Conservation of ParaHox genes' function in patterning of the digestive tract of the marine gastropod Gibbula varia
© Samadi and Steiner; licensee BioMed Central Ltd. 2010
Received: 17 February 2010
Accepted: 12 July 2010
Published: 12 July 2010
Presence of all three ParaHox genes has been described in deuterostomes and lophotrochozoans, but to date one of these three genes, Xlox has not been reported from any ecdysozoan taxa and both Xlox and Gsx are absent in nematodes. There is evidence that the ParaHox genes were ancestrally a single chromosomal cluster. Colinear expression of the ParaHox genes in anterior, middle, and posterior tissues of several species studied so far suggest that these genes may be responsible for axial patterning of the digestive tract. So far, there are no data on expression of these genes in molluscs.
We isolated the complete coding sequences of the three Gibbula varia ParaHox genes, and then tested their expression in larval and postlarval development. In Gibbula varia, the ParaHox genes participate in patterning of the digestive tract and are expressed in some cells of the neuroectoderm. The expression of these genes coincides with the gradual formation of the gut in the larva. Gva-Gsx patterns potential neural precursors of cerebral ganglia as well as of the apical sensory organ. During larval development this gene is involved in the formation of the mouth and during postlarval development it is expressed in the precursor cells involved in secretion of the radula, the odontoblasts. Gva-Xolx and Gva-Cdx are involved in gut patterning in the middle and posterior parts of digestive tract, respectively. Both genes are expressed in some ventral neuroectodermal cells; however the expression of Gva-Cdx fades in later larval stages while the expression of Gva-Xolx in these cells persists.
In Gibbula varia the ParaHox genes are expressed during anterior-posterior patterning of the digestive system. This colinearity is not easy to spot during early larval stages because the differentiated endothelial cells within the yolk permanently migrate to their destinations in the gut. After torsion, Gsx patterns the mouth and foregut, Xlox the midgut gland or digestive gland, and Cdx the hindgut. ParaHox genes of Gibbula are also expressed during specification of cerebral and ventral neuroectodermal cells. Our results provide additional support for the ancestral complexity of Gsx expression and its ancestral role in mouth patterning in protostomes, which was secondarily lost or simplified in some species.
The three ParaHox genes, Gsx, Xlox, and Cdx, were first described as a gene cluster in the invertebrate chordate Branchiostoma floridae (amphioxus) by the elegant work of Brooke et al. 1998 . ParaHox and Hox genes are believed to have evolved from a single ancient proto-Hox cluster composed of two to four genes prior to the divergence of cnidarians and bilaterians. Thus, they are considered evolutionary sister (or paralogue) clusters [1–7]. Vectorial expression of the ParaHox genes in anterior, middle, and posterior tissues of amphioxus and its distinct similarities to vertebrate ParaHox gene expression suggest that these genes may be responsible for axial patterning of the digestive tract [1, 3].
Expression of ParaHoxgenes in deuterostomes
ParaHox gene expression and genomic organisation have been studied extensively in deuterostomes. In Vertebrates, Gsh1 and Gsh2 genes are restricted to the central nervous system (CNS) [8–13]. Vertebrate Xlox is expressed both in CNS and the developing gut [14–20]. Cdx1 to Cdx4 genes of vertebrates are involved in posterior patterning, since they are expressed in posterior parts of CNS and gut [21–24]. Within invertebrate deuterostomes, apart from amphioxus, expression of ParaHox genes has been traced in the ascidian, Ciona intestinalis, the echinoderm, Strongylocentrotus purpuratus, and in a starfish, Archaster typicus [25–29]. In invertebrate deuterostomes, Gsx is expressed more anteriorly and only in the nervous system, while Xlox and Cdx are expressed within the gut primordium with Xlox anterior to Cdx [25–28]. In the sea star, however, the Aty-Xlox expression is found in the archenteron as well as in ectodermal cells near the vegetal region of early and mid-gastrula stages . This expression pattern is very different from those of Xlox homologues in other deuterostomes.
