Open Access

Unexpected co-linearity of Hox gene expression in an aculiferan mollusk

BMC Evolutionary Biology201515:151

https://doi.org/10.1186/s12862-015-0414-1

Received: 27 January 2015

Accepted: 8 June 2015

Published: 5 August 2015

Abstract

Background

Mollusca is an extremely diverse animal phylum that includes the aculiferans (worm-like aplacophorans and eight-shelled polyplacophorans) and their sister group, the conchiferans, comprising monoplacophorans, bivalves (clams, mussels), gastropods (snails, slugs), scaphopods (tusk shells) and cephalopods (squids, octopuses). Studies on mollusks have revealed an overall number of 11 Hox genes in seven out of eight molluscan “class”-level taxa, but expression data of key developmental regulators such as homeotic genes are only available for three gastropod and two cephalopod species. These show that Hox genes are involved in the formation of specific features including shell, foot, funnel or tentacles and not in antero-posterior body plan patterning as in most other bilaterian animals. The role of Hox genes in non-conchiferan (i.e., aculiferan) mollusks remains entirely unknown.

Results

Here we present the first data on the expression of seven Hox genes in apolyplacophoran mollusk, Acanthochitona crinita. In A. crinita the Hox genes Acr-Hox1-5, Hox7 and Post2 are expressed in a co-linear pattern along the antero-posterior axis, but not in molluscan-specific features such as the shell or the foot. The expression pattern is restricted to the post-trochal region and the transcripts are present in ecto-, endo- and mesodermal cell layers. Contrary to the situation in gastropods and cephalopods, we did neither find Hox gene expression in distinct neural subsets of A. crinita, nor in its developing shell plates.

Conclusions

Our analysis and comparison with other lophotrochozoans indicate that the basal role of Hox genes is in antero-posterior axis patterning in mollusks, similar to the vast majority of bilaterian animals, and that this role has been conserved in polyplacophorans, while co-option into patterning of evolutionary novelties emerged either at the base of Conchifera or independently in gastropods and cephalopods. These morphological innovations most likely contributed to the evolutionary success of its representatives, as exemplified by, e.g., the wide ecological range and species richness of gastropods.

Keywords

Body plan Development Evo-devo Evolution Mollusca Novelty Patterning Polyplacophora

Background

Developmental organization of each particular animal body plan is controlled by a conserved bilaterian-wide cluster of homeotic genes. One of these clusters, the Hox genes, with a conserved homeodomain sequence, possesses a distinctive genomic arrangement and it encodes a set of transcription factors that play a crucial role in organizing the antero-posterior (AP) body axis of many bilaterian animals during development [17]. A number of Hox gene expression studies have been published for various bilaterian clades, but most of them are confined to ecdysozoan or deutoterstome representatives (e.g., [818]), while data on lophotrochozoans outside the Annelida (e.g., [1618]) are scarce.

Mollusca is one of the most diverse animal phyla. According to recent phylogenomic analyses [1922], mollusks contain two monophyletic sister taxa, the primarily univalved Conchifera (Monoplacophora, Scaphopoda, Bivalvia, Gastropoda, Cephalopoda) and the non- or eight-shelled Aculifera (the aplacophoran taxa Neomeniomorpha or Solenogastres and Chaetodermomorpha or Caudofoveata, as well as the shell plate-bearing Polyplacophora). Thereby, recent developmental data have shown that the aculiferans most likely originated from a polyplacophoran-like ancestor, implying that the simple vermiform morphology of the aplacophorans evolved by secondary simplification [23].

Until today, an overall number of 11 Hox genes has been identified in seven out of the eight molluscan “class”-level taxa [2438], but expression studies on Hox genes are restricted to representatives of two, probably derived, clades of Mollusca that belong to the conchiferans, namely Gastropoda (snails and slugs) and Cephalopoda (squids and octopuses). The existing expression data on Mollusca are limited to larvae of the gastropods Haliotis asinina, H. rufescens and Gibbula varia, and to developing embryos of the cephalopods Euprymna scolopes and Sepia officinalis. These data show that the Hox genes are involved in the formation of specific morphological features such as the shell, the ganglionic nervous system, the tentacles and the funnel, and that they are - contrary to other bilaterians - expressed in a non-co-linear pattern [24, 26, 27, 3335, 38]. Furthermore, Hox transcription products could also be identified in sensory organs (e.g., in the apical organ and the statocyst in gastropod larvae) and in the light organ of squid embryos [27, 34]. As opposed to this, in most other bilaterians, Hox genes do show co-linearity and play a crucial role in organizing the antero-posterior body axis [4, 6, 7]. The gastropod and cephalopod expression data suggest that Hox genes may have been co-opted into the formation of novel molluscan morphological features [27, 3335, 38].

In contrast to gastropods and cephalopods, polyplacophorans possess a number of non-conchiferan characters such as serially arranged shell muscles, dorsal shell plates, as well as a non-ganglionic visceral and pedal nervous system with iterated commissures [23, 39, 40]. In order to assess potential functions of Hox genes in non-conchiferan mollusks, we here present the first data on Hox gene expression in an aculiferan mollusk, the polyplacophoran Acanthochitona crinita (Pennant, 1777).

Results

Hox gene orthologs and phylogenetic analysis

In the assembled transcriptome of Acanthochitona crinita (Acr), orthologous Hox gene sequences were identified. The sequences of the genes Acr-Hox1-5, Acr-Hox7 and Acr-Post2 contain a conserved homeo-domain amino-acid sequence and Acr-Hox1-2, Acr-Hox4-5 and Acr-Hox7 are flanked by the Y(P)WM-motif at the N-terminal (Fig. 1). All Acr-Hox protein sequences cluster together with their respective bilaterian orthologs, as revealed by the phylogenetic analysis (Fig. 2).
Fig. 1

Homeodomain alignment. Sequence alignment containing Hox gene homeo-domains (Hox1-5, Hox7 and Post2) and the flanking regions. The residues are bluish colored in each column according to the percentage of identity that agrees with the consensus sequence. Residues with less than 40 % of identity are not colored. Dashes represent missing data

Fig. 2

Phylogeny of Hox genes. Phylogenetic reconstruction of Hox genes families from amino-acid sequences present in the homeodomain and the flanking regions. The consensus tree was inferred through Bayesian phylogenetic analysis with MrBayes v3.2.2 discarding 25 % of the samples as burn-in. The branch support values are posterior probability values of Bayesian likelihood. Acr-Hox gene orthologs are highlighted by colored rectangles and the sequences used cluster with other appropriate Hox gene orthologs. The Hox1, Hox2, Hox4 and Post-2 families are strongly supported. Note that branches with posterior probability values less than 50 % are collapsed. The homeobox gene distalless was used as outgroup

Larval development of Acanthochitona crinita

Early trochophore larvae of A. crinita are less than 200 μm long (not shown) and have an apical tuft at the anterior pole. The well-developed prototroch divides the pre-trochal area (episphere) from the post-trochal region (hyposphere). Ventro-medially, posterior to the prototroch, the mouth opening (stomodaeum) is located.

