PATTERNS & PHENOTYPES

Somatic Muscle Development in Sepiaofficinalis (Cephalopoda - Mollusca): A NewRole for NK4Sandra Navet,1† Yann Bassaglia,1,2†* Sebastien Baratte,1 Madeleine Martin,1 and Laure Bonnaud1,3

Cephalopods are emerging as new developmental models. These lophotrochozoans exhibit numerousmorphological peculiarities among molluscs, not only regarding their nervous system but also regardingtheir circulatory system, which is closed and includes three hearts. However, the molecular control ofcardiac myogenesis in lophotrochozoans is largely unknown. In other groups, cardiac development dependson numerous different genes, among them NK4 seems to have a well-conserved function throughoutevolution. In this study, we assessed the expression pattern of SoNK4, the Sepia officinalis NK4 homologue,during Sepia officinalis development by whole-mount in situ hybridization. SoNK4 expression begins beforemorphogenesis, is not restricted to prospective cardiac muscles but above all concerns mesodermalstructures potentially rich in muscles such as arms and mantle. These results suggest an important role ofSoNK4 in locomotory (somatic) muscles development of Sepia officinalis, and thus a new role for NK4.Developmental Dynamics 237:1944–1951, 2008. © 2008 Wiley-Liss, Inc.

Key words: NK4/Tinman; Nkx2-5; Sepia officinalis; myogenesis; obliquely striated muscles; locomotory muscles

Accepted 14 May 2008

INTRODUCTION

Numerous vertebrate and ecdysozoananimal models are used in develop-mental biology. By contrast, only afew species have been studied in lo-photrochozoan groups. Cephalopodsare now emerging as novel lophotro-chozoan model systems; among them,Sepia officinalis is particularly suit-able because of its availability and re-garding its commercial value. Cepha-lopods have been essentially studiedfor the complexity and originality oftheir nervous system, as they show acentral nervous system presenting

both structural and functional conver-gences with vertebrates (Packard,1972; Hochner et al., 2006; William-son and Chrachri, 2007). However,other particularities are as remark-able, especially regarding the muscu-lar system. For example, the cephalo-pods cardiac muscular systeminvolves three hearts (at the base ofeach of the two gills, a branchial heartpumps blood from the body up to thegills where it is oxygenated; the axialsystemic heart pumps oxygenatedblood from both gills to the rest of thebody). However, despite of this particu-

larity, physiological convergences ofthe cephalopods’ closed circulatorysystem with the vertebrate one havebeen underlined years ago (reviewedin Schipp, 1987; Wells and Smith,1987), justifying the interest of thismodel in muscle structure and func-tion studies.

Different histological types of mus-cles are described in molluscs (Mill-man, 1967). Added to smooth andcross-striated muscles known in ver-tebrates, a third class, obliquely stri-ated muscles, is noticeable. In thesemuscle cells, the structures anchoring

1Museum National d�Histoire Naturelle, Departement Milieux et Peuplements Aquatiques, Laboratoire Biologie des Organismes Marins etEcosystemes, CNRS UMR5178 - MNHN USM 0401, Paris, France2Universite Paris 12, Creteil, France3Universite Paris Diderot - Paris 7, Paris, FranceGrant sponsor: Agence Nationale de la Recherche, ANR; Grant number: JC/JC 0043.†Drs. Navet and Bassaglia contributed equally to this work.*Correspondence to: Yann Bassaglia, MNHN, DMPA, BOME – CNRS UMR5178, 55 rue Buffon - CP 51. 75005 Paris, France.E-mail: bassaglia@mnhn.fr

DOI 10.1002/dvdy.21614Published online 21 June 2008 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 237:1944–1951, 2008

© 2008 Wiley-Liss, Inc.

actin filaments are not linked in con-tinuous Z-lines. Moreover, the con-tractile apparatus is organized instructures reminding of cross-striatedmuscle sarcomeres, but these “sarco-meres” are not laterally aligned; in-stead, the Z-line analogues arealigned obliquely to the longitudinalaxis of the cell (Gonzalez-Santanderand Socastro Garcia-Blanco, 1972).Obliquely striated muscles are presentin a few metazoan groups such as nem-atodes (Reger, 1964; Rosenbluth, 1965),annelids (Kawaguti and Ikemoto,1957a), and molluscs (cephalopods:Ballowitz, 1892; bivalve: Marceau,1904). Although they have often beenmisinterpreted as smooth muscle(Kawaguti and Ikemoto, 1957b; Lowyand Millman, 1962), almost all mus-cles in cephalopods are obliquely stri-ated. Cross-striated muscles have alsobeen described in tentacles (Kier,1985, 1991; Kier and Thompson, 2003;Kier and Schachat, 2008); the pres-ence of this type of muscle in the sys-temic heart is discussed (Schipp andSchafer, 1969; Kling and Schipp,1987; Budelmann, 1997).

