Staggered Hox expression is more widespread among molluscs than previously appreciated

(a) Hox and ParaHox gene expression in the scaphopod Antalis entalis

Hox and ParaHox genes have been previously identified based on a transcriptome screen of pooled developmental stages of the scaphopod An. entalis [19]. Thereby, all predicted Hox and ParaHox genes except for Hox7/Antp and Xlox were found. Unfortunately, we were unable to clone Lox2. Scaphopod Hox genes are expressed from the gastrula stage (not shown) to early postmetamorphic individuals (figures 2–5; electronic supplementary material, figures S3–20; adults were not investigated).

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(b) Early-stage trochophore larva

Hox and ParaHox genes are expressed predominately in the foot (Hox2, Hox3, Lox5, Lox4, Post1, Post2), the shell field (Hox1, Hox3, Post1), around the mouth (Hox2, Hox4), in the mesoderm (Hox3, Hox4, Hox5, Lox4), the pavilion (Post1, Post2), and in the region of the hindgut (Hox3, Lox5, Cdx) (figures 2–4; electronic supplementary material, figures S3A-C, S5A-C, S7A-C, S9A-C, S11A-C, S13A-C, S15A-C, S17A-C, S19A-C, S21A-C). In particular, Hox2 is expressed in the cerebral pits, which are Elav-expressing bilateral invaginations close to the apical organ that give rise to the cerebral ganglia (figure 4d–f; electronic supplementary material, figure S1A-D; for the location of cerebral pits, see [20]). Elav (embryonic lethal, abnormal visual system) is a marker of nascent neurons [21,22]. In addition, the nervous system of early-stage trochophore larvae as revealed by Elav-expression is composed of an apical organ, pedal and pleural ganglia, as well as neurons in a ganglion associated with the prospective posterior mantle opening (pavilion) (electronic supplementary material, figure S1A-D; [5]).

(c) Early mid-stage trochophore larva

Hox genes are expressed in a near-to staggered and partially overlapping fashion along the larval anterior–posterior axis (figures 5 and 6). Hox1 is co-expressed with Elav in the anlagen of the cerebral and pedal ganglia, cells of the apical organ, the anterior-most region of the foot and the pavilion (figure 5; electronic supplementary material, figures S1E-H; S3D-F). In addition, the anterior-most mantle margin expresses Hox1 (electronic supplementary material, figure S3D-F). The anterior-lateral ventral portion of the foot and the anterior mantle margin are Hox2 expression domains (figure 5; electronic supplementary material, figure S5D-F). Hox3 is expressed in lateral and ventral portions as well as in some dorsal portions of the mantle, in addition to the lateral–ventral trochoblasts and two lateral apical cells (figure 5; electronic supplementary material, figure S7D-F). Weak Hox3 expression is also present in the anterior and median region of the foot. Hox4 is expressed in the pedal ganglia and on the level of the statocysts, while Hox5 is expressed by cells on the level of the statocysts and slightly more posterior to the latter (figure 5; electronic supplementary material, figures S9D-F, S11D-F). The Lox5 expression domain extends posterior-laterally from the statocysts to the posterior-most region of the foot (figure 5; electronic supplementary material, figure S13D-F). Lox4 expressing cells are present ventrally to the statocysts and slightly more posterior to the latter (figure 5; electronic supplementary material, figure S15D-F). The posterior foot and the pavilion express Post1, while a domain extending from anterior to posterior to the statocysts, including the pedal ganglia, expresses Post2 (figure 5; electronic supplementary material, figures S17D-F, S19D-F). Cdx is expressed in the region of the hindgut (electronic supplementary material, figure S21D-F).

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(d) Late mid-stage trochophore larva

Hox1 is expressed in the cerebral and pedal ganglia, the anterior mantle opening, and the pavilion. Hox2 is expressed in the anterior mantle margin, slightly more posterior to the Hox1 expression domain, in the foot, and in the pedal ganglia (electronic supplementary material, figures S2A-D, S4A-C, S6A-C). The Hox3 expression domain is located in the anterior-median portion and in dorsal parts of the foot (electronic supplementary material, figure S8A-C). Hox4 is expressed in the pedal ganglia anterior to the statocysts, while Hox5 expression is located laterally in the foot on the level of the statocysts and anterior to the latter in two spots more medially and ventrally than the other Hox5 expression domain (electronic supplementary material, figures S10A-C, S12A-C). Lox5 expression is situated lateral, anterior and posterior to the statocysts in the foot and Lox4 is expressed in two spot-like domains anterior to the statocysts and in two domains posterior to the statocysts in the foot (electronic supplementary material, figures S14A-C, S16A-C). Post1 is expressed in the pavilion, two pedal domains anterior to the statocysts, and posterior to the latter (electronic supplementary material, figure S18A-C). In addition, a Post1 expression domain is present in the anterior-lateral and dorsal mantle margin and close to the anus. Post2 is expressed anterior to the statocysts in the foot and posterior to the statocysts with additional expression in the pavilion (electronic supplementary material, figure S20A-C). Cdx expression is only present in the posterior digestive tract similar to the early mid-stage trochophore (electronic supplementary material, figure S22A-C).

