Engrailed in cephalopods: a key gene related to the emergence of morphological novelties
The engrailed gene is a transcription factor required in numerous species for major developmental steps (neurogenesis, limb development, boundary establishment), and its evolution is known to be closely related to the evolution of the metazoan body plan. Cephalopods exhibit numerous morphological peculiarities among molluscs, such as a direct development, a complex sensory and nervous system (eyes, brain, giant axons), a reduced shell, a funnel, and a brachial crown. We assessed a potential recruitment of engrailed in the development of these derived traits and examined the expression pattern of engrailed during the organogenesis of the cuttlefish Sepia officinalis, by immunostaining. Engrailed was detected at the margin of the prospective internal shell, which is consistent with studies on molluscs having an external shell and confirms a conserved role of engrailed in delimitating the molluscan shell compartment. Interestingly, unexpected patterns were early detected in the emerging arms, funnel and optic vesicles and latter in tentacles and eye lids. We also identified an engrailed cognate in the squid Loligo, which provides new evidence that engrailed in molluscs is not restricted to a ‘shell function’ and has been recruited in the mollusc lineage for the emergence of morphological novelties in cephalopods.
KeywordsEngrailedSepiaShell differentiationGene co-optionDerived structures
A major issue in current biology is the evolution of the metazoan body plan. Widespread developmental processes, such as the establishment of the antero-posterior polarity or the formation of the mesodermal layer, are conducted by highly conserved groups of regulatory genes, whereas morphological variations and novelties often result from divergences (duplication, mutation) in gene sequences and/or in regulatory networks (Force et al. 1999; Gibert 2002). These networks often involve many homeobox genes acting as transcription factors regulating gene expression during developmental patterning or cell differentiation. Studying how they act and which feature they control has become a powerful way of understanding organism complexity. Among those genes, engrailed is one of the most relevant example demonstrating the evolvability and plasticity of gene function during evolution. This transcription factor was first shown in Drosophila to be a key gene in the establishment of segment polarity (Kornberg 1981; Fjose et al. 1985), in neurogenesis (Patel et al. 1989), and in appendage development (Raftery et al. 1991). Highly conserved in protostomes and deuterostomes, engrailed orthologues show similar roles in other arthropods (Patel et 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 taxa suggest that neurogenesis is likely the ancestral function of engrailed and that subsequent recruitments have increased engrailed contributions (Patel et al. 1989; Gibert 2002). In molluscs, however, there is no strong data for the involvement of engrailed in neural development. Instead, engrailed is expressed in cells at the margin of the future shell (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 polyplacophoran (Lepidochitona caverna, Jacobs et al. 2000). From engrailed role during shell development in molluscs, Nederbragt et al. (2002) proposed that its ancestral function is the formation of a compartment boundary as an alternative to the neurogenic hypothesis.
Among these studies on molluscs, a major group is lacking that could bring a larger and more complete overview of the molluscan taxa and of the lophotrochozoans in general. The cephalopods have not yet been investigated for engrailed, although they exhibit numerous derived traits among molluscs. As cephalopods undergo a direct development without a trochophore larval stage, investigating engrailed in a cephalopod provides a direct insight at the role of this gene in definitive organogenesis. In cephalopods, the typical molluscan foot is modified into eight to ten prehensile appendages (arms and tentacles) and a funnel. Both this funnel and a muscular mantle are implicated in jet propulsion. High capacities of cognition and reaction are permitted by a compact brain, by a peripheral nervous system allowing rapid nervous transmission (giant axons, stellate ganglia), and by sensory structures, such as complex camerular eyes (Boletzky 1988). Whereas one of the molluscan characteristic is an external shell secreted by the mantle, coleoid cephalopods, comprising all extant cephalopods except nautiluses, possess an internal shell, which may be regressed and sometimes absent.