Expression of ParaHoxgenes in ecdysozoans
In ecdysozoans, Gsx expression has been documented in the insects Drosophila and Tribolium [30, 31]. Insect Gsx (called ind) is expressed along a pair of medio-lateral neural columns and promotes neural precursor formation in the medial and intermediate columns of the CNS [30, 31]. The central ParaHox gene, Xlox, is lost in all insect genomes sequenced to date. Caudal has been known as a posterior patterning gene in several arthropods during segmentation [32–42]. Cdx is also a posterior patterning gene in the nematode Caenorhabditis elegans. Here, this gene is called pal-1 and patterns the precursor cells of alae and rays in the posterior of the worm . Gsx and Xlox orthologs are absent in the nematode .
Expression of ParaHoxgenes in lophotrochozoans
Within Lophotrochozoa, expression of the full complement of ParaHox genes has been described in the polychaetes Capitella teleta, Nereis virens, and Platynereis dumerilii [45–48]. In Capitella, Gsx is not expressed in the gut but in some neuroectoderm cells of the anterior brain . This is very different from the expression of Gsx in the nereid polychaetes, Nereis virens and Platynereis dumerilii [46, 48]. Nereid Gsx is first expressed in symmetrical bilateral domains in the dorso-medial episphere of the trochophore [46, 48]. Later this gene is expressed during formation of the midgut and the posterior foregut in both nereids [46, 48]. Xlox is expressed throughout the midgut in the polychaete Capitella . This is also true for the Xlox genes, named Lox3 in the leeches Helobdella triserialis and Hirudo medicinalis [49, 50]. Expression of Xlox is not reported in the nervous system of these annelids [45, 49, 50]. Nereid Xlox is also expressed in the midgut, but in contrast to Capitella and the leeches its expression is additionally detected in the CNS [46, 48]. As in arthropods, Cdx is a posterior patterning gene in the annelids. However in Platynereis, Nereis, Tubifex, and Capitella there are both anterior and posterior expression domains of Cdx [45–48, 51]. Capitella Cdx is expressed in the cerebral ganglia, Nvi-Cdx expression is detectable in the ventral nervous system, while Pdu-Cdx is not detected in the nervous system [45–48]. Expression of Cdx is also detected in more posterior parts of the gut [45–48]. Moreover, Cdx is expressed in the posterior ectodermal cells that form the pygidium epidermis of both nereids [46–48]. Additionally, Cdx expression can be traced in mesodermal cells in Capitella, Tubifex, and Platynereis [45, 47, 48, 51].
Little is known of ParaHox genes in other Lophotrochozoa than annelids [52–55]. The only available data are on the Cdx gene expression during the early development of the marine limpet, Patella vulgata . Pvu-Cdx is expressed at the onset of gastrulation in the ectodermal cells at the posterior edge of the blastopore and in the paired mesentoblasts . During trochophore larval stage, PvuCdx is expressed in the posterior neurectoderm of the larva, as well as in part of the mesoderm . Within Mollusca, a full complement of ParaHox genes has been shown for the chiton Nuttallochiton mirandus and the scallop Pecten maximus [54, 55]. However the information is limited to partial homeobox sequences, whereas expression patterns of Gsx and Xlox or chromosomal organisation of ParaHox genes have not been reported yet in any mollusc species.
Ancestral role of ParaHoxgenes
Holland (2001) elaborated the hypothesis of the ancestral role of ParaHox genes proposed by the original work of Brooke et al. 1998 [1, 3]. Holland's hypothesis proposes that the three ParaHox genes originated from the Proto Hox gene cluster and pattern anterior, middle and posterior gut regions in a colinear manner in basal animals . According to this hypothesis, a link of Gsx and anterior gut development existed in basal animals. However, Gsx is not expressed in the anterior gut of deuterostomes. This is explained by the loss of the primary mouth and formation of a secondary mouth in deuterostomes .
Gastropoda is undoubtedly the most successful taxon of the Mollusca, embracing more than 80% of all mollusc species . The vetigastropod Gibbula varia L. is a shallow subtidal top shell snail with encapsulated development. The lecithotrophic larval development is completed within the eggs. The juveniles leave the gelatinous egg masses only after metamorphosis. In order to elucidate the function of the ParaHox genes in molluscs and to gain broader insights into the evolution of the ParaHox genes in the Lophotrochozoa, we describe the sequences as well as expression patterns for all three ParaHox orthologues by whole mount in situ hybridization from embryonic through juvenile stages in the top shell Gibbula varia. This is the first report of expression patterns of the full ParaHox complement in a mollusc.