(e) Late-stage trochophore larva

Hox1 is expressed in the anterior mantle margin, the cerebral ganglia and probably in the palliovisceral ganglia (electronic supplementary material, figures S2E-H; S4D-F). Co-expression of Hox genes and Elav only occurs in the cerebral ganglia that are the only domains of the nervous system that express Elav during this larval stage. Hox2 expression domains are located close to the midgut gland, in the lateral mantle and in the region of the cerebral and visceral ganglia (electronic supplementary material, figure S6D-F). Hox3 is expressed in the cerebral and pedal ganglia (figure 4g–i), while Hox4 is expressed in the region of the visceral ganglia in late-stage trochophores (electronic supplementary material, figures S8D-F, S10D-F). Hox5 is expressed in the pedal and cerebral ganglia and the Lox5 expression domain is located between the pedal and the cerebral ganglia (electronic supplementary material, figures S12D-F, S14D-F). Lox4 is expressed faintly around the statocysts (electronic supplementary material, figure S16D-F). The cerebral ganglia and the anterolateral mantle margin express Post1, while Post2 is expressed in domains anterior and posterior to the statocysts and the visceral mass (electronic supplementary material, figures S18D-F, S20D-F). Cdx is expressed in the hindgut and in the foot (electronic supplementary material, figure S22D-F).

(f) Postmetamorphic/settled individuals

Settled animals express Hox2, Hox3 and Hox5 in portions of the mantle (electronic supplementary material, figures S6G-I, S8-G-I, S12G-I). Hox4 is expressed in both captacula anlagen, while Hox5 is expressed in the pedal ganglia (electronic supplementary material, figures S10G-I, S12G-I). Besides the pedal ganglia and the pavilion ganglion all other major ganglia such as the cerebropleural ganglia, the putative visceral ganglia, and bilateral lateral domains on the level between foot and intestine express Elav (figure 2i–l; electronic supplementary material, figure S2I-L). Lox5 is expressed in the anterior foot and Lox4 is expressed in the buccal region (electronic supplementary material, figures S14G-I, S16G-I). Post1 and Post2 are expressed in the anterior portion of the visceral mass, while Hox3, Hox4, Hox5, Lox5, Lox4, Post1 and Post2 are also expressed in the central portion of the visceral mass, probably in the region of the visceral ganglia (electronic supplementary material, figures S8G-I, S10G-I, S12G-I, S14G-I, S16G-I, S18G-I, S20G-I). Post2 is also expressed dorsally to the statocysts in the posterior region of the foot (electronic supplementary material, figure S20G-I). Cdx is expressed in the foot and in the region of the hindgut (electronic supplementary material, figure S22G-I).

(a) Traces of staggered Hox gene expression in molluscs

Until recently Hox genes have been thought to be expressed in a non-colinear fashion in the Mollusca as demonstrated for the gastropods Haliotis asinina, Gibbula varia and the cephalopod Euprymna scolopes [7,9–11]. All three conchiferan species have been reported to express Hox genes in distinct morphological structures rather than staggered along the anterior–posterior body axis as it is the case in the majority of ecdysozoans, deuterostomes, and lophotrochozoans investigated [23–33]. A recent study on the polyplacophoran Ac. crinita, however, revealed a staggered Hox expression in the trochophore larva of this aculiferan [16,17]. Moreover, another study indicated that staggered expression may also be found in the gastrula stage of a bivalve [34].

Our study demonstrates that the scaphopod An. entalis exhibits traces of staggered Hox expression during ontogeny. Seven of nine Hox genes are expressed in a staggered fashion along the larval anterior–posterior axis in early mid-stage trochophores (figures 5 and 6). The anterior-most expression domains are the anlagen of the cerebral ganglia, the apical organ and the anterior-most mantle margin that both express Hox1 (figure 5). While Hox2 is expressed slightly more posterior to Hox1 in the mantle margin, Hox2, Hox4, Hox5, Lox5, Lox4 and Post1 are predominantly expressed in the foot (figure 5). Hox3 clearly deviates from the staggered condition with expression domains in the trochoblasts, the foot, two lateral apical cells and large portions of the mantle (figures 5 and 6). In addition, Post2 expression is not in accordance with the staggered expression pattern because its pedal expression domain is located slightly more anterior to the one of Lox4 (figures 5 and 6). Notably, all anterior-most Hox3 expression domains of early mid-stage trochophores that deviate from the staggered expression, such as the trochoblasts and the apical cells, are in ‘larval’ structures that do not contribute to the prospective adult body plan. In the majority of bilaterians, Hox gene expression domains only comprise anlagen of ‘adult’ structures [25–33]. With exception of the annelid Platynereis dumerilii that expresses Hox1 in the larval episphere (same in An. entalis), molluscs constitute, so far, the only bilaterians that express Hox genes also in purely larval structures that do not persist through metamorphosis [28]. For instance, the gastropod G. varia expresses Lox5, Hox7, Lox4 and Lox2 in the velum or in the larval episphere, while only Lox5 is expressed in the episphere of the polyplacophoran Ac. crinita [7,11,16,17].

