- Research article
- Open Access
ParaHox gene expression in larval and postlarval development of the polychaete Nereis virens(Annelida, Lophotrochozoa)
© Kulakova et al; licensee BioMed Central Ltd. 2008
- Received: 10 December 2007
- Accepted: 29 May 2008
- Published: 29 May 2008
Transcription factors that encode ANTP-class homeobox genes play crucial roles in determining the body plan organization and specification of different organs and tissues in bilaterian animals. The three-gene ParaHox family descends from an ancestral gene cluster that existed before the evolution of the Bilateria. All three ParaHox genes are reported from deuterostomes and lophotrochozoans, but not to date from any ecdysozoan taxa, and there is evidence that the ParaHox genes, like the related Hox genes, were ancestrally a single chromosomal cluster. However, unlike the Hox genes, there is as yet no strong evidence that the ParaHox genes are expressed in spatial and temporal order during embryogenesis.
We isolated fragments of the three Nereis virens ParaHox genes, then used these as probes for whole-mount in situ hybridization in larval and postlarval worms. In Nereis virens the ParaHox genes participate in antero-posterior patterning of ectodermal and endodermal regions of the digestive tract and are expressed in some cells in the segment ganglia. The expression of these genes occurs in larval development in accordance with the position of these cells along the main body axis and in postlarval development in accordance with the position of cells in ganglia along the antero-posterior axis of each segment. In none of these tissues does expression of the three ParaHox genes follow the rule of temporal collinearity.
In Nereis virens the ParaHox genes are expressed during antero-posterior patterning of the digestive system (ectodermal foregut and hindgut, and endodermal midgut) of Nereis virens. These genes are also expressed during axial specification of ventral neuroectodermal cell domains, where the expression domains of each gene are re-iterated in each neuromere except for the first parapodial segment. These expression domains are probably predetermined and may be directed on the antero-posterior axis by the Hox genes, whose expression starts much earlier during embryogenesis. Our results support the hypothesis that the ParaHox genes are involved in antero-posterior patterning of the developing embryo, but they do not support the notion that these genes function only in the patterning of endodermal tissues.
- Expression Domain
- ParaHox Gene
- Trochophore Stage
- ParaHox Cluster
- Main Body Axis
Transcription factors that encode ANTP-class homeobox genes; NK, Rhox, Irx, and in particular the Hox and ParaHox genes, play crucial roles in determining body plan organization and specification of different organs and tissues of bilaterian animals [1–7]. The Hox and ParaHox genes are believed to descend from a cluster of two to four genes that duplicated before the divergence of the Cnidaria and the Bilateria [5, 8–13]: both sets of genes are ancestrally clustered on the genome, and individual genes within one cluster are paralogous with genes in the other cluster (but see  for a dissenting hypothesis).
Within the Bilateria Hox genes function to determine vectorial regionalization of the body along the antero-posterior axis and to specify body parts within this regionalization [1, 2, 4, 15–18]. The Hox genes are also characterized by a high degree of structural and functional conservation: they are often (and ancestrally) found as a single chromosomal cluster, and are expressed in canonical spatial and temporal modes during embryogenesis.
The ParaHox genes are also believed to have originated as an organized chromosomal cluster, but the evidence for spatially and temporally collinear expression in these genes is not as strong, partially due to lack of data [8, 12, 19, 20]. The three ParaHox genes; Gsx, Xlox, and Cdx, were first described as a paralogs to the Hox cluster in amphioxus (Branchiostoma floridae) by Brooke et al. . A full complement of ParaHox genes has been shown for a number of other deuterostomes: the sea urchin Strongylocentrotus purpuratus , the hemichordate Ptychodera flava , the ascidian Ciona intestinalis , and a number of mammals, as well as for some lophotrochozoans; the sipunculids Phascolion strombus and Nephasoma minuta , the polychaete annelid Capitella sp. I , the clitellate annelid Perionyx excavatus , and the chiton Nuttallochiton mirandus . However the full set of ParaHox genes has not yet been found in any ecdysozoan taxon, including two taxa for which complete genomes are available: the fruit fly Drosophila melanogaster has no Xlox ortholog [26, 27] while the nematode Caenorhabitis elegans has neither Gsh nor Xlox orthologs .
Of the organisms studied to date the linkage has been shown only in amphioxus, mouse, and human. The ParaHox genes are not linked in teleost fishes [7, 8], the ascidian C. intestinalis, or the sea urchin S. purpuratus [21, 23].
Brooke et al.  reported collinear expression of the ParaHox genes in anterior, middle, and posterior tissues of amphioxus, and suggested that these genes may be responsible for axial patterning in the digestive tract. This pattern may be ancestral for the ParaHox cluster [4, 8, 10]. Collinear expression of two ParaHox genes in the digestive tract has been described for other deuterostomes: mouse and human [29, 30], ascidians [31–34], and sea urchins . In all cases Xlox (= Pdx) and Cdx expression domains were found in the central and posterior parts of the gut, respectively. The anterior ParaHox gene, however, is not expressed in the anterior of the gut, but rather in the brains of these deuterostomes. Holland  suggested that Gsx may have lost its function in the anterior gut as a result of changes in the patterning mechanism of the position of the mouth in deuterostomes. If this is the case then all three genes may still participate in regionalization of the gut in the protostomes (Lophotrochozoa and Ecdysozoa) [4, 10].
Fröbius and Seaver  looked at expression of the ParaHox genes in a lophotrochozoan, the polychaete annelid Capitella sp. I, and found that Gsx is not expressed in the gut. This result does not support Holland's hypothesis that ParaHox genes are involved in gut regionalization in protostomes. However, Capitella sp. I is a single taxon, not basal within the annelids, and may have lost Gsx function independently of the deuterostomes.
In order to better understand the function of the ParaHox genes in the Lophotrochozoa we examined their expression in the polychaete annelid Nereis virens. Within the annelids Capitella sp. I and N. virens are evolutionarily remote [35, 36] so N. virens represents an opportunity to understand, or at least make hypotheses, regarding conservation of ParaHox expression among the polychaetes. In addition, errant nereids are believed to retain many ancestral characteristics of the annelids, including life history, developmental and genome characteristics, homonomy of segmentation, a simple reproductive system, gradual metamorphosis, absence of highly specialized larval structures, and high fecundity [37–40], so ParaHox expression in N. virens may be more likely to represent the ancestral state.