Mid-trochophore larvae are oval-shaped and approximately 280 μm in length (Fig. 3a and b). The hyposphere is more elongated, and posterior to the mouth opening the developing anlage of the foot extends in the ventral region. Dorso-laterally, on both sides of the hyposphere, a longitudinal row of epidermal and very prominent spicule-containing cells are discernible. Dorsally, an anterior transversal row of spicule-containing cells is present in the episphere and in the posterior region of the hyposphere. In this stage the anlagen of the seven dorsal shell plates are already visible in the hyposphere.
Fig. 3

Morphology of Acanthochitona crinita trochophore larvae. Anterior faces up. a, b Mid-trochophore larvae. Episphere and hyposphere are separated from each other by the prototroch. In the dorsal hyposphere region, the anlagen of the seven shells are surrounded by spicule-containing cells. c, d Late trochophore larvae. Seven differentiating dorsal shell plates are present. at apical tuft, ds dorsal shell plates, f foot, pt prototroch, sc spicule-forming cell(s)

Late trochophore larvae are about 360 μm in length and the hyposphere is considerably more elongated than in previous stages (Fig. 3c and d). On the dorsal side of the hyposphere, seven differentiating shell plates are present. At the end of the entirely lecithotrophic, i.e., non-feeding larval development, late trochophore larvae settle and undergo metamorphosis, which is characterized by considerable dorso-ventral flattening of the animals as well as loss of the apical ciliary tuft and the prototroch [41].

Hox gene expression

Acanthochitona crinita-Hox1

The expression of Acr-Hox1 in early and mid-trochophore larvae is restricted to the anterior region of the hyposphere, postero-lateral to the stomodaeum. Ventrally, the expression pattern is visible in two sub-epidermal cellular spots, lateral to the median body axis (Fig. 4a–f). Dorsally, a slight transversal band of Acr-Hox1 expression is detectable within the epidermal and sub-epidermal cell layer in the anterior region of the hyposphere (Fig. 4c–f). In later stages a weak Acr-Hox1 expression pattern is discernible in the epidermal and sub-epidermal cell layer (Fig. 4g–i).
Fig. 4

Acr-Hox1 expression pattern in Acanthochitona crinita. Anterior faces up. a, b Stereomicrographs and c light micrograph of mid-trochophore larvae showing the expression pattern of Acr-Hox1 next to the stomodaeum in the anterior hyposphere. df Autofluorescence (grey) of mid-trochophore larvae and specific transcription product distribution (yellow). d Single sagittal section and e, f ventral and ventro-lateral whole mount clipping plane projection showing the location of the Acr-Hox1 transcription products in the hyposphere. g, h Stereomicrographs and i light micrograph of the faint Acr-Hox1 expression pattern in late trochophores. ao apical organ, ds dorsal shell plates, f foot, mo mouth opening, pt prototroch, st stomatodaeum, trb trochoblast(s)

Acr-Hox2

Transcripts of Acr-Hox2 in early and mid-trochophore larvae are present in two sub-epidermal cell clusters, postero-lateral to the stomodaeum (Figs. 5a–c and 6a–c). The pattern is posteriorly more elongated than in Acr-Hox1 and each expression domain is also located lateral to the median body axis (Fig. 5a). In later stages, an indistinct, faint Acr-Hox2 expression pattern is found sub-epidermally and antero-posteriorly in the hyposphere (Fig. 7a–c).
Fig. 5

Acr-Hox2-5, Hox7 and Post2 expression in mid-trochophore larvae of Acanthochitona crinita. Anterior faces up. Left and middle column: stereomicrographs. Right column: light micrograph. ac Acr-Hox2 is expressed postero-laterally, next to the stomodaeum, in two ventral, short and parallel longitudinal strands. df Transcripts of Acr-Hox3 are present in two distinct ventral parallel longitudinal strands. gi Acr-Hox4 is expressed prominently in the hyposphere in two distinct domains which are antero-medially interconnected. jl The expression pattern of Acr-Hox5 is present in two ventral longitudinal strands in the posterior region. mo Acr-Hox7 transcripts are present in a ring-like arrangement in the posterior hyposphere. pr The expression pattern of Acr-Post2 is present at the posterior pole of the larva. Scale: 50 μm. ao apical organ, f foot, pt prototroch

Fig. 6

Distribution pattern of the Acr-Hox2-5, Hox7 and Post2 transcription products in mid-trochophore larvae of Acanthochitona crinita. Anterior faces up. Autofluorescence (grey) and specific transcription product distribution (yellow). Left column: single sagittal section. Middle and right column: ventral and ventro-lateral whole mount clipping plane projection. Transcripts of Acr-Hox2 are present sub-epidermally. Acr-Hox3 is ventrally expressed in sub-epidermal cell layers. Acr-Hox4 transcripts are present in the epidermal and sub-epidermal cell layers. Transcripts of Acr-Hox5 are present sub-epidermally. The posterior ring of Acr-Hox7 transcription products is present in sub-epidermal cell layers. Acr-Post2 transcription products accumulate in epidermal and sub-epidermal cell layers. ao apical organ, mo mouth opening, pt prototroch, rs radula sac, st stomatodaeum, trb trochoblast(s)

Fig. 7

Acr-Hox2-5, Hox7 and Post2 expression in late trochophore larvae of Acanthochitona crinita. Anterior faces up. Left and middle column: stereomicrographs. Right column: light micrograph. af Acr-Hox2 and Acr-Hox3 are indistinctly expressed along the antero-posterior larval body axis. gi Acr-Hox4 transcripts are ventrally present, posterior to the stomodaeum and around the lateral edges of the ventral foot. jl Two faint Acr-Hox5 strands are expressed in the mid-region of the hyposphere. mo Acr-Hox7 transcripts are indistinctly present in two postero-lateral expression strands. pr Acr-Post2 expressionis present in the posterior pole of the hyposphere. Scale: 50 μm. ao apical organ, ds dorsal shell plates, f foot, pt prototroch

Acr-Hox3

Acr-Hox3 expression pattern in early and mid-trochophore larvae is found in the hyposphere, in two parallel longitudinal sub-epidermal cellular strands (Figs. 5d–f and 6d–f). The pattern is posteriorly much more elongated than that of Acr-Hox2. Each strand of expression extends laterally to the median body axis. Dorsally, a slight transversal anterior ring of Acr-Hox3 transcripts is present in the hyposphere (Figs. 5e–f and 6e–f). Acr-Hox3 in later stages is only indistinctly expressed in the sub-epidermal layers (Fig. 7d–f).