Few data are available concerningthe genetic control of muscle develop-ment in cephalopods. In vertebrates,skeletal cross-striated muscle devel-opment depends of MRFs (myogenesisregulating factors), a highly conservedfamily of four transcription factors (re-view in Buckingham et al., 2003; Tap-scott, 2005): MyoD, myf-5, myogeninand MRF4 (myf-6). Using vertebrateanti-MRF antibodies, Grimaldi et al.(2004a,b) have shown myf5- andMyoD-like positive staining in musclecells of arms and tentacles in Sepiaofficinalis embryos from stage 26 (ac-cording to Lemaire, 1970) to hatching.

The development of cardiac muscle,the other type of striated muscle invertebrates, does not depend on MRF,but rather from numerous differenttranscription factors (review in Bru-neau, 2002; Brand, 2003; Buckinghamet al., 2005; Olson, 2006). Amongthem, NK4 is particularly interesting.This gene belongs to the NK gene clus-ter, which is probably the most an-cient homeobox gene cluster, dating atleast from the base of Metazoa (Lar-roux et al., 2007), and NK4 gene ho-mologues have been identified in nu-merous species (Harvey, 1996; Elliottet al., 2006). Moreover, the main func-

tion of these genes seems to be highlyconserved within Metazoa. The verte-brate NK4 homologue, Nkx2-5 (Lintset al., 1993), is implicated in heartdevelopment (review in Akazawa andKomuro, 2005; Olson, 2006). Tinman/NK4, the Drosophila founder memberof this family (Kim and Nirenberg,1989), is involved in mesoderm region-alization and is required for cell fatespecification of the dorsal vessel, theequivalent of the vertebrate heart(Bodmer et al., 1990; Bodmer, 1993;review in Cripps and Olson, 2002). InCaenorhabditis elegans, the tinmanhomologue ceh-22 is involved in pharyn-geal muscle differentiation (Okkemaand Fire, 1994; Okkema et al., 1997);these muscles are responsible for in-trinsic rhythmic contractions of thepharynx, and thus considered as ana-logs of vertebrate cardiac muscle (Pi-lon and Morck, 2005). In cephalopods,NK4 homologue expression in the sys-temic heart and the adjacent muscu-lar ink sac of Loligo paelii has beenpointed out in one late embryonicstage (Elliott et al., 2006). No data areavailable regarding the expression inearlier stages when muscle determi-nation and differentiation occur.

We determine in this study the ex-pression pattern of NK4 during Sepiaofficinalis development. We show bywhole-mount in situ hybridizationthat NK4 expression begins beforemorphogenesis, and is not restrictedto the prospective cardiac muscles butconcerns above all prospective locomo-tory muscles areas.

RESULTS

Sepia officinalis develops directlywithout metamorphosis. Organogene-sis proceeds during 2 to 3 weeks (at25°C), from stage 15 to hatching re-sulting in adult anatomy (Boletzky,2006). Zygote cleavage gives rise to adisk-shaped embryo at the animalpole of the egg, whereas the vegetalpole is made of thin layer of “extra-embryonic” ectoderm cells that coversthe yolk. After a disk-shaped phasewhere prospective organs start deli-neating, the embryo expands as allorgans gain volume. Describing thesesteps of development is often confus-ing due to an unusual orientation ofthe embryo when compared with otherwell-known models (Fig. 1): the stomo-

deum is dorsal, the oral (or anterior)pole of the future adult lies at the pe-riphery of the embryo (arm crown andstomodeum), whereas the future abo-ral (or posterior end of the adult) poleis central (mantle, gills, funnel). Thefinal adult arrangement is reached atstage 21, where the whole embryostraightens: eyes, mouth, and the armcrown are then located at the yolk side(cephalopodium) and the visceralmass, mantle cavity, and surroundingmantle (visceropallium) at the oppo-site side. The arm crown finally com-prises eight arms and two tentacles(which are numbered from the dorsalside); they are morphologically identi-cal until stage 22/23, after which ten-tacles (� “arms” IV) begin to show arecognizable tentacular club.