Two paralogues of engrailed have been found in Nautilus pompilius, a cephalopod with an external shell (Wray et al. 1995), whereas no en cognate was identified yet in the squid Loligo, a coleoid whose shell is internal and not calcified (chitinous gladius). As a consequence, Wanninger and Haszprunar (2001) have correlated the presence of engrailed with that of an external shell. In this paper, we present the first report of an engrailed gene expression pattern in a coleoid cephalopod, the cuttlefish Sepia officinalis. We show that it is expressed in the shell-forming cells in early stages of organogenesis. This supports the role of engrailed in molluscan shell formation to organisms with an internal shell. Moreover, we show that engrailed 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 widespread recruitment of engrailed in morphological novelties and diversification of metazoan body plan.
Materials and methods
Collection of Sepia embryos
During spring and summer (April to September), fertilized eggs were laid by captive S. officinalis females in the biological stations of Luc-sur-mer (France) and Banyuls-sur-mer (France). From egg batches, individual eggs were detached and embryos were taken out by removing the numerous surrounding envelopes using forceps in sea water. Then, embryos were visually staged using Lemaire’s (1970) system for S. officinalis. As we focused on organogenesis, embryos at stages 16 to 25 were selected for immunochemistry. After chorion removal, embryos were fixed for 1 h in 4% paraformaldehyde (PFA) at room temperature, washed in phosphate-buffered saline (PBS), gradually dehydrated in methanol, and stored at −20°C. Until stage 16, the chorion and embryo are in intimate contact, and an additional fixation for 1 h in PFA 4% was performed to strengthen the chorion and reduce rupture hazard.
Cloning of engrailed gene homeodomain
Genomic DNA of S. officinalis was extracted from adult brain using the DNeasy Tissue kit (Qiagen, Valencia, CA, USA). mRNAs of Loligo vulgaris (collected in Caen, France) were extracted from stage 30 embryos using Tri Reagent (MRC, Cincinnati, OH, USA), then converted into cDNA 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 to amplify a 106-bp homeodomain fragment in S. officinalis. Primers Eng-2 and Eng-4 (5′-TGRTTRRTANARNCCYTGNGCCAT-3′:MAQGLYN) were used to amplify a 189-bp homeodomain fragment in L. vulgaris. PCR conditions were: 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 for 1 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 GenBank BLASTn (BLAST, basic local alignment search tool).
The engrailed protein (En) was detected by using a monoclonal antibody Mab4D9 (Developmental Studies Hybridoma Bank, University of Iowa, USA) raised against a portion of the Drosophila En as primary antibody (Patel et al. 1989). It has been shown to selectively bind an engrailed protein in some molluscs: a gastropod and a chiton (Moshel et al. 1998; Jacobs et al. 2000), and we, therefore, assumed that it detected an engrailed-like protein in Sepia. Embryos were incubated in hydrogen peroxide (3% in pure methanol) for 1 h to inactivate endogenous peroxidases; 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 the Mab4D9 (1:200) in blocking solution overnight at 4°C (without Ab in controls). After five washes of 30 min in polybutylene terephthalate (PBT; PBS 1× + Triton X-100 0.1%), embryos were preincubated again for 1 h at room temperature in blocking solution and incubated with the 1:500 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-conjugated horseradish peroxidase (ABC kit) for 30 min, rinsed three times for 10 min in PBT, and MAb4D9-binding sites were finally revealed using 3,3-diaminobenzedine (DAB-nickel kit, 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, and embryos were fixed with PFA 4%–PBT.
Also deriving from the molluscan foot, the adult funnel is an unpaired organ made of two structures, a tube and a pouch, developing from distinct areas in the embryo. The funnel tube arises from two ventral bands that early separates from the arm crown at stage 16, close to arms 4 and 5 (Fig. 2, first column). At stage 20, they join as a cylinder that eventually closes at stage 22 (Fig. 2, second column). The funnel pouch, essentially providing the retracting muscles, develops from two narrow bands along the lateral mantle edges (Fig. 2, first column) that later connect the funnel tube (Fig. 2, second column). At stages 16 and 17, both tube and pouch areas show engrailed-expressing cells (Fig. 3a,b). The most noticeable change occurred at stage 18 when suddenly two intensely stained parallel lines included within a larger band of 4D9-positive cells 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 engrailed stopped being expressed.