Acr-Hox4

In comparison to the other genes, Acr-Hox4 shows the strongest expression pattern in early and mid-trochophore larvae. Acr-Hox4 transcripts are present in two prominent epidermal and sub-epidermal expression strands in the hyposphere (Figs. 5g–i and 6g–i). Ventro-medially, both elongated strands are interconnected with a slender expression band in the anterior hyposphere region. Laterally, the expression pattern is faint. A slight transversal ring of Acr-Hox4 expression is present antero-dorsally (Figs. 5h–i and 6h–i). Later stages retain a less prominent expression pattern in the ventral hyposphere (Fig. 7g–i).

Acr-Hox5

Transcripts of Acr-Hox5 in early and mid-trochophore larvae extend from the central to the posterior region of the hyposphere. Acr-Hox5 is expressed sub-epidermally, in two distinct longitudinal cellular strands (Figs. 5j–l and 6j–l). Antero-medially, both strands of expression are interconnected. In later stages, Acr-Hox5 is less strongly expressed, but still two weak, parallel, sub-epidermal cellular strands are present (Fig. 7j–l).

Acr-Hox7

Acr-Hox7 expression in early and mid-trochophore larvae is present in the posterior hyposphere, sub-epidermally in a ring-like pattern (Figs. 5m–o and 6m–o). Expression is ventrally and dorsally more prominent than in the lateral cell layers. Acr-Hox7 is expressed in later stages, indistinctly in the posterior region of the hyposphere (Fig. 7m–o).

Acr-Post2

Expression of Acr-Post2 in early and mid-trochophore larvae is in the posterior pole of the hyposphere. Acr-Post2 transcription products accumulate ventrally and dorsally in epidermal and sub-epidermal cell layers (Figs. 5p–r and 6p–r). In later stages, Acr-Post2 is less prominently expressed, but still discernible in the posterior pole of the larva (Fig. 7p–r).

Discussion

Hox gene expression in aculiferan and conchiferan mollusks

The existing expression data of three species of gastropods (Haliotis asinina, H. rufescens and Gibbula varia [24, 26, 3335]) and two cephalopod species (the squid Euprymna scolopes and the cuttlefish Sepia officinalis [27, 38] have shown that Hox genes are predominantly involved in the formation of the ganglionated central nervous system in these conchiferan mollusks. Other Hox gene transcripts are present in sensory organs such as the apical organ or the statocyst in the gastropod trochophore larvae and in the light organ of E. scolopes [27, 34]. Furthermore, Hox gene expression domains include the gastropod shell and the cephalopod brachial crown and funnel.

In the polyplacophoran Acanthochitona crinita, none of the seven investigated Hox gene orthologs (Acr-Hox1-5, Hox7 and Post2) show a comparable structural expression pattern during development. By contrast, the Hox genes are expressed in epidermal and sub-epidermal cell layers, in a co-linear manner along the antero-posterior axis in the hyposphere of early and mid-trochophore larvae of A. crinita (Figs. 4, 5, 6, 7 and 8).
Fig. 8

Schematic summary of Hox gene expression in Acanthochitona crinita. Each box shows the specific Acr-Hox1-5, Hox7 and Post2 gene expression patterns in mid (top) and late (bottom) trochophore larvae in ventral (left) and dorsal view (right), respectively

In early and mid-stage Acanthochitona crinita trochophores, Acr-Hox1 transcripts are found in two ventro-lateral cell clusters, next to the stomodaeum, and dorsally in a slender transversal band. In contrast, in the gastropod Gibbula varia, the Hox1 ortholog (Gva-Hox1, Fig. 9c) is expressed in the larval dorso-posterior shell anlage [33]. Acr-Hox2 transcripts are present in two ventral, short and parallel longitudinal strands, postero-lateral to the stomodaeum, and Acr-Hox3 expression is also present in two distinct ventral parallel longitudinal strands that extend farther in posterior direction than the Acr-Hox2 expression domain. The Acr-Hox4 ortholog is highly expressed in two domains which are antero-medially interconnected by a slender band. Gva-Hox2-4 and Has-Hox2-4 (Haliotis asinina-Hox2-4) transcripts are distributed in the ventral ectoderm, postero-laterally around the foot anlage [26, 33]. Acr-Hox5 is expressed in two ventral longitudinal strands in the posterior region of the hyposphere, whereas in gastropod trochophore larvae only a faint endodermal Gva-Hox5 expression adjacent to the foot rudiment was found [24, 26, 33, 34]. Acr-Hox7 is expressed in a ring-like pattern in the posterior hyposphere; this pattern differs strongly from that observed in the trochophores of the gastropod Gibbula varia, where Gva-Hox7 is exclusively expressed in the trochoblasts [34]. Acr-Post2 expression is present in the posterior pole of the hyposphere, while in Gibbula, a certain number of cells that surround the posterior foot anlage dorsally express Gva-Post2 [34].
Fig. 9

Comparison of Hox gene expression patterns in annelid (a), polyplacophoran (b) and gastropod (c) larvae. Antero-posterior Hox gene expression gradient in the larval body is indicated by colored vertical bars (Hox1-5, Hox7 and Post2) on the left side of each trochophore larva. The comparison reveals a relatively similar co-linear Hox gene expression pattern in Annelida and Polyplacophora, whereas in gastropods Hox gene transcripts are expressed non-co-linearly and are often confined to specific morphological features such as the prototroch or the shell field. Annelid data based on Nereis virens and Platynereis dumerilii [17] and gastropod data based on Gibbula varia [33, 34]. Black small circle illustrates the mouth opening, horizontal band the prototroch and ellipse the ventral foot

In late Acanthochitona crinita trochophore larvae, the expression pattern is faint at best or entirely absent (summarized in Fig. 8), while a distinct distribution of orthologous Hox gene transcripts is present in pre- and post-torsional veliger stages of the gastropods Gibbula varia and Haliotis asinina. Thereby, Gva-Hox1, Gva-Post2 and Has-Hox1 are expressed in the mantle and especially in the shell field [26, 33]. Gva- and Has-Hox2-4 transcripts are present in the operculum and in the foot anlage [26, 33]. However, the main expression domains of Gva-Hox2-5 and Has-Hox2-5, as well as that of Haliotis rufescens-Hox5 (Hru-Hox5) and Gva-Hox7 are in the cerebral, branchial, esophageal, pedal and pleural ganglia [24, 26, 33, 34]. Orthologs of Hox1, 3, 5 and 7 in the embryos of the cephalopod Euprymna scolopes (Esc-Hox1, 3, 5 and 7) are expressed in the developing cerebral, palliovisceral and pedal ganglia, and Esc-Hox1, 3 and Post2 in the stellar ganglion [27]. Esc-Hox1, 3, 5, 7 and Post2 are also expressed in cephalopod-specific structures such as the brachial crown and the funnel [27]. As in E. scolopes, in embryos of the European cuttlefish Sepia officinalis the ortholog of Sof-Hox3 is also expressed in distinct nervous system and locomotory structures. Sof-Hox3 transcripts are present in the sub-esophageal masses, brachial, buccal and stellar ganglia and in parts of the funnel [38].