SoNK4 expression has been de-tected at the earliest studied stage(early stage 15), before any visiblestructure emerged (Fig. 2-15b). Themantle area was stained, except in itscentral part that corresponds to theshell sac depression primordia. Theprospective arm areas were alsostained, except for arm III that isknown to appear later than the others.The intensity of expression was differ-ent between arms, being particularlyweaker in arm I. A weak but notice-able expression was observed as ananterior extension of arm II (earlystage 15, Fig. 2-15b) and I (late stage15, Fig. 2-15c), which could corre-spond to migrating cells, as it was notobserved on these arms in laterstages. As soon as the gill primordiawere discernable, they were NK4-pos-itive.

At stage 16, arm morphogenesis be-gan as an arm crown, over which armbuds grew. Arm I and III were notmorphologically perceptible. In thiscontinuous structure, SoNK4 expres-sion was still restricted to the futurearm buds, and as in stage 15, prospec-tive arm III was devoid of staining. Asarm buds grew, SoNK4 expression didnot concern all the bud structure, theventral part of the bud being devoid ofstaining (Fig. 2-16b). The mantle wasalso strongly underlined except a de-pression in the ventral median line;the interpretation of this structure(already signaled in morphologicalstudies by Naef, 1928) is not clear. Atthis stage, expression was clearly no-ticeable in the gill primordia. An ex-

NK4 IN SEPIA OFFICINALIS DEVELOPMENT 1945

Fig. 1.

Fig. 2.

Fig. 1. Orientation in Sepia officinalis embryo. The three axes (left–right, dorsal–ventral, andoral–aboral) are represented referring to the adult organization.

Fig. 2. SoNK4 expression in early stages of Sepia officinalis organogenesis. Whole-mount in situhybridization, stage 15 to 17 (Lemaire). Pictures are numbered according to the illustrated stage(e.g., 15 correspond to stage 15). The a-series correspond to fixed, nonhybridized embryos. Scalebar (unless otherwise specified) � 500 �m. 15b: Early stage 15 (no visible morphogenesis). 15c:Late stage 15. Due to lightening, the right arm I prospective area is barely visible. Arrow: SoNK4staining as an extension adjacent to arm prospective area. Arrowheads: gill primordia. 16b: Stage16, with arm buds emerging. Due to the labeling, the dual nature of the future bud is sometimealready discernable (see arm V). 16c: Detail of the ventral region of the mantle. The arrow points tothe prospective branchial heart area. Scale bar � 250 �m. 16d: Paraffin section of the mantle. Theepithelium (ectoderm) does not express SoNK4, which is exclusively mesodermic. Scale bar � 25�m. 17b: SoNK4 staining in the newly appeared arm III, with an adjacent extension (arrow). Insert:control. 17c: Dorsoventral paraffin section of the mantle area (the limit vitellus/embryo is under-lined). The shell sac is devoid of detectable SoNK4 expression Scale bar � 100 �m. 17d:Dorsoventral paraffin section of an arm bud (V). Only the dorsal part of the inner mass of the armbud express SoNK4. Legends: I, II, III, IV, V, arms; Do, dorsal; e, eye; fp, funnel pouch; ft: funneltube; g, gill; m, mantle; s, shell sac; st, statocyste; Ve, ventral. Scale bar � 20 �m.

1946 NAVET ET AL.

pression was also detectable at thebase of the gills, which corresponds tothe prospective branchial heart area(Fig. 2-16c).

At stage 17, all arm buds wereclearly SoNK4 positive, including armIII (Fig. 2-17b). Of interest, the stain-ing of this arm showed an anteriorextension similar to that observed forother arms at early stages. The al-ready stained structures were all stillunderlined.

Sections at these stages (16/17)showed in all cases a SoNK4 expres-sion below the epithelium, corre-sponding to the mesodermal layer(Fig. 2-16c, 2-17c), and confirmed arestricted expression in the dorsalpart of arm buds (Fig. 2-17d).