Engrailed and compartment boundaries
The engrailed expression patterns that we detected in the developing shell sac are consistent with observations made in previous studies on molluscs (Moshel et al. 1998; Jacobs et al. 2000; Wanninger and Haszprunar 2001; Nederbragt et al. 2002). As En was no longer detected in the shell sac when shell formation started (at around stage 21), we provide additional evidence that En in molluscs is not required for the shell formation (i.e., the skeletogenesis, Jacobs et al. 2000) but rather for delimiting the shell compartment boundary, as proposed by Nederbragt et al. (2002). This engrailed role, molecularly based on cell signaling together with the secreted Dpp protein, is similar to that observed in the formation of the antero-posterior boundary in arthropod parasegments (Gritzan et al. 1999) and of the dorso-ventral boundary in vertebrate limbs development (Hidalgo 1998). Our study contrasts with previously studied molluscs in that S. officinalis has no trochophore larval stage and no external shell. Therefore, we show that the role of engrailed also concerns adult shell development and can be extended to those molluscs whose shell sac invaginates leading to an internal shell.
The detection of En at the mantle edge in the first stages of organogenesis is a novel data among molluscs. Given that the mantle edge and the shell edge are two distinct boundaries in cephalopods, engrailed could have been co-opted in S. officinalis to set up the future mantle edge boundary in addition to the shell edge boundary. This would add a novel example of boundary delimitation triggered by the action of En. However, as the mantle edge secretes the shell edge in molluscs with an external shell, these two boundaries overlap, and it cannot be excluded that a ‘mantle delimitation’ function also exists in previously studied molluscs, not distinguishable from the ‘shell delimitation’ function.
Engrailed and cephalopod-derived structures
Another striking result is that engrailed turned out to be expressed in structures specific to cephalopods, such as eye lids and the funnel and arms, derived from the foot, suggesting a recruitment of engrailed for the development of major morphological novelties representing cephalopod synapomorphies.
The linear expression patterns observed in both the funnel and lid suggest that engrailed has been recruited for the delineation of novel compartment boundaries, similar to that found in mantle and shell. The delineation of two parallel lines of 4D9-positive cells predates the emergence of the funnel pouch that stands from stage 19 as a ‘wall’ at the surface of the embryo. Each line could correspond to an ectodermal boundary that later separates the funnel pouch from the mantle at one side and from the cephalic region at the other side. This engrailed expression stopped as the funnel pouch thickening visually appeared (stage 19). This suggests that engrailed is required for the establishment of these boundaries, but not for their maintenance. Along the arm pillars 4 and 2, lines of 4D9-positive cells predate the migration and the formation of the dorsal and ventral elements of the lids. Once again, this supports that engrailed has been co-opted to delineate the boundary of a novel structure.
We observed two distinct periods of expression of engrailed in the arms: sequential expression in arm buds during the first period (stages 17 to 19) and a linear expression in arms 4 during the second period (stages 24 to 25). The sequential expression of engrailed in the five pairs of arms reminds one of the unexpected expression patterns of 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 be sequentially expressed in arms of this sepiolid species. In S. officinalis, future tentacles are the only arms that express En at stages 24 and 25, in a linear pattern at their aboral side. This line of expression decreased in length and became restricted to the tentacular clubs, where suckers are exclusively located. As the other arms possess suckers all over their oral side, this differential expression of engrailed along the proximo-distal axis of arms 4 may be somehow connected to the tentacle-specific distribution of suckers. Further studies are thus required to (1) investigate whether engrailed is involved in the differentiation of tentacles vs arms and (2) show at the histological level whether engrailed expression is located at nervous structures or not.