In comparison to the Hox gene expression data on gastropods and cephalopods, the polyplacophoran A. crinita Hox gene transcripts are not restricted to particular subsets of the developing nervous system (although we do not rule out the presence of Hox transcripts during neurogenesis). This may be due to the medullary cord-like character of the polyplacophoran nervous system which, in contrast to the gastropods and cephalopods, lacks distinct ganglia [4143]. Data on polyplacophoran neurogenesis have shown that in early and mid-trochophore larvae of Ischnochiton hakodadensis and Mopalia muscosa the primary pedal neurons (derivatives of the ectoderm) develop and extend posteriorly [42, 43]. Interestingly, in the post-trochal region in early and mid-stage A. crinita trochophore larvae, the transcripts of the Acr-Hox1-5 genes are present in two parallel antero-posterior medial expression domains, which overlap with the region of the developing neurons of the future paired pedal nerve cords. In contrast, in the lateral post-trochal areas of the developing visceral nerve cords, no overlapping Acr-Hox gene expression pattern is present. However, in A. crinita, the overall expression pattern is not restricted to, e.g., the developing pedal nerve cords. Instead, the Acr-Hox gene transcripts are also found in more lateral and more central sub-epidermal tissues which clearly do not contribute to neural tissues (Figs. 46).

Developmental data on the muscular system have shown that in early and mid-trochophore larvae of Mopalia muscosa and Leptochiton asellus several muscle fibers start to develop in the post-trochal region (the future dorso-ventral muscles [23, 41]). The Hox gene transcripts in early and mid-stage A. crinita trochophore larvae are expressed in a region comparable to that where these (mesoderm-derived) muscles form. However, the Acr-Hox gene expression pattern is not restricted to individual muscular structures.

Henry and colleagues [44] labeled derivatives of the endoderm in the polyplacophoran species Chaetopleura apiculata via injected fluorescent markers in cell lineage experiments. Thereby, it was found that in early and mid-trochophore larvae of C. apiculata, third-quartet macromeres give rise to the developing body wall and the (endoderm-derived) gut in the post-trochal region. In early and mid-stage A. crinita trochophore larvae, the transcripts of anterior Hox genes (such as Acr-Hox1-4) are present post-trochally around the forming stomodaeum and adjacent to the central area of the forming digestive tract (as part of the endoderm).

Indeed, in the polyplacophoran Acanthochitona crinita, the Hox gene expression patterns are not restricted to distinct morphological structures (e.g., ganglia, shell plates, etc.). However, comparable developmental polyplacophoran data and also the Hox gene expression patterns indicate, that the Acr-Hox gene transcripts are seemingly present in tissues derived from all three germ layers (ectoderm, endoderm and mesoderm). These expression patterns differ significantly from the Hox gene expression pattern in gastropods and cephalopods (see above), indicating that Hox genes are involved in different, taxon-specific developmental processes in gastropods and cephalopods, but not in polyplacophorans. In contrast, the polyplacophoran transcripts Acr-Hox1-5, Hox7 and Acr-Post2 are distributed in a specific spatial antero-posterior pattern. Anterior Hox genes (e.g., Acr-Hox1-2) are only expressed in the anterior hyposphere, next to the stomodaeum, and posterior Hox gene transcripts (such as Acr-Post2) are only present in the posterior hyposphere. This antero-posterior mode of expression resembles that of the co-linear Hox gene expression in the vast majority of bilaterian animals, including Acoelomorpha, Annelida, Ecdysozoa and Deuterostomia (e.g., [14, 16, 17, 26, 34, 4554]) and thus appears to have been conserved in polyplacophoran mollusks.

In this study, the expression of seven out of the putatively 11 molluscan Hox genes was investigated in the polyplacophoran Acanthochitona crinita. Preliminary data on the yet missing genes Acr-Lox5, Lox4 and Lox2 (Acr-Post1 could not be identified) are in line with the antero-posterior Hox gene expression pattern described herein.

Antero-posterior Hox gene expression in Polyplacophora and Annelida

Comparing our results with those of Annelida, a taxon probably closely related to Mollusca (e.g., [19, 36, 5557]), important similarities in the spatial Hox gene expression patterns along the antero-posterior axis become obvious (Fig. 9a–b). Early trochophore larvae of the annelids Nereis virens and Platynereis dumerilii show a strikingly similar co-linear Hox gene expression pattern to that of Acanthochitona in the ventral hyposphere. In A. crinita, the Acr-Hox1 expression is located in the anterior hyposphere; orthologs of N. virens-Hox1 (Nvi-Hox1) and P. dumerilii-Hox1 (Pdu-Hox1) are similarly expressed in the anterior hyposphere, the future first parapodial segment [17]. The Acr-Hox2 expression postero-lateral to the stomodaeum is similar to the stomodeal Pdu-Hox2 expression [17]. The two lateral expression strands of Acr-Hox3 resemble the Nvi-Hox3 and Pdu-Hox3 expression strands [17]. Acr-Hox4 transcription products appear along the ventral and lateral hyposphere of the polyplacophoran, similar to the ventral and lateral expression of Nvi-Hox4 and Pdu-Hox4 in the developing second and third parapodial segments and the site of Acr-Hox5 expression is comparable to that of Nvi-Hox5 and Pdu-Hox5 expression in the developing second and third parapodial segment [17]. Acr-Hox7 transcripts are distributed posteriorly in a dorso-ventral ring-like pattern; Nvi-Hox7 and Pdu-Hox7 are also expressed posteriorly, namely in the developing third parapodial and pygidial segment [17]. Acr-Post2 expression is restricted to the posterior pole, similar to the pygidial expression of Nvi-Post2 and Pdu-Post2 [17]. In addition to the expression data in N. virens and P. dumerilii, a relatively similar AP expression pattern is also known from other early annelid developmental stages (e.g., Chaetopterus [16], Helobdella robusta and H. triserialis [46], Capitella sp. [51], Platynereis [53]).