Gibbula varialife history
Timing of developmental stages of Gibbula varia (at 22°C); different stages of larval development and metamorphosis of G. varia inside the gelatinous egg capsules before hatching.
Name of stage and approximate time of development (hpf)
Brief description of main features
Early Trochophore Larva (12 hpf)
The pretrochal cells are smaller than the posttrochal cells; prototroch starts to form by cilliation of trochoblasts; shell gland starts to evaginate; foot rudiment and stomodaeum are not completely formed.
Late Trochophore Larva (18-24 hpf)
The larva comprises a prototroch, shell field surrounded by mantle edge, a pedal rudiment, and stomodaeum.
Pretorsional veliger larva (36-48 hpf)
The mantle and mantle cavity form. The larva has a velum, apical organ marked by apical cilia, mouth opening, and pedal rudiment with anlage of operculum.
Post-torsional veliger larva (60 hpf)
The mantle lies over the back of the head and the velum gradually splits ventrally, the operculum apears.
Metamorphotic (competence) stage (72 hpf)
Eye rudiments and cephalic tentacles begin to form in the prevelar area. The anlage of the radula becomes visible.
Velum is completely lost; eyes and cerebral tentacles are formed.
Hatchling (96 hpf)
The encapsulated juvenile hatches and shell mineralization begins.
Development of gut in G. varia
The development of the digestive tract starts with the development of the stomodeum (future mouth opening) in the trochophore (additional file 1, Figure S1E). The mouth opens during the pretorsional veliger stage (additional file 1, Figure S1H, S1J) whereas the anus opens in the late posttorsional stage at the site of a few ciliated cells (anal markers). The development of the digestive tract is very similar to that described in G. cineraria and Haliotis tuberculata [57, 58]. The digestive gland begins to differentiate on the left side of the veliger just before torsion sets in [57, 58]. The gut develops from differentiated endodermal cells initially scattered within the yolk in the pretorsional veliger. They later migrate to the yolk boundaries to form the definitive midgut in the posttorsional veliger [57, 58]. Later, the hindgut develops from actively dividing cells of the digestive gland migrating to their final positions in the intestine [57, 58]. The competent larva's digestive system comprises a mouth opening and a bipartite oesophagus (the anterior part immediately behind the buccal cavity is not effected by torsion, the mid oesophagus includes a portion affected by the torsion), a stomach with the digestive gland, the hindgut leading to the anus that opens into the mantle cavity over the back of the head (additional file 1, Figure S2A and S2B). The radula anlage is a ventral differentiation of the foregut where mesenchym cells aggregate. The radula teeth become visible in the competent larva at the distal end of the radula sheath (additional file 1, Figure S2A and S2B).
ParaHoxgene expression in the trochophore larva
The expression pattern of Gva-Gsx is rather dynamic. The first signs of transcripts of Gva-Gsx are already detected at 12 hpf in early trochophore larvae, when a pair of intensive, bilateral expression domains appears in the dorso-medial episphere (Figure 2B). When viewed from the anterior, each pair of expression domains appears to be composed of 4-5 Gva-Gsx-positive cells, presumably in the area of future cerebral ganglia (Figure 2C). This pattern of expression continues in 18 hpf trochophores (Figure 2D and 2E). Here, the pattern of expression becomes considerably more complex. In addition to the paired expression domains in the dorso-medial episphere, Gva-Gsx transcripts can now be detected in a pair of cells at the tip of the developing apical sensory organ (Figure 2D and 2E). These two Gva-Gsx-positive cells at the tip of the apical organ do not bear any cilia or apical tuft in the trochophore stage of G. varia (Figure 2F). The expression of Gva-Gsx in the apical sensory organ is restricted to two groups consisting of three sensory cells (Figure 2G). Beside the expression in prospective neural or sensory tissues, Gva-Gsx transcripts are also detected around the stomodeum where they appear for the first time in trochophore 18 hpf in two intensely stained bilateral semicircular clusters located anteriorly at the sides of the mouth and a less intensely stained semicircular domain at the posterior part of the mouth (Figure 2D and 2H). Figures 2H and 2I show the trochophore stomodaeum and Gva-Gsx expression around it at 18 hpf. About 24 hpf, Gva-Gsx is expressed in a complete circle around the stomodeum (Figure 2J) and in three episphere domains: a pair of adjacent cells at the tip of the apical sensory organ, and two pairs of cell groups dorsolaterally marking presumptive sites of future cephalic neuroectodermal differentiation (Figure 2K).