From stage 18 to 20, expression wasstill strong in the mantle and in thearms (Fig. 3). Arm buds were clearlyseparated into two adjacent parts,with the dorsal-most part stained.This arm bud separation is underlinedby the venous system (Naef, 1928),and was not SoNK4-positive, suggest-ing that SoNK4 was not required forthe early development of this periph-eral vascular system. By contrast, thebranchial hearts as the gills were stillstained. A sparse but clear stainingwas also noticed in the funnel tubeprimordia that begin to take placefrom stage 16/17, but the funnel pouchprimordia remained negative: this dif-ference is in agreement with the facts(i) that these two structures areknown to differentiate independently(Naef, 1928) and (ii) that their embry-ological origins are really different,the funnel tube rudiments belongingto the cephalopodium, whereas thefunnel pouch has a pallio-visceral ori-gin (Boletzky, 1988b).

At stage 18 and 19, the anus arealocated between the gills was verytransiently stained as the stainingdisappeared after stage 19. A scat-tered expression was also noticed atstage 18 as a line in the middle of theforming eyes, which could correspondto the closing of the primary optic ves-icles. The contrast observed at laterstage in the eye was due to the pig-mentation of the retina, as the con-trols show similar staining (compareFig. 4-24 and Fig. 4-24T).

At stage 21 to 23, as the two funneltube primordia went in contact, gillsand funnel tube were no longer

stained (Fig. 3 insert). By contrast,expression in the mantle and in thearms persisted. It was progressivelyless intense in the arms. This dynam-ics of SoNK4 expression in arms wasunderlined by the fact that the latestappeared arms (III) presented at thisstage a higher and more regionalizedsignal than the others, in agreementwith its delayed morphogenesis (Fig.3-22b). As the arm bud extended,SoNK4 expression was restricted totheir distal end (Fig. 3-22�). No obvi-ous difference was noted betweenarms and tentacles (IV). The decreaseof the signal was also evident in man-tle. Sections showed that SoNK4 ex-pression was restricted to the futuremuscle layer (Fig. 3-23).

From stage 24 to 27, only the man-tle was stained; SoNK4 was not ex-pressed in the arms anymore (Fig. 4).Finally, in the later tested stages (28to hatching), all specific staining dis-appeared (not shown).

DISCUSSION

We show in this study that SoNK4 isexpressed during early developmentin Sepia officinalis not only in cardiactissue but also in muscle containingstructures such as the mantle, thefunnel, and the arms. To our knowl-edge, this is the first description ofNK4 expression during the develop-ment of a Mollusca.

In vertebrates, the NK4 homologueNkx2-5 is initially expressed in meso-derm and anterior endoderm (Lints etal., 1993), and the endodermic expres-sion has been linked to a role in thepharyngeal region patterning. By con-trast, the expression of NK4/Tinmanin Drosophila is strictly mesodermic(Bodmer et al., 1990; Bodmer, 1993).In Sepia officinalis, we did not detectany indication of SoNK4 expression inputative endodermic territories (suchas the so-called mesentodermic areas;Naef, 1928), except for a very tran-sient expression in the anal prospec-tive area at stages 18–19. The settingup of the digestive tract takes placerelatively late in the development asthe animal gains volume (Boletzky,1978); it becomes functional afterhatching. No expression has been de-tected in these tissues even in the lat-est stages (stages 25 to 30) where thetract is fully developed. Thus, it ap-

pears that SoNK4 is not implicated inthe development of anterior endoder-mic tissues in Sepia officinalis. As inDrosophila, its role seems to be re-stricted to mesodermic developmentin cephalopods.

The main accepted role of NK4 andits homologues is their implication incardiac/rhythmically contractile mus-cle development (Cripps and Olson,2002; Zaffran and Frasch, 2002; Aka-zawa and Komuro, 2005; Olson, 2006).According to Okkema and coworkers(Okkema and Fire, 1994; Okkema etal., 1997), the expression of ceh-22, theNK4 homologue in Caenorhabditis el-egans, is restricted to the rhythmi-cally contractile pharyngeal muscle,whereas bodywall (somatic) musclesdevelopment require other myogenicfactors such as MRF-related, MADS-related, or Hand-related factors(Baugh and Hunter, 2006; Fukushigeet al., 2006). In vertebrates, expres-sion of the NK4 homologue Nkx2-5 israpidly restricted to cardiac muscle(Olson, 2006); Nkx2.5 is not directlyinvolved in cardiac cells determina-tion but participates to the correctmorphogenesis of the heart (Lints etal., 1993; Lyons et al., 1995). In Dro-sophila melanogaster, NK4/Tinman isinvolved in the determination of car-diac cells (Bodmer, 1993) and it hasbeen shown recently that NK4 couldsuccessively intervene in cardiac cellsdetermination and differentiation(Zaffran et al., 2006). Thus, NK4 maybe responsible for cardiac cells deter-mination and/or differentiation, de-pending on the species.