We have cloned the full complement of ParaHox genes from the polychaete Nereis virens: Nvi-Gsh, Nvi-Xlox, and Nvi-Cad, and report larval and postlarval expression patterns for these genes in this study.
Nereis virenslife history
Timing of developmental stages in the larvae of Nereis virens (at 10.5°C)
Stage name and time boundaries (hours after fertilization)
Brief description of main features
ET, early trochophore, 44–62
trochoblasts ciliated; stomodaeum not formed
MT, middle trochophore, 63–85
larva perfectly spherical; stomodaeum fully formed; stomodaeum lies close to the further anal region; somatic plate weakly developed
LT, late trochophore 86–105
hyposphere slightly elongated, at posterior ciliated telotroch is formed; stomodaeum and further anal region lie more widely apart from each other; chaetal sacs become morphologically apparent
EM, early metatrochophore 106–122
larva starts to show external metamery; mesotrochs starts to form in posterior part of each segment; chaetae begin to develop in two anterior pairs of chaetal sacs, but do not protrude from the larval body yet
MM, middle metatrochophore 123–152
chaetae of the two anterior pairs of chaetal sacs protrude from the larval body, chaetae of the third chaetal sac start to form, segmental boundaries become distinct
LM, late metatrochophore 153–180
larval body gradually elongates; parapodial anlagen and pygidial (anal) lobe start to form
N, nectochaete 181–390
functional parapodia; distinct head with some head appendages (two antennae and two peristomial cirri); digestive tube completely form and larvae start to eat
juvenile worm 16–17 days of development
fourth trunk segment (first postlarval) begins to form
Expression of N. virens ParaHoxgenes in the prostomium
The prostomium of polychaetes is derived from the episphere of the trochophore. The cells of the episphere form from the first quartet of micromeres and include the head ectoderm, the cerebral ganglion, the larval and definitive eyes, the antennae, the palps, and other sensory organs of the head [41, 43–45]. We have observed the expression of two Nvi-ParaHox genes, Nvi-Gsh and Nvi-Xlox, in the prostomium of N. virens.
The first detectable expression of Nvi-Xlox is at the late trochophore stage when a pair of weak and transitory domains occurs in the lateral part of the episphere. These symmetrical zones do not coincide with the Gsh expression domains (Fig. 1g). Much later, at the nectochaete stage, Nvi-Xlox expression resumes in a pair of lobes in the brain (Fig. 1h, Fig. 4i). This disappears at the juvenile worm stage.
Expression of N. virens ParaHoxgenes during ventral nervous system formation
The ventral (somatic or vegetative) plate of polychaetes is derived from the 2d blastomere (the first somatoblast), which originates in the second quartet micromere. The descendants of this cell (the ectoteloblasts of Wilson ) intensively proliferate in the dorsal region of the embryo during gastrulation and at the trochophore stage. The lateral domains of the somatic plate become wider, move towards the ventral side and join each other between the future mouth and anus to form the ventral midline. This process leads to gradual movement of the mouth under the prototroch away from the vegetative pole that marks the position of the future anus. The vegetative pole position is stable during all of larval development. The ventral regions of the somatic plate form the neuroectoderm of the ventral neural cord and probably also the parapodial ganglia. The lateral regions of the somatic plate form the chaetae sacs, and ectoderm of the larval segments as well as the neurons of the peripheral nervous system . The larval trunk of N. virens consists of four segments – one peristomial and three parapodial. The peristomial segment has no chaetal sacs and probably no neuromere , but it forms two pairs of peristomial cirri with a pair of pedal ganglia .
All three N. virens ParaHox genes are expressed in the ventral neuroectodermal cells. The activation of these genes occurs at the late trochophore/early metatrochophore stage in several symmetrical pairs of cells. The external morphological markers of segments are absent at this stage; hence the position of ParaHox-positive cells cannot be determined with precision. However we can compare the expression domains of Nvi-ParaHox genes and some Nvi-Hox genes. We have previously described Nvi-Hox1, Nvi-Hox4, and Nvi-Lox5 expression domains during early developmental stages, and these can be use as positional markers of the first, second, and third parapodial segments respectively .
During later developmental stages morphological markers of segments (mesotrochs) appear in the posterior of each segment. The position of ParaHox-positive cells can easily be compared with these markers. Note that the boundaries of segments and neuromeres in the adult worm do not coincide: the anterior boundary of the neuromeres is shifted ahead one third of a segment length . This offset probably also exists for larval segments and forming neuromeres. If this is so then the anterior part of the neuromere lies exactly under the mesotroch of the preceding segment and its posterior part is at the level of its own segment mesotroch (Fig. 7).
Expression of Nvi-Gshduring formation of the ventral nervous system
Middle stage metatrochophore larvae have well developed mesotrochs in the posterior part of each segment. At this stage Nvi-Gsh positive cells occur in the posterior part of the first and second parapodial segments parallel to or slightly posterior to the mesotroch (Fig. 2c). The Nvi-Gsh positive cells are thus localized in the anterior of the forming second and third neuromeres, and Nvi-Gsh positive zones are closer to the midline than previously as a result of rearrangement of the ventral plate cells. Nvi-Gsh expression in the neuromere of the first segment is no longer detected at this time (Fig. 2c). During the course of metatrochophore development the domains of Nvi-Gsh expression become wider in the second and third neuromere (Fig. 2d, e). Occasionally, single Nvi-Gsh positive cells can be found more laterally (not shown). At the late metatrochophore and early nectochaete stages the level of Nvi-Gsh expression gradually decreases. Still later the expression completely disappears in the second and then also in the third neuromeres (Fig. 2f, g). By the beginning of formation of the first postlarval segment Nvi-Gsh expression can be detected again in the posterior part of the third larval segment (Fig. 2h). This can be considered the expression in the first postlarval neuromere.