Engrailed and neurogenesis
Immunostaining with Mab4D9 did not revealed any expression of engrailed in the nervous system. In annelids, engrailed is expressed in the peripheral neurons of leech embryos (Wedeen and Weisblat 1991) and in various nerve cells (bilateral pairs and apical tuft sensorial cells) of the Chaetopterus trochophore larva (Seaver et al. 2001). In molluscs, engrailed expression has only been found in sensorial cells near the apical tuft in the Patella trochophore larva (Nederbragt et al. 2002) and in the ladder-like nervous system of Lepidochitona (Jacobs et al. 2000). It has been assumed that loss of an engrailed role in neurogenesis predates the Cambrian radiation of molluscs, including cephalopods (Gibert 2002). We thus postulate that engrailed has not been recruited either for ganglia condensation, which characterizes cephalopods within molluscs, or for the innovations of their peripheral nervous system (e.g., the stellate ganglia). Alternatively, engrailed might be involved at earlier stages of neurogenesis.
Successive detections of the En protein in the optic vesicle (Fig. 5b–e) suggest a potential involvement of engrailed in the development of the camerular eye of S. officinalis. Interestingly, the Engrailed protein has been shown to possess a paracrine activity and to be involved in guiding and linking the retinal axons to the optic tectum of vertebrate brains (Friedman and O’Leary 1996; Logan et al. 1996; Brunet et al. 2005). This result provides additional evidence about the fascinating convergence between eyes of cephalopods and vertebrates (Harris 1997). A putative co-factor could be the Pax6 gene required in eye development 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 is somewhat similar to that of Pax6 observed in L. opalescens (Tomarev et al. 1997) and E. scolopes embryos (Hartmann et al. 2003). As in chicken, Engrailed down-regulates the expression of Pax6 (Plaza et al. 1997); Pax6 might also be a target gene of Engrailed in the regulatory pathway of eye development in cephalopods. Further studies are currently conducted to assess this question. and a S. officinalis fragment of Pax6 has been recently sequenced (accession number: AM422131).
Engrailed and evolutionary issues
Assuming that engrailed in molluscs possesses a unique function (skeletogenesis) in a unique structure (the shell), Wanninger and Haszprunar (2001) suggested that the lack of a mineralized shell in Loligo (squid) was a valuable explanation for a lack of an engrailed cognate in Loligo species (Wray et al. 1995). Our identification of the first engrailed cognate in a squid and the previous characterization of engrailed in E. scolopes (with a rudimentary chitinous gladius) definitely obliterate this assumption. Besides, numerous studies now provide evidence for a widespread evolvability and plasticity of engrailed, which casts doubt on any reasoning based on the absence of a single trait. In S. officinalis, engrailed is expressed during organogenesis of derived structures shared by cuttlefishes and 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 engrailed expression pattern in L. vulgaris should be similar to that observed here in S. officinalis.
From an evolutionary perspective, we think that the loss of the external shell, which provides a physical protection against predators, could not occur without any earlier adaptations, anatomical, neural, or behavioral, allowing a better protection. The evolutionary success and diversification of the cephalopod group might result from a win–win evolutionary mechanism: Reduction in the external shell drawbacks (weight and volume) may have facilitated the expansion of both mantle and foot and their recruitment for new functions, such as prey capture or escape, which consequently decreased predation pressure and thus allowed further shell reduction. Recruitment of engrailed in the development of novel structures as we detected in the present paper may have played an important role in this evolutionary scenario.
We would like to thank S.v. Boletzky and the Observatoire Océanologique of Banyuls (Université Pierre et Marie Curie, Paris 6), L. Dickel, C. Alves and the Station Marine of Luc/Mer (Université of Caen) for providing biological material. We are grateful to M. Martin for technical help. We especially thank J. S. Deutsch and S.v. Boletzky for reading the manuscript and critical comments. The 4D9 anti-engrailed/invected antibody developed by Goodman was obtained from the Developmental studies Hybridoma Bank developed under the auspices of the NICHD and maintained at the University of Iowa, Department of Biological Sciences, Iowa City, IA52242.