In contrast to all other developmental stages of the scaphopod An. entalis, late mid-stage trochophores also show traces of staggered Hox gene expression, although during this stage Lox5 and Post1 (and not Hox3 and Post2 as in early mid-stage trochophores) disrupt staggered Hox expression (electronic supplementary material, figures S4A-C, S6A-C, S8A-C, S10A-C, S12A-C, S14A-C, S16A-C, S18A-C, S20A-C). Contrary to early mid-stage trochophores, late mid-stage trochophores do not exhibit any Hox expression in larval structures.

Interestingly, a closer look at the cephalopod E. scolopes indicates that all studied Hox genes except for Lox5 and Post1 are expressed in a staggered fashion in the stage 19/20 embryo, the earliest developmental stage that exhibits distinct anlagen of all major organ system such as the eyes, mantle, funnel and arm crown (figure 6b; [10]). Cephalopod Hox genes are typically expressed in the arm crown with additional expression domains in the funnel, the pedal ganglia, the palliovisceral ganglia or the light organ [10]. Morphological studies suggest that the cephalopod arm crown evolved from the anterior portion of the foot, while the funnel is probably a derivative of the posterior foot ([35–38]; but see [39–41] for a different view). Bearing this topology in mind, Hox1, Hox3, Hox5, Hox7, Lox4 and Post2 are expressed in a staggered fashion from anterior (the first arm) to posterior (the funnel; figure 6b). By contrast, earlier and later cephalopod developmental stages do not show obvious traces of staggered Hox expression. Interestingly, Lox5 deviates from the staggered expression pattern in the cephalopod by being expressed more anteriorly, a condition that is also found in polyplacophorans and gastropods but not in scaphopods (figure 6; [7,10,17]). Lox5 belongs to one of the six posterior Hox genes that are not expressed in a staggered fashion in the gastropod pre-torsional veliger larvae [7,11]. By contrast, all anterior Hox genes, i.e. Hox1, Hox2, Hox3, Hox4 and Hox5 are expressed in a staggered fashion in the mantle and in the pedal and pleural ganglia of the gastropod pre-torsional veliger (figure 6a–d of present study; [7,9,11]). Notably, staggered Hox expression is neither found in early gastropod and scaphopod trochophores nor in the early stage 18 cephalopod embryo. Remainders are only present in developmental stages with a well-developed foot (scaphopods and gastropods) or arm crown and the funnel (cephalopods) (present study; [7,9–11]). This may be different in bivalves that (putatively) exhibit staggered Hox expression in the gastrula prior to foot formation [34].

(b) Conchiferan molluscs express Hox genes in distinct morphological structures

Although traces of a staggered Hox expression are present during the ontogeny of all investigated molluscs, our data on the scaphopod An. entalis also support the notion that Hox genes pattern morphological structures in the Conchifera [4,10]. In all scaphopod developmental stages, Hox genes are expressed in distinct features such as the shell field (figure 3b: Hox1), the foot (figure 3h: Post1), ganglia (figure 4: Hox1-3), as well as in trochoblasts and cells of the apical organ (figure 4a: Hox1; figure 5: Hox3). Hox genes are usually not expressed in the anterior-most part of the adult bilaterian nervous system, i.e. the cerebral ganglia in molluscs [10,42]. Therefore, it is surprising that Hox1-3 are expressed in the developing scaphopod cerebral ganglia, a condition that has only been reported for very few bilaterian species so far, including the polychaete annelid Capitella teleta [29] and the cephalochordate Branchiostoma lanceolatum [43].