Expression of Nvi-Xloxduring formation of the ventral nervous system
Expression of Nvi-Xlox begins in the neuroectoderm at the early metatrochophore stage (Fig. 2j, 112 h). Low levels of expression can first be detected in several bilaterally symmetrical surface pairs of cells at the level of the first and second parapodial segments (Fig. 2j, k). Later Nvi-Xlox is also expressed in the third parapodial segment (Fig. 2l). Nvi-Xlox expression domains in parapodial segments become wider and stronger at middle and late metatrochophore stages. Weaker and short-term expression can also be seen in the peristomial segment at these stages (not shown). In all segments the expression domains are localized anterior to the mesotrochs and thus are in the central region of the neuromeres. In some larvae Nvi-Xlox positive cells also occur in the lateral regions of segments (Fig. 2l). This is analogous to expression of Nvi-Gsh at the same stages. Intensive expression of Nvi-Xlox is maintained for much longer than that of Nvi-Gsh (Fig. 2n, o). Nvi-Xlox expression gradually diminishes during the nectochaete stage (Fig. 2o), and is not detected in the neuroectoderm of the late nectochaete (Fig. 2p).
During postlarval segmentation Nvi-Xlox expression occurs in the forming neuromeres of newly formed segments. This surface expression can be seen in the anterior half of segments below segmental borders (Fig. 3c, d). These segments already have parapodial anlagen and cuticular furrows near nephridial pores. Expression gradually decreases in older segments that have chaetae and more developed parapodia. The youngest newly formed segments show no Nvi-Xlox expression.
Expression of Nvi-Cadduring formation of the ventral nervous system
In the late trochophore stage pairs of cells in the ventral neuroectoderm express Nvi-Cad at the position where the second and third parapodial segments will be formed (Fig. 2q, 100 h). At this time the neuroectoderm consists of one or two cell layers. The duration of neuroectodermal Nvi-Cad expression is shorter compared to that of Nvi-Gsh and Nvi-Xlox: Nvi-Cad expression is not detected after 112–115 h of development (Fig. 2s, u–x). However at the middle trochophore stage weak surface expression was visible in the third segment in a single larva from among 200–300 analyzed (Fig. 2t). In these rare larvae the position of the Nvi-Cad expression domain can be localized because morphological markers are already formed and expression patterns of all three Nvi-ParaHox genes in the third forming neuromere can be compared at this stage. Using this marker we can see that Nvi-Cad is expressed at the level of the third parapodial segment mesotroch, Nvi-Xlox is expressed immediately anterior of this region, and Nvi-Gsh is in turn expressed in cells just anterior to the Nvi-Xlox expression domain (Fig. 2d, l, t). At later stages Nvi-Cad is not expressed in the larval neuroectoderm.
Expression of Nvi-Cad resumes during postlarval development in newly formed segments, where pairs of Nvi-Cad positive cells appear on the surface of the new segment. Expression levels are very low and the Nvi-Cad positive cells are better detected in TO-PRO-1 stained larvae. Bilaterally symmetrical Nvi-Cad positive cell groups are situated close to the center, or slightly to the posterior, of each segment, i.e. in the posterior part of the neuromere (Fig. 3e). We observed this expression pattern in only a few animals, and think it likely that this expression is transient. We found that new segments are formed faster during the regeneration of posterior parts of the growing worm. In this case the frequency of animals expressing Nvi-Cad is higher and transcripts can be detected more easily (Fig. 3f).
To summarize: along the zone of newly formed segments short-term and weak expression of Nvi-Cad occurs in a few segments, expression of Nvi-Gsh is stronger and occurs in older segments, and Nvi-Xlox expression occurs in more developed segments and lasts longer than that of Cad or Gsh (Fig. 3). Thus, the spatio-temporal dynamics of postlarval Nvi-ParaHox expression are very similar to those of larval stages (Fig. 2; Fig. 3). Moreover the antero-posterior positions of Nvi-ParaHox positive cells in each postlarval segment are also very similar to the pattern of these genes in the second and third larval segments (Fig. 2d, l, t; Fig. 3).
Expression of N. virens ParaHoxgenes during formation of the digestive tract
Overview of gut formation
The digestive tract of N. virens is not complete until late in the nectochaete stage. Prior to this the larvae are lecithotrophic.
The foregut of polychaetes is derived from stomatoblasts [41, 47]. During blastopore closure, the stomatoblasts of Nereis virens divide rapidly and form first the stomodaeal arch, and then the stomodaeal plate. The stomodaeal arch transforms later into the mouth, the stomodaeal plate forms the stomodaeal cavity or stomodeum by invagination. Later several sections are formed in the stomodeum: the buccal capsule, pharynx, and esophagus; the latter is connected with two large esophageal glands.
The hindgut (proctodaeum) is very short and connects the anus and the midgut. Anlagen of the hindgut, pygidium, and growth zone are determined during the trochophore stage from different parts of the somatic plate. These three zones are located near each other at the vegetative pole and are surrounded by the telotroch.
Expression of Nvi-Gshduring formation of foregut and midgut
By the late trochophore stage Nvi-Gsh expression is activated in wide lateral domains of the forming stomodeum (Fig. 2a; Fig. 4a). The intensity of expression rises until the early metatrochophore stage, when these zones become wider (Fig. 2b; Fig. 4b). During the metatrochophore and nectochaete stages the Nvi-Gsh expression level gradually decreases (Fig. 2d, e; Fig. 4c, d). Expression in the stomodeum completely disappears in later nectochaete stages (Fig. 4e).
Expression of Nvi-Xloxduring midgut formation
The first endodermal cells can be seen at the middle trochophore stage (80–85 h). They form small cell groups inside the vegetative and animal zones of larvae. Weak Nvi-Xlox expression was found only in the vegetative zone 20 hours later at the late trochophore/early metatrochophore stage (Fig. 4f, g). During the metatrochophore and nectochaete stages this gene is expressed only in the posterior part of the forming midgut (Fig. 4h, i).
As postlarval stages begin Nvi-Xlox expression becomes stronger throughout the midgut, and is strongest in the central and posterior part of the intestine except at the most posterior end (Fig. 4j). This pattern resembles the future postlarval one. During postlarval development in the anterior half of the intestine an antero-posterior expressional gradient forms; in the posterior half there is a double gradient in which expression is highest in the center of the domain and decreases towards each end (Fig. 5c, d). In the most posterior part of the gut separate single Nvi-Xlox positive cells can be detected (Fig. 5d).