In Sepia officinalis, NK4 expressionis observed at early stages (stages 16to 18) in the gills and in the branchialhearts prospective areas. Branchialhearts differentiate earlier than thearterial (systemic) heart. In a rela-tively close species (Octopus), they be-gin to pulse at stage 22–23 (before thegills function) irregularly then regu-larly. At the end of this period, thesystemic heart begins to contract andadopts a synchronous contractionrhythm with branchial hearts atstages 24–25, as arterial and venouscircuits are established (Boletzky,1987). At early stages (16–18), the gillrudiments are not yet vascularized inSepia officinalis (Schipp, 1987) and novascular/heart muscle structure arepresent. After stage 20, SoNK4 ex-

NK4 IN SEPIA OFFICINALIS DEVELOPMENT 1947

pression has been detected neither inbranchial or systemic hearts nor gills.In a late stage of development (proba-bly stage 26, personal communica-tion), Elliott et al. (2006) reported anexpression of SoNK4 in the systemicheart but not in branchial hearts ofLoligo paelii. These results confirmthat expression of SoNK4 in gills andbranchial hearts prospective areas istransient and restricted to the earlierstages of development in cephalopods.Therefore, SoNK4 could play a role inbranchial heart determination, butdoes not seem to take part inbranchial heart morphogenesis. Bycontrast, the expression observed insystemic heart of Loligo, late in devel-opment, suggests that NK4 could in-tervene in the final morphogenesis asthe circulatory system takes placeonly at the end of the development.Even if we cannot exclude thatbranchial hearts and the systemicheart do not have identical muscle cellstructure (Schipp and Schafer, 1969;Kling and Schipp, 1987; Budelmann,1997), it appears clearly that the pul-satory system development takesplace by the way of different geneticmechanisms, suggesting a NK4 role attwo different developmental stages,one for the branchial hearts (the ve-

Fig. 3.

Fig. 4.

Fig. 3. SoNK4 expression in Sepia officinalisorganogenesis. Whole-mount in situ hybridiza-tion, stage 18 to 22 (Lemaire). Same legends asFigure 2. Scale bar � 500 �m in a-series, 250�m in b-series. 18b: Existing structures (eye,funnel tube) show a sparse SoNK4 expression,as opposed to the continuous aspect observedin already expressing structures (mantle, armbuds, gills). 20b: As they progress to the me-dian line, the funnel tube primordia expressSoNK4 (arrowhead). This expression is stoppedwhen they come into contact (insert). The com-pact staining of the arm tends to disrupt. 22b:SoNK4 staining is reduced in arms, except inarm III. 22�: Late stage 22. SoNK4 staining isessentially restricted to the distal part of thegrowing arms and the mantle. Scale bar � 500�m. 23: mantle section. The different prospec-tive layers are depicted (ep�d: epidermis anddermis, mu: muscle). mc, mantle cavity. Scalebar � 100 �m.

Fig. 4. SoNK4 expression in Sepia officinalisorganogenesis. Whole-mount in situ hybridiza-tion, stage 24 and 26 (Lemaire). Same legendsas Figure 2. Scale bar � 1 mm. 24: stage 24,SoNK4. 24T: Stage 24, control. 26a: Stage 26,SoNK4 (left lateral view). 26b: stage 26, SoNK4(ventral view). 26T: Stage 26, control (left lateralview). Only the mantle is SoNK4-positive.