Gva-Xlox transcription begins later than Gva-Gsx expression. No expression is detectable until 24 hpf when Gva-Xlox transcripts appear in a group of cells located ventrally in the hyposphere and in a pair of symmetrical expression domains in the medio-ventral episphere of the trochophore larva (Figure 2L and 2M). These symmetrical expression areas are located ventrally of the more intensely stained Gva-Gsx expression domains in the pretrochal area. Gva-Xlox is also expressed in the hyposphere in 8-9 cells forming a semicircle around the anal marker (Figures 2N and 2O). These weakly stained Gva-Xlox-positive cells are probably part of ventral neuroectoderm.
Gva-Cdx transcripts are first detected in the early trochophore larva (12 hpf). It is expressed at 12 and 18 hpf in two domains in the ventral vegetal plate: one in an area of presumptive posterior neuroectoderm, the other in a bilateral pair of cells in the interior of the larva (Figure 2P and 2Q). Using Patella vulgata as a reference, the latter expression of Pvu-Cdx probably marks the left and right primary mesentoblasts (green arrows in Figure 2P and 2Q). Gva-Cdx-positive neuroectodermal cells are first observed as a patch of cells expressing this gene in varying intensities (Figure 2P). Gradually they migrate to the boundary of the expression area (Figure 2Q) so that they from a circle of Gva-Cdx-expressing cells around the anal marker at 24 hpf (Figure 2R and 2S). The expression of Gva-Cdx around the anal marker at 24 hpf partly overlaps with the expression of Gva-Xlox in the ventral area at this stage, which is visible as a semicircle located ventrally around the anal marker (Figure 2N and 2R).
ParaHoxgene expression in the pretorsional veliger larva
Expression of ParaHoxgenes in veliger and competent larvae
Post-larval ParaHoxgene expression
Is ParaHoxgene expression colinear during patterning of gut?
It has been proposed that the origin of the three germ layered animals, the Bilateria, is associated with the innovation of several gene clusters of the ANTP family, with the Hox-cluster genes participating mainly in patterning of the neuroectoderm, the NK-cluster genes in formation of the mesodermal layers, and ParaHox in colinear regionalisation of the endoderm [1, 3]. Of the animals studied to date, the chromosomal linkage of ParaHox genes has been shown only in amphioxus, mouse, and human [1, 3]. The ParaHox genes are not linked in teleost fishes, the ascidian or the sea urchin [27, 28, 59]. The only description of the expression patterns of all three ParaHox genes for lophotrochozoans in relation to their genomic organisation is for the polychaete P. dumerilii . Here, Gsx and Xlox are clustered and Cdx is separated, without clear evidence of colinear expression.
There also seems to be a temporal colinearity in expression of ParaHox genes in the gradual formation of the digestive system. In the trochophore larva, development of the digestive system begins with the formation of the stomodeum involving Gva-Gsx expression only (Figure 6). Gva-Xlox and Gva-Cdx are expressed at later stages in the more posterior parts of the gut. When the patterning of the gut is completed in the hatchling, expressions of Gva-Xlox and Gva-Cdx cease while Gva-Gsx continues to be involved in the patterning of the radula (Figure 6). During postlarval development, Gva-Gsx is expressed in the paired odontoblastic cushions of Gibbula (Figure 5). The gradient of Gva-Gsx expression from posterior to anterior in the odontoblastic cushions suggests that this gene is associated with mitotic features of these cells and their ability to divide and replace the odontoblasts, rather than direct involvement in secretion of radula teeth.