Expression of Nvi-Cadin posterior regions of the digestive gut and pygidium
During almost the whole of embryogenesis and larval development Nvi-Cad is intensively expressed in the ectodermal anlagen of the pygidial lobes, proctodaeum, and growth zone. These zones are surrounded by the telotroch. The pygidial anlage is situated near the ventral side and this domain is characterized by the earliest Nvi-Cad expression (40 h). Much later (100–105 h) the expression of Nvi-Post2 (an Nvi-Hox cluster posterior gene) occurs in the same zone . The proctodael anlage is near the dorsal side of the embryo and does not express Nvi-Post2. The invagination of the proctodaeum starts during the late trochophore stage and coincides with intensive expression of Nvi-Cad in this zone (Fig. 4k, l). At the end of the metatrochophore stage the process of midgut formation is ongoing, the midgut joins with the proctodaeum, and Nvi-Cad transcription is activated in midgut cells (Fig. 4m). Nvi-Cad expression in the posterior part of the midgut and in the proctodaeum is maintained and intensified during the nectochaete stage (Fig. 4n).
In juvenile worms and during postlarval development Nvi-Cad is expressed in the same zones (Fig. 4o; Fig. 5e). In the posterior part of the gut an Nvi-Cad expression gradient forms with highest expression most posteriorly. This is the reverse of the gradient of Nvi-Xlox expression (Fig. 4j, o; Fig. 5c).
Holland  and Garcia-Fernandez  have suggested that the origin of the Bilateria, with three germ layers, was associated with the origin of several gene clusters in the ANTP family: the Hox-cluster genes participated mainly in regionalization of the neuroectoderm, the NK-cluster genes took part in patterning the mesodermal layers, and ParaHox patterned the endoderm. However, previous to our work on N. virens, no bilaterians are known in which all three ParaHox genes participate in patterning of the gut.
In deuterostomes the patterning of the digestive system is clearly different from the ancestral state since the mouth forms in a different position in deuterostome embryos, thus changes in the function of genes controlling development of the gut are not unexpected. In deuterostomes all three ParaHox genes are found, but Gsx is not, or is no longer, expressed in the anterior of the gut .
All three ParaHox genes have been found in a number of lophotrochozoan taxa [20, 24, 25, 50], but expression of the full set has to date been described only for the polychaete Capitella sp. I . In this species Gsx is also not expressed in the gut, but is, as in many other bilaterians, associated with brain development.
Are ParaHoxgenes expressed ancestrally in the brain?
Gsx is expressed in the brain during development of all bilaterians studied to date, including N. virens. Nvi-Gsx is activated early, during the middle trochophore stage, in dorso-lateral regions of the head neuroectoderm. Expression of this gene is intensive, prolonged, and dynamic (Fig. 1). Maps of presumptive brain anlagen for a related species, Platynereis dumerilii  can be correlated with development in N. virens and allow us to locate Nvi-Gsx expression domains with optic areas and possibly with regions of prospective antennae. In another polychaete, Capitella sp. I, CapI-Gsx is also expressed in the head neuroectoderm, and is indeed the only expression domain for this gene in that species .
In D. melanogaster the Gsx homologue, ind, is expressed in a subset of brain neuroblasts . Ci-gsx in the ascidian Ciona intestinalis was found in the sensory vesicle, which is thought to be homologous to the vertebrate forebrain and midbrain . AmphiGsx is expressed in the cerebral vesicle in amphioxus . Gsh1 and Gsh2 of the mouse take part in patterning of the telencephalon, particularly in the lateral ganglionic eminence and the olfactory bulb [52–54]. Gsh-1 of the mouse and Ol-Gsh1 of the medaka, Oryzias latipes, have similar expression patterns in the optic tectum, dorsal diencephalon, hypothalamus, and rostral telencephalon [55, 56]. Thus Gsx expression has been observed in the brain of all three major branches of the Bilateria.
The Gsx homologue anthox2 in Nematostella vectensis (Anthozoa), which is a basal representative of Cnidaria, is expressed around the oral pole in scattered ectodermal cells that are probably neural cells [14, 57]. In another diploblast, the coral Acropora millepora, the Gsx homologue cnox-2Am is also expressed in ectodermal (probably neural) cells of the oral region . Thus in Bilateria and Cnidaria Gsx is expressed in neural cells, and may have an ancient role in patterning the neural system of the ancestral bilaterian [14, 57, 58].
We also observed that very weak and transitory Nvi-Xlox expression occurs in the episphere of the trochophore and in the cerebral ganglion of the N. virens nectochaete. These early expression domains do not correlate with any identified brain anlagen or with zones of Nvi-Gsh expression (Fig. 1).
In the polychaete Capitella sp.I Cap-Xlox expression is not detected in head neuroectoderm or any other derivatives of the brain . The Xlox homologue of the rat, IDX1/IPF is expressed in several areas of the developing brain, including the cortex, ganglionic eminence, hypothalamus, and inferior colliculus [59, 60]; and Ci-IPF1, the Xlox homologue in the acsidian Ciona intestinalis, is expressed in the larval sensory vesicle .
Nvi-Cad is not expressed in the brain of Nereis virens at any studied developmental stage. In Capitella sp.I CapI-Cdx expression was detected in multiple domains of the developing larvae. CapI-Gsh and CapI-Cad areco-expressed in neuroectoderm during the early stages of brain development .
In sum, ParaHox genes are expressed in the brains of all studied bilaterian animals but in none of them are all three genes expressed. Expression in the head neuroectoderm is always found for Gsx and is probably the ancestral characteristic for the last common bilaterian and cnidarian ancestor. The two other ParaHox genes are not always expressed in the head neuroectoderm: Xlox is expressed in a few studied species, and Cdx in only one, the polychaete Capitella sp. I; we cannot at present make any hypotheses regarding their ancestral expression patterns in the brain, and await further data.