Overall, the relative spatial antero-posterior Hox gene expression pattern in early developmental stages of Nereis virens and Platynereis dumerilii, as well as other annelids, is comparable to that of the polyplacophoran Acanthochitona crinita. In particular, the spatial antero-posterior Hox gene expression pattern in early polyplacophoran trochophores is also found in the ecto-, endo- and mesodermal cell layers and is thus not restricted to specific organs. Annelid Hox gene expression in the nervous system appears for the first time in meta-trochophore larvae [16, 17, 46, 51].

Conclusion

The relatively similar co-linear Hox gene expression signature in Polyplacophora, Annelida and other bilaterians demonstrates that the ancestral role of Hox genes in Mollusca was also in patterning of the antero-posterior body axis. After the aculiferan-conchiferan split, the Hox genes were secondarily recruited into novel functions in gastropods and cephalopods (either individually or at the base of Conchifera), but data on bivalves, scaphopods and monoplacophorans are needed to further substantiate this assumption. Overall, the plasticity of Hox gene functions in mollusks may well explain the high morphological variability exhibited among the various molluscan sub-lineages and may thus have been an important driving force for the evolutionary success of gastropods and cephalopods.

Methods

Rearing and larval development of Acanthochitona crinita

Adult A. crinita were collected at the Biological Station Roscoff, Brittany, France. Spawning was induced by alternating hot (30–40 °C) and cold (3–6 °C) shocks as well as sunlight exposure. After spawning, one to two drops of concentrated sperm solution was used to fertilize the eggs in 150–200 ml seawater for 30 min at 21–23 °C. After spawning and fertilization, developmental stages were reared in filtered seawater. First cleavage stages appeared 3–5 h after fertilization. First trochophore larvae hatched nine to 10 h after fertilization and post-metamorphic animals appeared three to 4 days after fertilization.

Fixation and RNA extraction

Developmental stages were fixed in 4 % paraformaldehyde in MOPS buffer (0.5 M Mops, 10 mM MgSO4, 5 mM EGTA, 2.5 M NaCl) for 45 min at room temperature, stepped into 100 % methanol and stored in 100 % methanol at −20 °C. To extract RNA with the RNeasy mini Kit (#74104,Qiagen), a pool of representative developmental stages was used. RNA was sequenced by Illumina technology and reads were assembled into contigs by Trinity [58].

Orthology assignment and phylogenetic analysis of Hox genes

The assembled sequences from the Acanthochitona crinita transcriptome were used in local similarity searches using the program tblastn [59] against known and well-curated Hox sequences retrieved from GenBank non-redundant protein database. The top 10 BLAST hits of each similarity search were individually and manually analyzed. The Hox domain-containing sequences were assigned according to the presence of diagnostic residues/motif in the homeodomain as well as in the flanking regions and through Bayesian molecular phylogenetic analysis (Fig. 1). The program Jalview 2 [60] was used to illustrate the multiple sequence alignment of the Hox genes. All sequences of species used in the phylogenetic analysis and corresponding accession numbers are listed in Table 1. The Bayesian analyses were carried out with MrBayes v3.2.2 software [61] with Jones-Taylor-Thornton model of amino-acid substitution [62], gamma-distributed rates, 25,000,000 generations and sampling frequency of 1000.
Table 1

Species, genes and GenBank accession numbers used in the phylogenetic analysis

Species name/Abbreviation

Phylum

Gene Name

Accession Number

Acanthochitona crinita/Acr

Mollusca

Acr_Hox1

KR190463

Acr_Hox2

KR190464

Acr_Hox3

KR190465

Acr_Hox4

KR190466

Acr_Hox5

KR190467

Acr_Hox7

KR190468

Acr_Post2

KR190469

Branchiostoma floridae/Bfl

Chordata

Bfl_Hox1

BAA78620

Bfl_Hox2

BAA78621

Bfl_Hox4

BAA78622

Bfl_Hox5

ABX39489

Bfl_Hox7

ABX39491

Bugula turrita/Btu

Bryozoa (Ectoprocta)

