Staggered Hox expression is more widespread among molluscs than previously appreciated

Abstract

Hox genes are expressed along the anterior-posterior body axis in a colinear fashion in the majority of bilaterians. Contrary to polyplacophorans, a group of aculiferan molluscs with conserved ancestral molluscan features, gastropods and cephalopods deviate from this pattern by expressing Hox genes in distinct morphological structures and not in a staggered fashion. Among conchiferans, scaphopods exhibit many similarities with gastropods, cephalopods and bivalves, however, the molecular developmental underpinnings of these similar traits remain unknown. We investigated Hox gene expression in developmental stages of the scaphopod Antalis entalis to elucidate whether these genes are involved in patterning morphological traits shared by their kin conchiferans. Scaphopod Hox genes are predominantly expressed in the foot and mantle but also in the central nervous system. Surprisingly, the scaphopod mid-stage trochophore exhibits a near-to staggered expression of all nine Hox genes identified. Temporal colinearity was not found and early-stage and late-stage trochophores, as well as postmetamorphic individuals, do not show any apparent traces of staggered expression. In these stages, Hox genes are expressed in distinct morphological structures such as the cerebral and pedal ganglia and in the shell field of early-stage trochophores. Interestingly, a re-evaluation of previously published data on early-stage cephalopod embryos and of the gastropod pre-torsional veliger shows that these developmental stages exhibit traces of staggered Hox expression. Considering our results and all gene expression and genomic data available for molluscs as well as other bilaterians, we suggest a last common molluscan ancestor with colinear Hox expression in predominantly ectodermal tissues along the anterior-posterior axis. Subsequently, certain Hox genes have been co-opted into the patterning process of distinct structures (apical organ or prototroch) in conchiferans.

Keywords: Lophotrochozoa; Polyplacophora; cephalopod; gastropod; gene expression; nervous system.

Figure 1.

Current view of molluscan phylogeny based on Smith et al. [3]. Aculifera and Conchifera are sister groups. Aculiferans comprise the eight-shelled Polyplacophora with its sister group composed of the worm-shaped Solenogastres and Caudofoveata. Conchiferans are an assemblage of cephalopods/monoplacophorans and a sister group comprising bivalves as well as a scaphopod/gastropod clade.

Figure 2.

Mesodermal Hox gene expression in the early-stage trochophore larva of the scaphopod Antalis entalis. The mouth is labelled with an asterisk and all views are lateral with the ventral side facing to the left. Hox3 (a), Hox4 (b), Hox5 (c) and Lox4 (d) are expressed in internal mesodermal domains (arrowheads) that are located between the endoderm and ectoderm. sf, shell field. Scale bars, 50 µm.

Figure 3.

Hox gene expression in the scaphopod shell field. Blastopore and mouth are labelled with asterisks in differential interference contrast images. All lateral views. (a) Sketch of early-stage trochophore with shell field (green), digestive tract (dashed red line), and prototroch (dark grey). (b,d) Hox1 expression in shell field (sf) and shell-secreting mantle margin (m) of early-stage (b) and early mid-stage trochophore (d). (c) Sketch of an early mid-stage trochophore with the shell-secreting mantle margin (green), digestive track (dashed red line) and prototroch (dark grey). (e) Hox2 expression in the mantle of an early mid-stage trochophore. (f,g) Hox3 expression in the shell field and mesoderm of an early-stage trochophore (f) and in the mantle and foot of an early mid-stage trochophore (g). (h,i) Post1 expression in foot and shell field of early-stage trochophore (h) and the foot, mantle, pedal ganglia (pg), pavilion (pv), and anus (an) of late mid-stage trochophore (i). pt, prototroch. Scale bars, 50 µm.

Figure 4.

Hox gene expression in the scaphopod nervous system. Blastopore and mouth are labelled with asterisks. Differential interference contrast image (DIC) of Hox gene expression (left column), confocal reflection scan of Hox expression (red) (middle column), DIC image of Elav expression in developing neurons (right column). (a,b) Hox1 expression in the anlagen of the cerebral (cg) and pedal ganglia (pg), cells of the apical organ (ao), and the pavilion (pv) of an early mid-stage trochophore. (d,e) Hox2 expression in the anlagen of the cerebral ganglia (cerebral pits). (g,h) Hox3 expression in the cerebral ganglia of a late-stage trochophore. (h) Dashed line marks digestive tract. dt, digestive tract; o, oesophagus; f, foot; plg, pleural ganglia; pt, prototroch; stc, statocyst. Scale bars: 50 µm.

Figure 5.

Expression of Hox genes in the early mid-stage trochophore of the scaphopod Antalis entalis. Differential interference contrast images (first and third rows) of Hox gene expression and sketches as summary of expression (second and fourth rows). Digestive tract (dashed red lines) and statocysts (dashed red circles) are indicated. All lateral views and ventral faces to the left. Hox genes are expressed in a near-to staggered fashion in the scaphopod's mid-stage trochophore with only Hox3 and Post2 deviating from this pattern. Note the weak unspecific staining on the prototroch and mantle (Post1). Scale bars, 100 µm.

Figure 6.

Traces of staggered Hox expression in molluscan developmental stages. Coloured bars indicate anterior–posterior extension of individual Hox gene expression domains and underlying grey shading highlights genes that are expressed in a staggered fashion. Digestive tract (stippled red lines), shell/shell plates (dashed black line), blastopore/mouth (asterisks) and prototroch/velum (shaded in dark grey) are indicated. (a) Late-stage trochophore of the polyplacophoran Acanthochitona crinita. (b) Stage 19/20 embryo of the cephalopod Euprymna scolopes. (c) Early mid-stage trochophore of the scaphopod Antalis entalis. (d) Pre-torsional veliger larva of the gastropod Gibbula varia. (e) Gastrula of the bivalve Patinopecten yessoensis. (f) Inferred trochophore larva of the last common molluscan ancestor. a, anterior; ar, arm; d, dorsal; ep, episphere; f, foot; fn, funnel; m, mantle; v, ventral; p, posterior; pg, pedal ganglion; pvg, palliovisceral ganglion; sf, shell field; ys, yolk sac.