Expression of ParaHoxgenes in the trunk neuroectoderm
Expression of all three ParaHox genes in ventral, or dorsal, neuroectoderm has not been demonstrated in any studied species. Mice, humans, and probably Xenopus as well, have only one ParaHox cluster, Gsh1, Ipf1, (= Pdx1, = Xlox), and Cdx2; as well as two additional orphan Cdx genes (Cdx1 and Cdx4) and one orphan Gsx gene (Gsh2) [7, 61]. Gsh-1 of the mouse (and Ol-Gsh1 of the medaka fish) is expressed in the hindbrain [55, 56]. It has been shown that Gsh1 as well as Gsh2 take part in dorso-ventral patterning [27, 62]. The Gsx homologue in D. melanogaster, ind, also takes part in dorsoventral specification of the nerve cord . IDX1/IPF1 (Xlox) of the rat is also expressed in the hindbrain at embryonic day 15 .
All three mouse Cdx genes are expressed in the posterior of the neural tube, and their expression domains overlap considerably [63, 64]. In zebrafish there is no Cdx expression in the progenitor cells of the hindbrain, but loss of function of Cdx1 and Cdx4 or gain of function of Cdx4 result in displacement of the hindbrain-spinal cord boundary (i.e. a homeotic transformation). These genes may determine axial regionalisation in the trunk neural cord .
In amphioxus AmphiGsh is not expressed in the trunk neuroectoderm, AmphiXlox is expressed in pigment cells, and AmphiCdx in the most posterior region of the neural cord . Ci-IPF1 (Xlox) of Ciona intestinalis is expressed in the visceral ganglia, which are thought to be homologous to the vertebrate hindbrain/spinal cord region . In another ascidian, Herdmania curvata, Hec-Cdx is expressed in the posterior of the neural tube .
All three ParaHox genes are found in the polychaete Capitella but only one gene, Capl-Cdx, is expressed in posterior neuroectodermal cells . Platynereis dumerilii (a polychaete) also has a full set of ParaHox genes (our unpublished data for Pdu-Xlox; ref.  for Pdu-Gsh; and ref.  for Pdu-Cad)). Pdu-Gsx is expressed in a central part of the larval ventral neuroectoderm (Pdu-nk2.2 positive domain) in which somatic serotonergic motoneurons are identified and differentiated. Pdu-Gsx is also expressed at late stages of neuroectoderm formation and is associated with cell differentiation. An ordered disposition of different zones along the apicobasal axis has been shown for P. dumerilii: there is a zone of proliferation (more apically), a progenitor zone of postmitotic cells in which neuronal identity genes are expressed, and a zone of differentiation (more basally) . Platynereis dumerilii and Nereis virens develop similarly and show identical patterns of Hox gene expression . In N. virens ParaHox positive cells are situated in the zone of proliferation and in the progenitor zone (Fig. 2), which we suggest indicates that these genes take part in cell division and/or cell patterning processes. The expression of the ParaHox genes in order along the antero-posterior axis also suggests that these genes have a patterning function in N. virens.
Axial pattern of Nvi-ParaHoxgene expression in the forming neuromeres
To date, the polychaete Nereis virens is the only studied animal in which all the ParaHox genes are expressed in the ventral neural system. Nvi-ParaHox gene expression is so far unique in being metameric: the expression patterns of each ParaHox gene are found in repeating cell groups of each larval and postlarval segment, but at different times during development. Exceptions are the peristomial and the first parapodial segments of the larva, where expression of Nvi-Cad is not detected (Fig. 2; Fig. 3). These particular segments will be included in the head of the adult worm, and their ganglia will differ morphologically and probably functionally from the rest of the neuromeres.
During larval development
By contrast, the Nvi-ParaHox genes do not exhibit temporal collinearity of expression. Nvi-Cad is activated in the neuroectoderm a little earlier than Nvi-Gsh or simultaneously with it, and Nvi-Xlox is activated later than the two other genes (Fig. 2). Interestingly, larval Nvi-Hox genes also lack temporal collinearity of expression: the posterior gene Nvi-Post2 is expressed earlier then the genes of the central group: Nvi-Hox7, Nvi-Lox4, and Nvi-Lox2, and Nvi-Hox3 is expressed before Nvi-Hox1 .
During postlarval development
All Nvi-ParaHox genes are expressed in each segment, but in different cell groups of the forming neuromere and at different times during segment morphogenesis. In every neuromere the spatial domains of Nvi-ParaHox expression are shifted relative to each other and specify different groups of neurons along the antero-posterior axis in apparent accord with the linear position of the genes in the Nvi-ParaHox cluster  (Fig. 7). In this case, however, collinearity can be seen in every neuromere but not in the whole trunk.
Nvi-Cad demonstrates the earliest and most transient expression in the two or three youngest postlarval segments. Expression of Nvi-Gsh and Nvi-Xlox is activated in older segments, but there are more Nvi-Xlox expressing segments than Nvi-Gsh ones, suggesting that Nvi-Xlox expression is longer lasting than that of Nvi-Gsh. We did not observe simultaneous expression of all three ParaHox genes in the same postlarval segment. During postlarval development a dynamic morphogenetic system is formed that includes an active growth zone and many segments at various stages of maturity. The difference in time of activation and down regulation of different ParaHox genes leads to full segmental separation of their expression domains.
Axial patterning of ParaHoxgene expression in the digestive system
During larval development Nvi-Gsh is first expressed in the foregut, where it is later downregulated and then expressed in basal cells of the midgut (Fig. 4; Fig. 8). During postlarval development the number of basal cells increases towards the posterior end of the midgut and the expression pattern looks like a gradient. We will abbreviate this type of antero-posterior gradient as a-P. Nvi-Gsh expression in the epithelial layer of the posterior domain is a two sloped gradient p-P-p (Fig. 5; Fig. 8). During larval development Nvi-Xlox marks the posterior endodermal cells of the forming midgut, and in postlarval development the expression forms an A-a-p-P-p pattern along the intestine (Fig. 4; Fig. 5; Fig. 8). Nvi-Cad is expressed in the forming proctodaeum before and after its invagination. Later this gene is also expressed in the posterior of the larval midgut and at this time a p-P expression gradient in the midgut starts to form. This pattern is retained during postlarval development (Fig. 4; Fig. 5; Fig. 8).