Btu_Hox2

AAS77225

Btu_Hox3

AAS77226

Btu_Hox4a

AAS77227

Btu_Hox4b

AAS77228

Btu_Post2

AAS77230

Caenorhabditis elegans/Cel

Nematoda

Cel_Hox1

CAA34929

Ciona intestinalis/Cin

Chordata

Cin_Hox1

NP_001122333

Cin_Hox2

CAD59668

Cin_Hox4

NP_001027781

Cin_Hox5

NP_001027665

Drosophila melanogaster/Dme

Arthropoda

Dme_Hox1

CAB57787

Dme_Hox2

CAA45271

Dme_Hox3

P09089

Dme_Hox4

P07548

Dme_Hox5

NP_524248

Dme_Hox7

CAA27417

Dme_Dll

NP_726486

Euprymna scolopes/Esc

Mollusca

Esc_Hox1

AAL25804

Esc_Hox3

AAR16188

Esc_Hox5

AAR16189

Esc_Hox7

AAL25809

Esc_Post2

AAL25812

Flaccisagitta enflata/Fen

Chaetognatha

Fen_Hox1

ABS18809

Fen_Hox3

ABS18810

Fen_Hox4

ABS18811

Fen_Hox5

ABS18812

Gibbula varia/Gva

Mollusca

Gva_Hox1

ACX84671

Gva_Hox2

ADJ18233

Gva_Hox3

ADJ18232

Gva_Hox4

ACX84672

Gva_Hox5

ADJ18234

Gva_Hox7

ADJ18235

Gva_Post2

ACX84674

Haliotis asinina/Has

Mollusca

Has_Hox3

AAK17185

Has_Hox4

AAK11240

Has_Hox5

AAF78248

Lingula anatina/Lan

Brachiopoda

Lan_Hox1

AAD45587

Lan_Hox3

AAD45588

Lan_Hox5

AAD45589

Lan_Hox7

AAD45590

Lan_Post2

AAD45595

Lithobius atkinsoni/Lat

Arthropoda

Lat_Hox1

AAL36907

Lat_Hox2

AAL36908

Lat_Hox3

AAL36906

Lat_Hox4

AAL36902

Lat_Hox5

AAL36909

Lat_Hox7

AAL36901

Lineus sanguineus/Lsa

Nemertea

Lsa_Hox1

CAA76295

Lsa_Hox3

CAA76296

Mus musculus/Mmu

Chordata

Mmu_Hox1

NP_034579

Mmu_Hox2

NP_034581

Mmu_Hox3

NP_034582

Mmu_Hox4

NP_032291

Mmu_Hox5

NP_034583

Mmu_Hox7

NP_034585

Nymphon gracile/Ngr

Arthropoda

Ngr_Hox1

ABD46723

Ngr_Hox2

ABD46725

Ngr_Hox5

ABD46729

Ngr_Hox7

ABD46732

Nereis virens/Nvi

Annelida

Nvi_Hox1

AAD46166

Nvi_Hox2

AAD46167

Nvi_Hox4

AAD46169

Nvi_Hox5

AAD46170

Nvi_Hox7

ABD04657

Nvi_Post2

AAD46176

Platynereis dumerilii/Pdu

Annelida

Pdu_Hox3

ABD04656

Pdu_Hox4

ABD04658

Pdu_Hox5

ABD04660

Pdu_Post2

ABD04651

Pdu_Dlx1

CAJ38799

Sacculina carcini/Sca

Arthropoda

Sca_Hox1

ABB46347

Sca_Hox2

AAD00340

Sca_Hox4

AAD00345

Sca_Hox5

AAM50457

Saccoglossus kowalevskii/Sko

Hemichordata

Sko_Hox1

AAP79296

Sko_Hox2

ABK00018

Sko_Hox3

AAP79286

Sko_Hox4

AAP79297

Sko_Hox5

ABK00019

Sko_Hox7

AAP79287

Symsagittifera roscoffensis/Sro

Acoelomorpha

Sro_Hox1

AAN11404

Tribolium castaneum/Tca

Arthropoda

Tca_Hox1

NP_001107762

Tca_Hox3

AAK16424

Tca_Hox4

AAK16423

Tca_Hox5

AAK16422

Primer design and probe synthesis

Acr-Hox gene-specific primer design was performed with the sequence assembling software Geneious 6.1.6 (Biomatters Limited) and primers were purchased from Life Technologies Company (Thermo Fischer Scientific). First strand cDNA was synthesized by reverse transcription of RNA pooled from representative developmental stages covering the entire larval and early post-metamorphic development (cDNA synthesis kit, #04379012001, Roche Diagnostics). Hox gene sequences were amplified with the gene-specific primers via standard PCR. PCR products were cloned by insertion into pGEM-T easy vectors (#A1360, Promega) and plasmid minipreps were purified with Qia-miniprep kit (#27106, Qiagen). Antisense and sense probes from linearized plasmid sequences were synthesized with a DIG-labeling kit (#11277073910, Roche Diagnostics).

Whole mount in situ hybridization

Acanthochitona crinita trochophore larvae were decalcified in ME (90 % MeOH and 50 mM EGTA) for 10 min and in PPE (4 % PFA, 1 × PBS, 50 mM EGTA) for 45 min and subsequently washed in PBT (1 × PBS, 0.1 % Tween 20). Larvae were incubated in a 60 μg/ml proteinase-K solution at 37 °C for 10 min and then washed in 0.2 % glycine in PBT. To reduce charged probe binding, larvae were put in 1 % triethanolamine and 0.5 % acetic anhydride solution, washed in PBT and post-fixed for 45 min in 4 % PFA. Afterwards, larvae were stepped into 100 % hybridization buffer and pre-incubated at 60 °C in a water bath overnight. Larvae were hybridized with antisense probes which are complementary to the transcript sequence or with sense probes as negative controls (0.25–0.7 ng/μl probe concentration) at 60 °C for 48 h. After hybridization, larvae were washed several times in 4 × Wash (25 % formamide, 4 × SSC, 0.1 % Tween 20), 2 × Wash (with 2 × SSC) and 1 × Wash (with 1 × SSC). Afterwards, specimens were stepped into and washed several times in 1 × SSC, then several times in MAB (100 mM maleic acid, 150 mM NaCl, 0.02 % Tween 20). Larvae were washed in 2 % blocking solution (#11096176001, Roche) in MAB for 2–3 h and incubated in 1:5000 DIG antibody solution (#11093274910, Roche) at 4 °C overnight, then washed several times in PBT, incubated twice in an AP-buffer (1× alkaline phosphatase buffer, 0.1 Tween 20) and twice in an AP-buffer with MgCl2. RNA transcripts were visualized with a color reaction buffer (1× AP-buffer, 7.5 % polyvinylalcohol, 2 % NBT/BCIP (#11681451001, Roche)); the reaction was stopped with PBT washing steps and then larvae were post-fixed in 4 % PFA at 4 °C overnight. Larvae were cleared in 1:1 benzylalcohol:benzylbenzoate mixture and mounted on glass slides.

Stained larvae were documented with a Nikon Eclipse E800 microscope and a Nikon Fi2-U3 camera. Additionally, larvae were scanned with a Leica DMI6000 CFS confocal laser scanning microscope equipped with a Leica TCS SP5 II scanning system. Specific NBT/BCIP stainings were detected with 633 nm laser wavelength reflection scanning [63]. Confocal stacks were edited using IMARIS 7.3.1 (Bitplane AG) and figure plates were assembled using Coral Graphic Suite X3 (Corel Corporation).

Declarations

Acknowledgements

We thank Daniel J. Jackson (University of Göttingen, Germany) for helpful hybridization protocols and advice. This work was funded by the German Science foundation (Deutsche Forschungsgemeinschaft, DFG; project FR 3392/1-1 to MF) and the ASSEMBLE program (Grant Agreement n°227799 to MF). We gratefully acknowledge support by the Faculty of Life Sciences, University of Vienna and the staff of the Biological Station de Roscoff (France) for logistic support. André Luiz de Oliveira gratefully acknowledges the financial support of CAPES and the Brazilian program Science without Borders (CsF – project 6090/13-3).