Expression of ParaHoxgenes in cephalic neural and neurosensory cells
Gastropod larvae are well provisioned with multicellular sensory structures, but only the apical sensory organ is typically present in both plankton-feeding and non-plankton-feeding veligers . This suggests that information detected by the apical sensory organ is important during the entire larval stage, regardless of the length of larval life or capacity for feeding. Moreover, the apical sensory organ disappears at metamorphosis in species where this has been studied . Therefore the apical sensory organ has functions restricted to the larval stage. During larval development in Gibbula, Gva-Gsx exhibits a complex pattern of expression in potential cephalic neural cells and in the apical organ. This pattern shows distinct similarity to Pdu-Gsx expression in the trochophore stage in which Pdu-Gsx expression is detectable in flask-shaped sensory-neurosecretory cells in the medial forebrain . Prior to torsion, the Gva-Gsx pattern is spotted in the paired apical tufts and several neurosecretory cells or sensory cups of the apical organ (Figure 3 and 6). This gene also appears to be involved in the formation of parts of the cerebral ganglia from the apical sensory organ in competent larvae. This compares well to the polychaetes Capitella, Nereis, and Platynereis, where Gsx is expressed in the cerebral ganglia [45, 46, 48]. Our results may lend further support to the theory of complex ancestral expression of Gsx that was secondarily simplified in several lineages. In addition to Gva-Gsx expression in the dorsal episphere of the trochophore, Gva-Xlox is detected in a pair of expression domains located more ventrally. It is possible that these cells contribute to neural cells of future cerebral ganglia. However this pattern of expression is transient and is lost in later developmental stages.
Possible expression of ParaHoxgenes in the trunk neuroectoderm
Expression of ParaHox genes in ventral or dorsal neuroectoderm has been demonstrated in several species. Within Lophotrochozoa Capl-Cdx is expressed in posterior neuroectodermal cells in the polychaete Capitella. In Platynereis, Pdu-Gsx is expressed in a central part of the larval ventral neuroectoderm in which somatic serotonergic neurons are identified [45, 48]. Nereis is the only species studied so far in which all three ParaHox genes are known to be involved in patterning of the trunk neuroectoderm . In Gibbula, Gva-Xlox and Gva-Cdx are expressed around the anal marker in the trochophore larvae. It has been shown that these cells express SoxB in the prospective neuroectoderm of the trunk in Patella . Therefore, it is likely that these cells expressing Gva-Xlox and Gva-Cdx contribute to the trunk neuroectoderm. Temporary expression of Gva-Cdx in ventral neuroectoderm earlier during development, and expression of Gva-Xlox in overlapping regions at a later stage (Figure 6), may suggest that Gva-Cdx contributes to patterning of ventral neuroectoderm upstream of Gva-Xlox.
Hypothetical ancestral ParaHoxgene expression
Comparative analyses across the animal kingdom show conservation of ParaHox gene expression domains in distinct tissues. Comparing Platynereis ParaHox gene expression to that of the orthologues in deuterostomes and ecdysozoans, Hui et al. 2009 confirmed Holland's hypothesis about the ancestral role of ParaHox genes, suggesting that the pattern of Gsx expression in the protostome-deuterostome ancestor was complex, with Gsx domains in several structures of the nervous system, and was secondarily reduced to small patches of expression in the anterior CNS in several lineages . Holland's model further suggests that Gsx was expressed in the mouth region of the last bilaterian ancestor . Lack of Gsx expression in the anterior gut of deuterostomes is explained by loss of the primary mouth and evolution of a new secondary mouth . If this be the case, protostomes should maintain Gsx expression in anterior gut structures. Capitella results do not support such a model since CapI-Gsx expression is limited to a restricted region of the forming brain. The expression of Nvi-Gsh, Pdu-Gsx, and Gva-Gsx described here provides further support to the ancestral mouth patterning role of Gsx [46, 48].
Xlox is expressed during midgut development in annelids [45, 46, 48–50]. Pdu-Xlox and Nvi-Xlox are also expressed in the nervous system. In Gibbula, Gva-Xlox pattern is detected in the digestive gland and ventral neuroectoderm, and expression in potential cephalic nerve cells is transient. Therefore, our results provide additional support that the expression of Xlox may reflect an ancestral function in central regions of the gut as well as a role in the nervous system. If this hypothesis is true, however, it would once more imply secondary simplification and loss of neural Xlox expression in several lineages . However, the possibility that ancestral Xlox expression was simple and has become more complicated in different lineages cannot be ruled out since Xlox is expressed in ventral neuroectoderm in Nereis and Gibbula, in addition to cerebral ganglia, but is lacking in all other protostomes studied to date .