During postlarval development, then, the spatial expression pattern of Nvi-ParaHox genes in the digestive system does not substantially change. As the worm becomes longer the length of the digestive tract is also increased and along it all the established gradients are maintained (Fig. 5; Fig. 8). During this period the function of the Nvi-ParaHox genes may be related to the maintainence of regional specificity (i) and cell differentiation (ii) along the antero-posterior axis of the gut.
(i) The very rapid reorganization of the Nvi-Cad endodermal expression pattern during regeneration (Fig. 6) points to a regional patterning function of this gene. We detected upregulation of this gene in the most posterior part of the bisected worm as soon as 4 h after amputation. This is not enough time for dedifferentiation of old cell types and the differentiation of new ones: we suggest that this is evidence that new positional information is created in old differentiated cells.
(ii) It is possible that Nvi-ParaHox genes take part in cell differentiation. The gradient distribution of different cell types along the intestine  probably coincides with the gradient expressional patterns of Nvi-ParaHox genes (Fig. 5). For example, the number of secretory and absorptive cells in the epithelial layer gradually decreases in the posterior part of the gut . This is correlated with the gradual diminishing of Nvi-Gsh and Nvi-Xlox expression and very high expression levels of Nvi-Cad in the posterior of the gut; the number of basal cells gradually increases in the posterior of the intestine, and is correlated with the p-P gradient of Nvi-Cad and a-P gradient of Nvi-Gsh in basal cells of the intestine (Fig. 5; Fig. 8).
These expression patterns are similar to patterns of Pdx-1 (Xlox) and Cdx-2 along the small intestine of the adult mouse. Fang et al.  showed that these genes form two opposite and overlapping expression gradients in this part of the gut, and that the transcription of some digestive enzymes is activated by Cdx-2 and repressed by Pdx-1. Thus two overlapping gradients of upstream regulators take part in functional regionalization of the small intestine .
During mouse embryogenesis Pdx-1 also takes part in the patterning of the pancreas and the anterior part of the duodenum. Pdx-1 inactivation results in atrophy of the pancreas and defects in the anterior part of the duodenum . Inactivation of Cdx-2 results in homeotic transformation: stomach tissue is induced in the midgut and hindgut . Moreover Cdx2 is essential for axial elongation in the mouse . Interestingly, all ParaHox genes are expressed in the pancreas of the adult mouse .
During development in two deuterostomes; amphioxus and the sea urchin, only two ParaHox genes, Xlox and Cdx, take part in patterning and specification of different regions of the digestive tract [8, 21]. Holland  suggested that this function has been lost for Gsh because the position of the mouth has changed in this branch of the Bilateria. Our results, showing that all three ParaHox genes are expressed in a lophotrochozoan, support the hypothesis that Gsh patterning of the gut has been lost in at least some of the deuterostomes.
Fröbius and Seaver  described the expression patterns of the ParaHox genes in the polychaete Capitella sp. I. They did not observe CapI-Gsx expression in the digestive tract, but did see brief expression of CapI-Xlox in the forming midgut and expression of CapI-Cad in the posterior part of the gut. Thus expression of ParaHox genes in Capitella and their expression in N. virens are very different: this is not surprising since the two species are phylogenetically distant from one another [35, 36].
Errant nereids are thought to retain many ancestral polychaete characters [37–40]. If this is also true of gene expression then the involvement of all three ParaHox genes in the development of N. virens may more closely reflect the ancestral use of these genes, thus supporting the idea that the three ParaHox genes, like the Hox genes, were ancestrally involved in antero-posterior patterning during bilaterian development.
In the Bilateria the role of Hox genes in antero-posterior axial regionalization is well established, and the ancestral function of these genes has been in patterning and specification of ectodermal and neuroectodermal anlagen. The ancestral role of the ParaHox genes is less well established; it has been suggested that these genes might have a parallel role in antero-posterior patterning in the endodermal layer of the developing embryo [4, 10].
This hypothesis has been partially supported by studies in deuterostomes and ecdysozoans, but to date there are no taxa from either group for which all three ParaHox genes are expressed in developing endodermal tissues: because one or more ParaHox genes have been either lost or coopted for other functions. However, in the sole lophotrochozoan taxon studied to date, Capitella sp. I, one of three genes (CapI-Gsh) has no function in endodermal patterning. In aggregate, these studies show that all three ParaHox genes are expressed in some endodermal tissues in at least some bilaterians, but in no single taxon are all three genes expressed in endodermal or antero-posterior patterning.
In this paper we describe the expression patterns of the three ParaHox genes in another polychaete, Nereis virens, during formation of the digestive system and the ventral nerve system. We found that all three ParaHox genes are expressed during antero-posterior regionalization of the gut, and that these genes are transcribed along the main body axis in a collinear order that involves both ectodermal (foregut and hindgut) and endodermal (midgut) regions of the digestive tract. During ventral nerve cord formation the ParaHox genes are expressed during specification of some cells in the ganglia of larval and postlarval segments. The well-ordered expression of these genes occurs in larval development in accordance with the position of these cells along the main body axis. During postlarval development these genes are expressed in order in accordance with the position of cells in ganglia along the antero-posterior axis of each segment. In none of these tissues are the three ParaHox genes expressed following the rule of temporal collinearity.
The expression of ParaHox genes during antero-posterior development of the digestive system (ectodermal foregut and hindgut, and endodermal midgut) suggests that these genes are actively involved in antero-posterio specification in the N. virens gut. Similarly, the segmental expression of Nvi-ParaHox genes in each neuromere except in the first parapodial segment, suggests that these genes also take part in axial specification of ventral neuroectodermal cell domains. We hypothesize that these expression domains may be predetermined and directed on the antero-posterior axis by the Hox genes, whose expression starts much earlier during embryogenesis, and along the mediolateral axis by the BMP-regulatory system of the Pax and NK genes [18, 46]. Our results lend support to the hypothesis that the ParaHox genes, like their sister Hox genes, are involved in antero-posterior patterning of the developing embryo, but they do not support the notion that these genes function only in the patterning of endodermal tissues. Confirmation of the function of the ParaHox genes awaits additional descriptive data from other taxa, as well functional studies that manipulate gene expression during development in a lophotrochozoan taxon.