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Integrative Zoology, Faculty of Life Sciences, University of Vienna

References

  1. Lewis EB. A gene complex controlling segmentation in Drosophila. Nature. 1978;276:565–70.View ArticlePubMedGoogle Scholar
  2. Scott MP, Tamkun JW, Hartzell III GW. The structure and function of the homeodomain. Biochem Biophys Acta. 1989;989:25–48.PubMedGoogle Scholar
  3. McGinnis W, Krumlauf R. Homeobox genes and axial patterning. Cell. 1992;68:283–302.View ArticlePubMedGoogle Scholar
  4. Caroll SB. Homeotic genes and the evolution of arthropods and chordates. Nature. 1995;376:479–85.View ArticleGoogle Scholar
  5. Ferrier DE, Holland PW. Ancient origin of the Hox gene cluster. Nat Rev Genet. 2001;2:33–8.View ArticlePubMedGoogle Scholar
  6. Wray GA, Hahn MW, Abouheif E, Balhoff JP, Pizer M, Rockman MV, et al. The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol. 2003;20:1377–419.View ArticlePubMedGoogle Scholar
  7. Garcia-Fernàndez J. Hox, ParaHox, ProtoHox: facts and guesses. Heredity (Edinb). 2005;94:145–52.View ArticleGoogle Scholar
  8. Wang BB, Müller-Immergluck MM, Austin J, Robinson NT, Chisholm A, Kenyon C. A homeotic gene cluster patterns the anteroposterior body axis of C. elegans. Cell. 1993;74:29–42.View ArticlePubMedGoogle Scholar
  9. Orii H, Kato K, Umesono Y, Sakurai T, Agata K, Watanabe K. The planarian Hom/Hoxhomeobox genes (Plox) expressed along the anteroposterior axis. Dev Biol. 1999;210:456–68.View ArticlePubMedGoogle Scholar
  10. Averof M, Akam M. Hox genes and the diversification of insect and crustacean body plans. Nature. 1995;376:420–3.View ArticlePubMedGoogle Scholar
  11. Averof M, Patel NH. Crustacean appendage evolution associated with changes in Hox gene expression. Nature. 1997;388:682–6.View ArticlePubMedGoogle Scholar
  12. Prince VE, Price a L, Ho RK. Hox gene expression reveals regionalization along the anteroposterior axis of the zebra fish notochord. Dev Genes Evol. 1998;208:517–22.View ArticlePubMedGoogle Scholar
  13. Peterson MD, Rogers BT, Popadić a, Kaufman TC. The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect. Dev Genes Evol. 1999;209:77–90.View ArticlePubMedGoogle Scholar
  14. Hughes CL, Kaufman TC. Hox genes and the evolution of the arthropod body plan. Evol Dev. 2002;499:459–99.View ArticleGoogle Scholar
  15. Lowe CJ, Wu M, Salic A, Evans L, Lander E, Stange-Thomann N, et al. Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell. 2003;113:853–65.View ArticlePubMedGoogle Scholar
  16. Irvine SQ, Martindale MQ. Expression patterns of anterior Hox genes in the polychaete Chaetopterus: correlation with morphological boundaries. Dev Biol. 2000;217:333–51.View ArticlePubMedGoogle Scholar
  17. Kulakova M, Bakalenko N, Novikova E, Cook CE, Eliseeva E, Steinmetz PRH, et al. Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa). Dev Genes Evol. 2007;217:39–54.View ArticlePubMedGoogle Scholar
  18. Bakalenko NI, Novikova EL, Nesterenko AY, Kulakova M a. Hox gene expression during post larval development of the polychaete Alitta virens. EvoDevo. 2013;4:13.View ArticlePubMed CentralPubMedGoogle Scholar
  19. Kocot KM, Cannon JT, Todt C, Citarella MR, Kohn AB, Meyer A, et al. Phylogenomics reveals deep molluscan relationships. Nature. 2011;477:452–6.View ArticlePubMed CentralPubMedGoogle Scholar
  20. Smith SA, Wilson NG, Goetz FE, Feehery C, Andrade SCS, Rouse GW, et al. Resolving the evolutionary relationships of molluscs with phylogenomic tools. Nature. 2011;480:364–7.View ArticlePubMedGoogle Scholar
  21. Sutton MD, Briggs DEG, Siveter DJ, Siveter DJ, Sigwart JD. A Silurian armoureda placophoran and implications for molluscan phylogeny. Nature. 2012;490:94–7.View ArticlePubMedGoogle Scholar
  22. Osca D, Irisarri I, Todt C, Grande C, Zardoya R. The complete mitochondrial genome of Scutopus ventrolineatus (Mollusca: Chaetodermomorpha) supports the Aculifera hypothesis. BMC Evol Biol. 2014;14:197.PubMed CentralPubMedGoogle Scholar
  23. Scherholz M, Redl E, Wollesen T, Todt C, Wanninger A. Aplacophoran mollusks evolved from ancestors with polyplacophoran-like features. Curr Biol. 2013;23:2130–4.View ArticlePubMed CentralPubMedGoogle Scholar
  24. Giusti AF, Hinman VF, Degnan SM, Degnan BM, Morse DE. Expression of a Scr/Hox5 gene in the larval central nervous system of the gastropod Haliotis, a non-segmented spiralian lophotrochozoan. Evol Dev. 2000;2:294–302.View ArticlePubMedGoogle Scholar
  25. Callaerts P, Lee PN, Hartmann B, Farfan C, Choy DWY, Ikeo K, et al. HOX genes in the sepiolid squid Euprymna scolopes: implications for the evolution of complex body plans. Proc Natl Acad Sci U S A. 2002;99:2088–93.View ArticlePubMed CentralPubMedGoogle Scholar
  26. Hinman VF, O’Brien EK, Richards GS, Degnan BM. Expression of anterior Hox genes during larval development of the gastropod Haliotis asinina. Evol Dev. 2003;5:508–21.View ArticlePubMedGoogle Scholar
  27. Lee PN, Callaerts P, de Couet HG, Martindale MQ. Cephalopod Hox genes and the origin of morphological novelties. Nature. 2003;424:1061–5.View ArticlePubMedGoogle Scholar
  28. Canapa A, Biscotti MA, Olmo E, Barucca M. Isolation of Hox and ParaHox genes in the bivalve Pecten maximus. Gene. 2005;348:83–8.View ArticlePubMedGoogle Scholar
  29. Pérez-Parallé ML, Carpintero P, Pazos AJ, Abad M, Sánchez JL. The Hox gene cluster in the bivalve mollusk Mytilus galloprovincialis. Biochem Genet. 2005;43:417–24.View ArticlePubMedGoogle Scholar
  30. Iijima M, Akiba N, Sarashina I, Kuratani S, Endo K. Evolution of Hox genes in molluscs: a comparison among seven morphologically diverse classes. J Molluscan Stud. 2006;72:259–66.View ArticleGoogle Scholar
  31. Pernice M, Deutsch JS, Andouche A, Boucher-Rodoni R, Bonnaud L. Unexpected variation of Hox genes’ homeodomains in cephalopods. Mol Phylogenet Evol. 2006;40:872–9.View ArticlePubMedGoogle Scholar
  32. Biscotti MA, Canapa A, Olmo E. Hox genes in the Antarctic polyplacophoran Nuttallochiton mirandus. J ExpZool. 2007;308B:507–13.Google Scholar