Cdx shows a complex, dynamic pattern of expression in cells of the ectoderm, endoderm and possibly mesoderm, extending to extremely anterior regions in all annelids studied so far [45–48, 51]. This anterior expression of Cdx was also recently described in the acoel flatworm, Convolutriloba longifissura . ClCdx is expressed in the commissures posterior to the statocyst, following the paths of nerve tracks and extending anteriorly. ClCdx is also expressed in an area surrounding the eyes, forming direct connections to the brain commissure . Cdx anterior expression seems to be the case in the limpet Patella as well, in which the gene is expressed in posterior ectoderm during gastrulation. The posterior ectodermal expression starts to fade in the trochophore, while expression extends anteriorly in the shape of an incomplete equatorial ring of ectodermal cells that corresponds to some cells of the prototroch . Later in the young free swimming trochophore, Pvu-Cdx expression in the prototroch disappears. The gene is also transiently expressed in the stomodeum . Gva-Cdx expression differs from that of Pvu-Cdx by being absent during gastrulation. In addition, we did not detect any sign of Gva-Cdx expression in the trochophore prototroch or stomodeum. In contrast, the detection of Cdx in mesentoblasts and in ectodermal cells situated on the posterior most part of the ventral side of the trochophore is a common feature in Gibbula and Patella. These are some of the cells that also express SoxB, a neurectodermal marker . Therefore, Cdx seems to pattern the ventral neuroectoderm as well as mesentoblasts in gastropods. Anterior expression of Cdx was not observed during the larval development of Gibbula at any stage. This can be either interpreted as secondary loss of the anterior function of Cdx in Gibbula, or as a gain of function for this gene in several tissues in other species. The first possibility has been favoured since it can be explained by the separation of the gene from the cluster . Nonetheless, variety in the pattern of expression of Cdx in different animals can serve as another example for the plasticity of gene expression during evolution. Whether the expression of the ParaHox genes in nervous systems is related to their function in the gut, i.e. innervation of different parts of the gut and/or to feeding behaviour, awaits future research. Gene function experiments, therefore, would be desirable to give us better understanding of how these genes are employed.
The expression of ParaHox genes during anterior-posterior development of the digestive system (with Gsx patterning the mouth and foregut, Xlox patterning the midgut or digestive gland, and Cdx patterning the hindgut) suggests that these genes are involved in anterior-posterior specification of the G. varia gut. Our results support Holland's hypothesis that ParaHox genes are involved in gut regionalization and offer further support to the ancestral mouth patterning role of Gsx in protostomes. All three ParaHox genes of G. varia are involved in patterning of the nervous system. Gva-Gsx and Gva-Xlox are expressed in neural precursors of cerebral ganglia, the expression domain of these two genes does not coincide in the episphere and fades away in the case of Gva-Xlox in later larval stages. Additionally, Gva-Gsx patterns the neurosensory cells of the apical organ. Gva-Xlox and Gva-Cdx pattern the ventral neuroectoderm with Cdx possibly acting upstream of Xlox. During postlarval development, Gva-Gsx transcripts are detected in the precursor cells of odontoblasts at the base of the radula sac. This is probably a molluscan novelty related to radula evolution. Further research in other molluscan classes and use of experimental tools, e.g. RNAi, are required to improve our understanding of gene functions and enable a sound reconstruction of their ancestral role.
The adults of Gibbula varia (L.) were collected in Crete, Greece and cultured in 150-200 liter aquariums in artificial sea water at 22°C (salinity 28°). Copulation was induced by lowering the salinity a few degrees by adding fresh water to the aquariums at 17°C (personal observation of Achim Meyer, The Johannes Gutenberg University of Mainz).