Adult Nereis virens were collected at the Chupa Inlet Marine Biological Station of St. Petersburg State University, on the White Sea. Mature animals were caught with a hand net at the water surface during the spawning period (June and July). Artificial fertilization and cultivation of the embryos were carried out at 10.5°C (Dondua, 1975).
Cloning of short homeobox-containing fragments of Nvi-ParaHox genes
We have previously described the use of degenerate primers complementary to the most conserved motifs in the homeodomain to amplify Hox genes from N. virens [18, 66]. These screens, using forward primers GARCTIGARAARGARTT (1798C), CTIGARCTIGARAARGA (Fbam), YTIGARYTIGARAARGART (AN1); and reverse primers ICGRTTTTGRAACCA (Rxba), CGYTTRTTTTGRAACCA (Rend), and ATTCATICKICKRTTYTGRAACCAIATYT (AN2), produce PCR amplicons that are mixtures of different homeobox-containing fragments. We isolated individual sequences by cloning the PCR products into pBluescript SK+ at the EcoRV restriction site, then growing and sequencing individual clones. These clones included the eleven Hox genes that we have previously reported, and the ParaHox genes that we report herein. It is important to note that each primer pair produces a different suite of amplified genes, so thorough screens of this type benefit from the use of many primer pairs. Of the nine different primer combinations that we used we recovered ParaHox sequences from only four: Nvi-Gsh was obtained with the 1798C/AN2 primer pair, Nvi-Xlox with AN1/Rxba and AN1/AN2, and Nvi-Cad with AN1/AN2 and Fbam/Rend.
The short gene fragments were identified using Wu-BlastX searches against GenBank. Gene identities were confirmed by additional Blast searches after longer sequences became available. The sequences have been deposited in GenBank with accession numbers AY117546, DQ206221, and DQ366677.
Cloning of long Nvi-Gsh, Nvi-Xlox, and Nvi-Cadfragments using inverse PCR and 3' RACE
We used inverse PCR (iPCR) and 3' RACE to extend the ParaHox gene sequences outside of the short fragments obtained using degenerate primers. We have described iPCR in detail elsewhere . Briefly, Nereis virens DNA was digested with the following restriction endonucleases: EcoRI, BamHI, HindIII, and AluI, and the DNA fragments obtained were ligated under conditions that ensured self-ligation, resulting in circularized DNA. This DNA was then used as template for PCR. Pairs of nested primers for each small fragment of ParaHox gene were constructed in such a way that the ends of the amplified iPCR fragment were in the middle of an earlier known sequence. Amplified products were cloned in the T Easy vector (Promega), then sequenced.
For 3' RACE total RNA was isolated from Nereis virens at various stages of development using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. In every case RNA was isolated from about 5000 larvae. Mixed RNA from different larval stages was used as a template for a reverse transcriptase reaction with an oligo-dT-universal primer. Semi-nested PCR was performed using the product of the reverse transcriptase reaction as a template with pairs of gene specific forward primers and a reverse universal primer. Amplified products were cloned in the T Easy vector (Promega) and sequenced. Primers designed using these assembled long sequences were used to generate probes for in situ hybridization by RT-PCR.
Whole-mount in situ hybridization (WMISH)
Samples were fixed with 4% PFA in 1.75×PBS. A detailed protocol is available upon request. Dig-labeled RNA probes were prepared according to the manufacturer's protocol (Roche). Hybridization was carried out at 68°C. BM-purple (Roche) was used as a chromogenic substrate to localize the hybridized probe [18, 48]. The results were imaged on a DMRXA microscope (Leica) with a Leica DC500 digital camera under Nomarsky optics.
The authors thank the group "Chromas," at the Biological Institute of Saint-Petersburg University for allowing the use of core facility equipment. This work was supported by the Russian Foundation for Basic Research Grant no. 06-04-49654-a, by the Biotechnology and Biological Sciences Research Council of the UK grant no. 8G14526, and by a BBSRC Underwood Fund grant to CEC and TFA. Neither funded body had any role in study design; in data analysis, collection, or interpretation; or in the decision to submit the manuscript for publication.
- Akam M: Hox genes and the evolution of diverse body plans. Phil Trans R Soc Lond B. 1995, 349: 313–319-10.1098/rstb.1995.0119.View ArticleGoogle Scholar
- Akam M: Hox genes, homeosis and the evolution of segment identity: no need for hopeless monsters. International Journal of Developmental Biology. 1998, 42: 445-451.PubMedGoogle Scholar
- Slack J, Holland P, Graham C: Development of the zootype: Reply. Nature. 1993, 363: 307-10.1038/363307c0.View ArticleGoogle Scholar
- Garcia-Fernàndez J: The genesis and evolution of homeobox gene clusters. Nat Rev Genet. 2005, 6: 881-892.View ArticlePubMedGoogle Scholar
- Maclean JA, Chen MA, Wayne CM, Bruce SR, Rao M, Meistrich ML, Macleod C, Wilkinson MF: Rhox: a new homeobox gene cluster. Cell. 2005, 120: 369-382. 10.1016/j.cell.2004.12.022.View ArticlePubMedGoogle Scholar
- Holland PW, Takahashi T: The evolution of homeobox genes: Implications for the study of brain development. Brain Research Bulletin. 2005, 66: 484-490. 10.1016/j.brainresbull.2005.06.003.View ArticlePubMedGoogle Scholar