Although scaphopod Hox genes are predominantly expressed in ectodermal derivatives such as the nervous system and the anlagen of the shell field/the shell secreting mantle margin of progressive developmental stages, there are also endodermal and mesodermal expression domains (figure 2). The mesoderm of scaphopods is of dual origin and derives from the mesentoblasts during gastrulation and progressive development [20,44]. Mesodermal cells that will eventually differentiate, among others, into muscle cells are already located in the region between the developing mantle (ectoderm) and the digestive system (endoderm) in the early-stage trochophore larva (fig. 12 in [20,44]). While Hox3-5 and Lox4 are expressed in some parts of the mesoderm of scaphopod early-stage trochophores, no mesodermal Hox expression was observed in progressive developmental stages (figure 2; electronic supplementary material, figures S3-S20). Hox genes have been reported to pattern mesodermal derivatives in a variety of bilaterians including molluscs [7,10,11,16,17,45]. Hox genes are expressed to a lesser degree in mesodermal structures of conchiferans than in polyplacophoran aculiferans. As An. crinita and representatives of several other bilaterian phyla express individual Hox genes broadly throughout all three germ layers, it may be assumed that a similar condition was also present in the last common ancestor of all molluscs.

Five out of nine Hox genes are expressed in the scaphopod shell field and the shell-secreting mantle margin of progressive developmental stages (Hox1-4, Post1; electronic supplementary material, table S1). While in polyplacophorans nine of 10 Hox genes (i.e. all except for Hox5) are expressed in the shell fields, gastropods only express four of 11 Hox genes in their shell field (Hox1, Hox4, Post2, Post1) and cephalopods none (electronic supplementary material, table S1). Scaphopods express all nine and polyplacophorans all 10 Hox genes in their foot in contrast to gastropods with only three of the 10 Hox genes showing expression in the foot (Hox2-4; electronic supplementary material, table S1). As the cephalopod foot homologues, the arms express all seven Hox genes investigated in E. scolopes and the funnel four of the seven Hox genes (electronic supplementary material, table S1). All Hox genes are expressed in the nervous system of polyplacophorans, scaphopods and cephalopods, while in gastropods only three of 10 Hox genes are neural (electronic supplementary material, table S1). The cephalopod arms contain the anlagen of the brachial nerve cords and Hox expression domains in the arms are probably also neuroectodermal [10]. Considering a staggered Hox expression as ancestral for Bilateria, the polyplacophoran anterior–posterior expression pattern is closest to this plesiomorphic condition (electronic supplementary material, table S1; [16,17]). Interestingly, the cephalopod Hox expression profile of the nervous system and the foot homologues resembles that of the scaphopod and polyplacophoran more than that of gastropods (electronic supplementary material, table S1). This suggests that scaphopods have retained Hox genes as players acting in anterior–posterior patterning as opposed to their close conchiferan allies but more similar to their distant aculiferan relatives.

(c) A last common molluscan ancestor with colinear Hox expression?

For the Mollusca, spatial colinear Hox expression has only been suggested for the gastrula of the bivalve Patinopecten yessoensis [34]. No genomic information on the organization of Hox loci has been published for any of the other above-mentioned molluscs for which expression data based on in situ hybridization are available. Accordingly, for these species it is impossible to decide whether Hox genes are arranged in a clustered or a non-clustered fashion on the genome. Nevertheless, several molluscan genomes have been sequenced and the one of the patellogastropod Lottia gigantea shows a clustered arrangement of the Hox genes [46]. By contrast, the genomes of the bivalves Crassostrea gigas and Pinctada fucata and that of the cephalopod Octopus bimaculoides do not show a continuous Hox cluster [47,48]. Temporal colinearity, i.e. the progressive expression of Hox genes according to their location on the chromosome, is probably not present in the scaphopod An. entalis and it has not been reported for any other mollusc so far.

A look at other bilaterians shows that a categorization into ‘colinear’ versus ‘non-colinear’ Hox gene expression patterns is not as clear-cut as it is exemplified by Hox expression of fruit fly or mouse embryos in textbooks [23,24]. The cephalochordate B. lanceolatum possesses an intact Hox cluster but no colinear Hox expression [43]. The platyhelminth Schmidtea mediterranea and the leech Helobdella robusta exhibit signs of staggered Hox expression, however both show a disrupted cluster [49,50]. The polychaete annelid C. teleta as well as the brachiopod Terebratalia transversa have a split Hox cluster but while the polychaete exhibits staggered Hox expression, this is not the case in the brachiopod [29,33]. If a colinear Hox expression and genomic clustering were ancestral for Bilateria, then the above-mentioned examples show that staggered Hox expression, clustered genomic organization and temporal colinearity would have been lost independently in several lophotrochozoan taxa. This substantiates the notion that scaphopods, cephalopods and gastropods lost colinear Hox expression to varying degrees independently of each other. If true, then Hox genes were not ‘recruited independently for development’ of the cephalopod arm crown and funnel as previously assumed [10], but show a rather conserved function in anterior–posterior patterning of these cephalopod foot homologues. The fact that the Hox cluster of one cephalopod species is disrupted does not necessarily argue against this scenario [48]. This is exemplified by the ascidian Oikopleura dioica that exhibits staggered Hox expression while having a disintegrated cluster [26]. Its chordate ancestor, however, presumably showed colinear Hox expression.