Cloning of ParaHoxgenes
DNA extraction was performed using the PeqGOLD Tissue DNA kit (PEQLAB Biotechnologie GmbH, Polling, Austria) according to the manufacturer's instructions. Homeobox fragments of ParaHox genes were obtained by polymerase chain reaction (PCR) from genomic DNA using Hox degenerate primers described previously [64, 65]. These primers produce PCR amplification products that are mixtures of different fragments containing homeobox. The PCR fragments were purified using peqGOLD MicroSpin Cycle-Pure Kit (PEQLAB Biotechnologie GmbH, Polling, Austria). Purified PCR products were cloned with the TOPO TA Cloning Kit (Invitrogen GmbH, Karlsruhe, Germany). In total 255 clones were sequenced and all eleven Hox genes (Samadi and Steiner, unpublished data) and the three ParaHox genes were recovered. RNA was extracted from blastula and gastrula stages, trochophore, veliger, and competent larvae, and encapsulated juveniles using RNeasy Mini Kit (QIAGEN Vertriebs GmbH, Vienna, Austria). The cDNA from each developmental stage was synthesized using SuperScript® III reverse transcriptase (Invitrogen GmbH, Karlsruhe, Germany). The homeobox fragments were used to design primers for rapid amplification of cDNA ends (RACE). The RACE was performed with modifications according to Schramm et al. 2000 . For further details on RACE protocol see supplementary data of . The RACE products were cloned by the Topo-TA cloning kit (Invitrogen GmbH, Karlsruhe, Germany) and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and run on an ABI 3130xl DNA analyser automated capillary sequencer.
Orthology assignment and phylogenetic analyses
The initial orthology of the ParaHox genes was tested by searching against GenBank non-redundant protein databases using the BlastX algorithm. The genes were named Gva-Gsx, Gva-Xlox, and Gva-Cdx and deposited in GenBank under accession numbers HM136802, HM136803, HM136804, respectively. Orthology assignment of the genes was made based on phylogenetic analysis. The phylogenetic analyses were carried out using amino acid sequences. We compiled a ParaHox gene alignment including representatives of bilaterians. Sequences were aligned using the program ClustalX v.2.0.10. First the homeobox region was aligned, then, using the homeobox as an anchor, the flanking regions were aligned and subsequent trimming carried out manually. Bayesian inference on amino acid data using MrBayes version 3.1.1 was applied for orthology analysis, with 2 × 4 Markov chains under the Jones amino acid substitution model . Chains were run for five million generations with a sampling frequency of 1000 generations and the burnin set to 5000 generations.
Whole-mount in situ hybridization
The Maxiscript T7 and SP6 RNA polymerase kit (Ambion, Austin, USA) was used to synthesize the sense and anti-sense probes that were labelled by the Dig RNA labelling kit (Roche Molecular Biochemicals, Vienna, Austria). WMISH was performed with few modifications after Lespinet et al. 2002 . DIG-labelled riboprobes were detected colourimetrically with NBT/BCIP substrates. The details of modifications can be found in . For WMISH, embryos were mounted in 70% glycerol and the expression patterns were documented. For serial-sectioned in situ hybridization, embryos were embedded in Epoxy resin after in situ hybridization according to the standard protocols, and sectioned with a microtome at a thickness of 2 μm. Sections were stained with Eosin using standard histological protocols.
Larvae were fixed in 4% paraformaldehyde (PFA) in 0.1 M saline phosphate buffer (PBS) for 4 h at room temperature or overnight at 4°C, washed three times for 15 min in PBS containing 0.1% sodium azide (NaN3), postfixed in osmium tetroxide (1% in distilled water for 2 h at room temperature), followed by three washes in distilled water, and dehydrated in a graded ethanol/acetone series. Drying was performed either by critical point dryer or chemical drying with HMDS (Hexamethyldisilazane). After drying, the samples were mounted on scanning electron microscopy (SEM) stubs, sputter-coated with gold, and observed with a LEO 1430VP scanning electron microscope.
central nervous system
hours post fertilization
nitro blue tetrazolium chloride
open reading frame
rapid amplification of cDNA ends
scanning electron micrograph
whole-mount in situ hybridization.
The authors are grateful to two anonymous reviewers for their helpful comments. LS was supported by an EC fellowship within the MOLMORPH network under the 6th Framework Programme "Marie Curie Fellowships for Early Stage Research Training (EST) (Contract number MEST-CT-2005 - 020542). We are greatly indebted to Mariti Steiner for the considerable improvement of the English.
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