1948 NAVET ET AL.

nous system) and one for the systemicheart (the arterial system). Thus,NK4 expression in cephalopods doesnot correlate with its proposed generalrole in pulsatory cell determination(Olson, 2006), and NK4 should be in-terpreted as part of a pulsatory genenetwork rather than as a pulsatorymaster gene. This is also the case inother species. In C. elegans embryoCeh22 (NK4 homologue) is not ex-pressed in all the putative contractivepharyngeal muscular cells and re-quires later other genes to specify thecontractile characteristic of these cells(Okkema and Fire, 1994). The expres-sion domain of tinman in Drosophilais much broader than the region ofheart precursor formation, but its ec-topic expression does not produce ec-topic cardiac tissue (Yin and Frasch,1998); other genes like the GATA fac-tors family are required to promotethe cardiac cell lineage (Gajewski etal., 1999).

The main expression of SoNK4 inSepia officinalis is essentially locatedin structures known to be particularlyrich in muscles, namely arms andmantle. The arms’ buds are positivefrom stage 16 to 19 and the mantlefrom stage 15 to 27. Taken together,these results suggest that a transientexpression of SoNK4 could be impli-cated in early myogenesis of noncar-diac muscle in Sepia officinalis. Thisearly expression of SoNK4, even be-fore any detectable morphogenesis,suggests a role in muscle cell determi-nation. As no expression is detected atlater stages (stages 27 to 30), a puta-tive role in muscle differentiation(which occurs after stage 26 at least intentacles, Grimaldi et al., 2004b) orfunction can be discarded.

Cardiac, arm, and mantle muscula-ture of Sepia officinalis are composedof obliquely striated muscles, suggest-ing that SoNK4 expression in cepha-lopods somatic muscle could be linkedto this particular type of muscle cell.We also found that tentacles (armsIV), like the arms (arms I, II, III, andV), are positive for SoNK4. It has beenshown by Kier and coworkers (Kier,1985; Kier and Schachat, 2008) thatadult cephalopods tentacles arelargely composed of transversal cross-striated muscles, but striated musclesof the tentacles are known to appearafter embryonic development, around

hatching time in Sepia officinalis(Grimaldi et al., 2004b), and 4 to 5weeks after hatching in Loligo paelii(Kier, 1996). Thus, other gene(s) arelikely to be implicated in the differen-tiation of cross-striated muscle in thisspecies, and MRF are obviously goodcandidates: we are currently exploringthis possibility. However, it should beemphasized that the muscular wall ofthe buccal mass and the oesophagus,as the plexiform layer of the stomachof Sepia officinalis have also been de-scribed as obliquely striated (Amsel-lem and Nicaise, 1980). We were un-able to detect any SoNK4 expressionin these structures during Sepia offi-cinalis development. Therefore, it isunlikely that SoNK4 expression isstrictly related to the structural typeof muscles in Sepia officinalis.

NK4 gene homologues expressionhas been strongly linked to rhythmi-cally contractile muscle development,but it is clear from our results thatSoNK4 is not strictly linked to thisspecific muscular function, namely in-trinsic rhythmic contractility. By con-trast, it is noteworthy that structuresstrongly expressing SoNK4 during Se-pia officinalis development are in-volved in the locomotory function, in-cluding part of the muscularhydrostat (Kier, 1989; Kier andThompson, 2003). Locomotory mus-cles in cephalopods (such as mantle,funnel, or arm muscles) can be func-tionally assimilated to skeletal mus-cles of vertebrates or body wall muscleof Drosophila, which do not expressNK4. A role in specific parts of somaticmuscle development has also beenproposed for Tinman in Drosophila(Bodmer et al., 1990; Azpiazu andFrasch, 1993; Bodmer, 1993). Al-though SoNK4 expression is notstrictly restricted to muscular struc-tures in Sepia officinalis, suggestingan implication of SoNK4 in differentmesodermal areas, it is clearly prefer-entially linked to the locomotory sys-tem, thus underlying this model as aparticularly original one.

Last but not least, our results aboutSoNK4 expression in a cephalopod al-low evolutionary inference. Indeed,arms and funnel are synapomorphiesof cephalopods among mollusks, as de-rived from the foot of a mollusc puta-tive ancestor (Naef, 1923; Boletzky,1988a). In association with the funnel,

the mantle is also modified for a loco-motory function (the jet propulsion) incephalopods. NK4 could have been re-cruited in this clade for the apparitionof morphological novelties implicatedin the locomotory function. Alterna-tively, NK4 recruitment could be inter-preted as a molluscan synapomorphyimplicated in the foot morphogenesis.To our knowledge, no data are currentlyavailable regarding the expression ofNK4 homologues in other molluscanspecies. This could be an interestingperspective to explore.