  33. Samadi L, Steiner G. Involvement of Hox genes in shell morphogenesis in the encapsulated development of a top shell gastropod (Gibbula varia L.). Dev Genes Evol. 2009;219:523–30.View ArticlePubMedGoogle Scholar
  34. Samadi L, Steiner G. Expression of Hox genes during the larval development of the snail, Gibbula varia (L.) - further evidence of non-colinearity in molluscs. Dev Genes Evol. 2010;220:161–72.View ArticlePubMedGoogle Scholar
  35. Samadi L, Steiner G. Conservation of ParaHox genes’ function in patterning of the digestive tract of the marine gastropod Gibulla varia. BMC Dev Biol. 2010;10:74.View ArticlePubMed CentralPubMedGoogle Scholar
  36. Simakov O, Marletaz F, Cho S-J, Edsinger-Gonzales E, Havlak P, Hellsten U, et al. Insights into bilaterian evolution from three spiralian genomes. Nature. 2013;498:526–31.Google Scholar
  37. Biscotti MA, Canapa A, Forconi M, Barucca M. Hox and ParaHox genes: a review on molluscs. Genesis. 2014;52:935–45.View ArticlePubMedGoogle Scholar
  38. Focareta L, Sesso S, Cole AG. Characterization of homeobox genes reveals sophisticated regionalization of the central nervous system in the European cuttlefish Sepia officinalis. PLoS One. 2014;9:10.View ArticleGoogle Scholar
  39. Ponder WF, Lindberg DR. Phylogeny and Evolution of the Mollusca. Berkely, Los Angeles, London: University of California Press; 2008.View ArticleGoogle Scholar
  40. Faller S, Rothe BH, Todt C, Schmidt-Rhaesa A, Loesel R. Comparative neuroanatomy of Caudofoveata, Solenogastres, Polyplacophora, and Scaphopoda (Mollusca) and its phylogenetic implications. Zoomorphology. 2012;131:149–70.View ArticleGoogle Scholar
  41. Wanninger A, Haszprunar G. Chiton myogenesis: perspectives for the development and evolution of larval and adult muscle systems in molluscs. J Morphol. 2002;251:103–13.View ArticlePubMedGoogle Scholar
  42. Friedrich S, Wanninger A, Brückner M, Haszprunar G. Neurogenesis in the mossy chiton, Mopalia muscosa (Gould) (Polyplacophora): evidence against molluscan metamerism. J Morphol. 2002;253:109–17.View ArticlePubMedGoogle Scholar
  43. Voronezhskaya EE, Tyurin SA, Nezlin LP. Neuronal development in larval chiton Ischnochiton hakodadensis (Mollusca: Polyplacophora). J Comp Neurol. 2002;38:25–38.View ArticleGoogle Scholar
  44. Henry JQ, Okusu A, Martindale MQ. The cell lineage of the polyplacophoran, Chaetopleura apiculata: variation in the spiralian program and implications for molluscan evolution. Dev Biol. 2004;272:145–60.View ArticlePubMedGoogle Scholar
  45. Nardelli-Haefliger D, Bruce AEE, Shankland M. An axial domain of HOM/Hox gene expression is formed by morphogenetic alignment of independently specified cell lineages in the leech Helobdella. Development. 1994;120:1839–49.PubMedGoogle Scholar
  46. Kourakis MJ, Master V a, Lokhorst DK, Nardelli-Haefliger D, Wedeen CJ, Martindale MQ, et al. Conserved anterior boundaries of Hox gene expression in the central nervous system of the leech Helobdella. Dev Biol. 1997;190:284–300.View ArticlePubMedGoogle Scholar
  47. Coates MI, Cohn MJ. Fins, limbs and tails: outgrowths and axial patterning in vertebrate evolution. Bio Essays. 1998;20:371–81.Google Scholar
  48. Dictus WJAG, Damen P. Cell-lineage and clonal-contribution map of the trochophore larva of Patella vulgata (Mollusca). Mech Dev. 1997;62:213–26.View ArticlePubMedGoogle Scholar
  49. Nowicki JL, Burke AC. Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm. Development. 2000;127:4265–75.PubMedGoogle Scholar
  50. Balavoine G, de Rosa R, Adoutte A. Hox clusters and bilaterian phylogeny. Mol Phylogenet Evol. 2002;24:366–73.View ArticlePubMedGoogle Scholar
  51. Fröbius AC, Matus DQ, Seaver EC. Genomic organization and expression demonstrate spatial and temporal Hox gene colinearity in the lophotrochozoan Capitella sp. I. PLoS One. 2008;3:e4004.View ArticlePubMed CentralPubMedGoogle Scholar
  52. Hejnol A, Martindale MQ. Coordinated spatial and temporal expression of Hox genes during embryogenesis in the acoel Convolutriloba longifissura. BMC Biol. 2009;7:65.View ArticlePubMed CentralPubMedGoogle Scholar
  53. Steinmetz PRH, Kostyuchenko RP, Fischer A, Arendt D. The segmental pattern of otx, gbx, and Hox genes in the annelid Platynereis dumerilii. Evol Dev. 2011;13:72–9.View ArticlePubMedGoogle Scholar
  54. Holland LZ, Carvalho JE, Escriva H, Laudet V, Schubert M, Shimeld SM, et al. Evolution of bilaterian central nervous systems: a single origin? Evo Devo. 2013;4:1–27.Google Scholar
  55. Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, et al. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature. 2008;452:745–9.View ArticlePubMedGoogle Scholar
  56. Riesgo A, Andrade SCS, Sharma PP, Novo M, Pérez-Porro AR, Vahtera V, et al. Comparative description of ten transcriptomes of newly sequenced invertebrates and efficiency estimation of genomic sampling in non-model taxa. Front Zool. 2012;9:33.View ArticlePubMed CentralPubMedGoogle Scholar
  57. Weigert A, Helm C, Meyer M, Nickel B, Arendt D, Hausdorf B, et al. Illuminating the base of the annelid tree using transcriptomics. Mol Biol Evol. 2014;31:1391–401.View ArticlePubMedGoogle Scholar
  58. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol. 2011;15:644–52.View ArticleGoogle Scholar
  59. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421.View ArticlePubMed CentralPubMedGoogle Scholar
  60. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:189–1191.View ArticleGoogle Scholar
  61. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61:539–42.View ArticlePubMed CentralPubMedGoogle Scholar
  62. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8:275–82.PubMedGoogle Scholar
  63. Jékely G, Arendt D. Cellular resolution expression profiling using confocal detection of NBT/BCIP precipitate by reflection microscopy. Biotechniques. 2007;42:751–5.View ArticlePubMedGoogle Scholar

Copyright

© Fritsch et al. 2015

Advertisement