the formation of the dorsal and ventralelements of the lids. Once again, this supports thatengrailed has been co-opted to delineate the boundary ofa novel structure.

We observed two distinct periods of expression ofengrailed in the arms: sequential expression in arm budsduring the first period (stages 17 to 19) and a linearexpression in arms 4 during the second period (stages 24 to25). The sequential expression of engrailed in the five pairsof arms reminds one of the unexpected expression patternsof Hox genes found in Euprymna scolopes (Lee et al.2003). Besides their conserved role in setting up the antero-posterior axis, seven Hox genes were shown to besequentially expressed in arms of this sepiolid species. InS. officinalis, future tentacles are the only arms that expressEn at stages 24 and 25, in a linear pattern at their aboralside. This line of expression decreased in length andbecame restricted to the tentacular clubs, where suckersare exclusively located. As the other arms possess suckersall over their oral side, this differential expression ofengrailed along the proximo-distal axis of arms 4 may besomehow connected to the tentacle-specific distribution ofsuckers. Further studies are thus required to (1) investigatewhether engrailed is involved in the differentiation oftentacles vs arms and (2) show at the histological levelwhether engrailed expression is located at nervous struc-tures or not.

Engrailed and neurogenesis

Immunostaining with Mab4D9 did not revealed any expres-sion of engrailed in the nervous system. In annelids,engrailed is expressed in the peripheral neurons of leechembryos (Wedeen and Weisblat 1991) and in various nervecells (bilateral pairs and apical tuft sensorial cells) of theChaetopterus trochophore larva (Seaver et al. 2001). Inmolluscs, engrailed expression has only been found insensorial cells near the apical tuft in the Patella trochophorelarva (Nederbragt et al. 2002) and in the ladder-like nervous

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system of Lepidochitona (Jacobs et al. 2000). It has beenassumed that loss of an engrailed role in neurogenesispredates the Cambrian radiation of molluscs, includingcephalopods (Gibert 2002). We thus postulate that en-grailed has not been recruited either for ganglia condensa-tion, which characterizes cephalopods within molluscs, orfor the innovations of their peripheral nervous system (e.g.,the stellate ganglia). Alternatively, engrailed might beinvolved at earlier stages of neurogenesis.

Successive detections of the En protein in the opticvesicle (Fig. 5b–e) suggest a potential involvement ofengrailed in the development of the camerular eye of S.officinalis. Interestingly, the Engrailed protein has beenshown to possess a paracrine activity and to be involved inguiding and linking the retinal axons to the optic tectum ofvertebrate brains (Friedman and O’Leary 1996; Logan et al.1996; Brunet et al. 2005). This result provides additionalevidence about the fascinating convergence between eyesof cephalopods and vertebrates (Harris 1997). A putativeco-factor could be the Pax6 gene required in eye develop-ment in a large variety of organisms including cephalopods(Quiring et al. 1994; Tomarev et al. 1997; Hartmann et al.2003; Gehring 2005). Interestingly, the pattern of 4D9-reactive cells we observed in the eye S. officinalis issomewhat similar to that of Pax6 observed in L. opalescens(Tomarev et al. 1997) and E. scolopes embryos (Hartmannet al. 2003). As in chicken, Engrailed down-regulates theexpression of Pax6 (Plaza et al. 1997); Pax6 might also bea target gene of Engrailed in the regulatory pathway of eyedevelopment in cephalopods. Further studies are currentlyconducted to assess this question. and a S. officinalisfragment of Pax6 has been recently sequenced (accessionnumber: AM422131).

Engrailed and evolutionary issues

Assuming that engrailed in molluscs possesses a uniquefunction (skeletogenesis) in a unique structure (the shell),Wanninger and Haszprunar (2001) suggested that the lackof a mineralized shell in Loligo (squid) was a valuableexplanation for a lack of an engrailed cognate in Loligospecies (Wray et al. 1995). Our identification of the firstengrailed cognate in a squid and the previous characteriza-tion of engrailed in E. scolopes (with a rudimentarychitinous gladius) definitely obliterate this assumption.Besides, numerous studies now provide evidence for awidespread evolvability and plasticity of engrailed, whichcasts doubt on any reasoning based on the absence of asingle trait. In S. officinalis, engrailed is expressed duringorganogenesis of derived structures shared by cuttlefishesand squids: the shell sac before shell formation, the funnel,the ten arms and the four lid elements. As a consequence,the presence of engrailed in cephalopods is not linked to

the presence of a calcified shell, and the engrailedexpression pattern in L. vulgaris should be similar to thatobserved here in S. officinalis.

From an evolutionary perspective, we think that the lossof the external shell, which provides a physical protectionagainst predators, could not occur without any earlieradaptations, anatomical, neural, or behavioral, allowing abetter protection. The evolutionary success and diversifica-tion of the cephalopod group might result from a win–winevolutionary mechanism: Reduction in the external shelldrawbacks (weight and volume) may have facilitated theexpansion of both mantle and foot and their recruitment fornew functions, such as prey capture or escape, whichconsequently decreased predation pressure and thusallowed further shell reduction. Recruitment of engrailedin the development of novel structures as we detected in thepresent paper may have played an important role in thisevolutionary scenario.

Acknowledgments We would like to thank S.v. Boletzky and theObservatoire Océanologique of Banyuls (Université Pierre et MarieCurie, Paris 6), L. Dickel, C. Alves and the Station Marine of Luc/Mer(Université of Caen) for providing biological material. We are gratefulto M. Martin for technical help. We especially thank J. S. Deutsch andS.v. Boletzky for reading the manuscript and critical comments. The4D9 anti-engrailed/invected antibody developed by Goodman wasobtained from the Developmental studies Hybridoma Bank developedunder the auspices of the NICHD and maintained at the University ofIowa, Department of Biological Sciences, Iowa City, IA52242.

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