EXPERIMENTALPROCEDURES

Collection of Sepiaofficinalis Embryos

During spring and summer (April toSeptember), fertilized eggs were laidby captive Sepia officinalis femalesmaintained in the biological stationsof Luc-sur-mer (France) and Banyuls-sur-mer (France). Eggs were kept inartificial see water (Red Sea) with aer-ation at room temperature (RT). De-velopment occurred into the chorion, atough secreted membrane that sur-rounds the egg. In these conditions,the development was normal, as as-sessed by its timing and the morpho-logical aspect of the embryos. Fromeggs batches, individual eggs were de-tached and embryos were taken out byremoving some of the numerous sur-rounding envelopes using forceps insea water. Then, embryos were visu-ally staged using Lemaire’s (1970)system for Sepia officinalis. As we fo-cused on organogenesis, embryos atstages 15 to 30 were selected for insitu hybridization (ISH). Embryoswere fixed one night within the cho-rion in 3.7% paraformaldehyde (PFA)in phosphate buffered saline (PBS),then dechorionated. A second step offixation (3.7% PFA, 24 hr) followed.After three rinses in PBS, embryoswere dehydrated in increasing concen-trations of methanol (25% to 100%).They were conserved at �20°C untiluse.

In Situ Hybridization

The full-length SoNK4 (Sepia officina-lis NK4) cDNA (EMBL accessionnumber AY298768) was kindly pro-

NK4 IN SEPIA OFFICINALIS DEVELOPMENT 1949

vided by Dr Richard P. Harvey fromthe Developmental Biology unit of Vic-tor Chang Cardiac Research Institute,Sydney Australia.

RNA probes (1,150 bp) were ob-tained with the Sp6-T7 kit from Rocheas recommended by the manufac-turer. Antisense probes were obtainedwith Sp6 polymerase. Sense probesobtained with the T7 polymerase wereused as a control.

ISH was done in hemolyze tubes,each containing one embryo in largevolumes of solution (approximately 2ml), and under agitation. At least 3embryos from each studied stage havebeen treated, and controls were donefor each stage. Progressive rehydra-tion of the embryos was done by suc-cessive immersions (5 min, RT) inPTW/methanol solution (PBS, Tween201%, methanol from 60% to 0%). Em-bryos were then treated by proteinase Kin PTW (0.05%, 20 to 50 min, dependingon the stage of the embryos). They werethen post-fixed 1 hr in PFA.

A prehybridization step was done inSH solution (formamide 50%, stan-dard saline citrate 20�, Tween20 1%,sodium dodecyl sulfate 2.5%) allowingprogressive impregnation of the tis-sues. Embryos were then incubated 1night at 65°C in this solution to whichthe probe had been added. Control em-bryos were incubated with a senseprobe. Excess of probe was eliminatedby five rinses (30 min, 65°C) in SHsolution.

Embryos were impregnated withthe buffer of blocking solution (BS:Maleic acid 100 mM, NaCl 150 mM,pH 7.5, Tween20 1%). Saturation wasthen done in blocking solution (BS, BB4% Roche, fetal bovine serum [FBS]15%, 1 hr, RT), followed by incubation1 night at 4°C with anti-digoxigeninantibodies (Roche) coupled to alkalinephosphatase (AP) in blocking solution(BS, BB 2.4%, FBS 20%).

Revelation of AP activity was doneusing NBT-BCIP (Roche) as a sub-strate. After complete development,the reaction was stopped by severalwashing in PTW solution. A final fix-ation in PFA (2 hr, RT) was then re-alized. To obtain more precise local-izations, some of these embryos wereincluded in paraffin using standardprotocols, and cut in 7-�m sections.

Observations were done with a

Leica M16 2F binocular stereomicro-scope and Leica microscope.

ACKNOWLEDGMENTSWe thank the biological stations ofLuc sur mer, Banyuls sur mer, JoelHenry (Universite de Caen), LudovicDickel (Universite de Caen) for pro-viding Sepia brood, Aude Andouchefor help in Sepia officinalis embryosdissection, and referees for helpfulcommentaries.

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NK4 IN SEPIA OFFICINALIS DEVELOPMENT 1951