- Mulley JF, Chiu C, Holland PWH: Breakup of a homeobox cluster after genome duplication in teleosts. Proceedings- National Academy of Sciences USA. 2006, 103: 10369-10372. 10.1073/pnas.0600341103.View ArticleGoogle Scholar
- Brooke NM, Garcia-Fernandez J, Holland PW: The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature. 1998, 392: 920-922. 10.1038/31933.View ArticlePubMedGoogle Scholar
- Finnerty JR, Martindale MQ: Ancient origins of axial patterning genes: Hox genes and ParaHox genes in the Cnidaria. Evol Dev. 1999, 1: 16-23. 10.1046/j.1525-142x.1999.99010.x.View ArticlePubMedGoogle Scholar
- Holland PW: Beyond the Hox: how widespread is homeobox gene clustering?. Journal of Anatomy. 2001, 199: 13-24.View ArticlePubMed CentralPubMedGoogle Scholar
- Hill A, Wagner A, Hill M: Hox and paraHox genes from the anthozoan Parazoanthus parasiticus. Molecular Phylogenetics and Evolution. 2003, 28: 529-535. 10.1016/S1055-7903(03)00062-9.View ArticlePubMedGoogle Scholar
- Garcia-Fernàndez J: Hox, ParaHox, ProtoHox: facts and guesses. Heredity. 2005, 94: 145-152. 10.1038/sj.hdy.6800621.View ArticlePubMedGoogle Scholar
- Chourrout D, Delsuc F, Chourrout P, Edvardsen RB, Rentzsch F, Renfer E, Jensen MF, Zhu B, de Jong P, Steele RE, Technau U: Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements. Nature. 2006, 442: 684-687. 10.1038/nature04863.View ArticlePubMedGoogle Scholar
- Ryan JF, Mazza ME, Pang K, Matus DQ, Baxevanis AD, Martindale MQ, Finnerty JR: Pre-bilaterian origins of the Hox cluster and the Hox code: evidence from the sea anemone, Nematostella vectensis. PLoS ONE. 2007, 2: e153-10.1371/journal.pone.0000153.View ArticlePubMed CentralPubMedGoogle Scholar
- Davidson EH, Peterson KJ, Cameron RA: Origin of bilaterian body plans: evolution of developmental regulatory mechanisms. Science. 1995, 270: 1319-1325. 10.1126/science.270.5240.1319.View ArticlePubMedGoogle Scholar
- Davidson EH: Genomic Regulatory Systems: Development and Evolution. 2001, San Diego, Academic PressGoogle Scholar
- Erwin DH, Davidson EH: The last common bilaterian ancestor. Development. 2002, 129: 3021-3032.PubMedGoogle Scholar
- Kulakova M, Bakalenko N, Novikova E, Cook CE, Eliseeva E, Steinmetz PR, Kostyuchenko RP, Dondua A, Arendt D, Akam M: Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa). Development Genes and Evolution. 2007, 217: 39-54. 10.1007/s00427-006-0119-y.View ArticlePubMedGoogle Scholar
- Ferrier DE, Minguillon C: Evolution of the Hox/ParaHox gene clusters. Int J Dev Biol. 2003, 47: 605-611.PubMedGoogle Scholar
- Fröbius AC, Seaver EC: ParaHox gene expression in the polychaete annelid Capitella sp. I. Dev Genes Evol. 2006, 216: 81-88. 10.1007/s00427-005-0049-0.View ArticlePubMedGoogle Scholar
- Arnone MI, Rizzo F, Annunciata R, Cameron RA, Peterson KJ, Martinez P: Genetic organization and embryonic expression of the ParaHox genes in the sea urchin S. purpuratus: insights into the relationship between clustering and colinearity. Dev Biol. 2006, 300: 63-73. 10.1016/j.ydbio.2006.07.037.View ArticlePubMedGoogle Scholar
- Peterson KJ: Isolation of Hox and Parahox genes in the hemichordate Ptychodera flava and the evolution of deuterostome Hox genes. Molecular Phylogenetics and Evolution. 2004, 31: 1208-1215. 10.1016/j.ympev.2003.10.007.View ArticlePubMedGoogle Scholar
- Ferrier DE, Holland PW: Ciona intestinalis ParaHox genes: evolution of Hox/ParaHox cluster integrity, developmental mode, and temporal colinearity. Mol Phylogenet Evol. 2002, 24: 412-417. 10.1016/S1055-7903(02)00204-X.View ArticlePubMedGoogle Scholar
- Park BJ, Cho SJ, Tak ES, Lee BE, Park SC: The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus. Development Genes and Evolution. 2006, 216: 551-553. 10.1007/s00427-006-0071-x.View ArticlePubMedGoogle Scholar
- Barucca M, Biscotti MA, Olmo E, Canapa A: All the three ParaHox genes are present in Nuttallochiton mirandus (Mollusca: polyplacophora): evolutionary considerations. J Exp Zoolog B Mol Dev Evol. 2006, 306: 164-167. 10.1002/jez.b.21082.View ArticleGoogle Scholar
- Macdonald PM, Struhl G: A molecular gradient in early Drosophila embryos and its role in specifying the body pattern. Nature. 1986, 324: 537-545. 10.1038/324537a0.View ArticlePubMedGoogle Scholar
- Weiss JB, Von Ohlen T, Mellerick DM, Dressler G, Doe CQ, Scott MP: Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity. Genes Dev. 1998, 12: 3591-3602. 10.1101/gad.12.22.3591.View ArticlePubMed CentralPubMedGoogle Scholar
- Ruvkun G, Hobert O: The taxonomy of developmental control in Caenorhabditis elegans. Science. 1998, 282: 2033-2041. 10.1126/science.282.5396.2033.View ArticlePubMedGoogle Scholar
- Grapin-Botton A: Antero-posterior patterning of the vertebrate digestive tract: 40 years after Nicole Le Douarin's PhD thesis. Int J Dev Biol. 2005, 49: 335-347. 10.1387/ijdb.041946ag.View ArticlePubMedGoogle Scholar
- Fang R, Olds LC, Sibley E: Spatio-temporal patterns of intestine-specific transcription factor expression during postnatal mouse gut development. Gene Expr Patterns. 2006, 6: 426-432. 10.1016/j.modgep.2005.09.003.View ArticlePubMedGoogle Scholar
- Katsuyama Y, Sato Y, Wada S, Saiga H: Ascidian tail formation requires caudal function. Developmental Biology. 1999, 213: 257-268. 10.1006/dbio.1999.9403.View ArticlePubMedGoogle Scholar
- Hinman VF, Becker E, Degnan BM: Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis. Development Genes and Evolution. 2000, 210: 212-216. 10.1007/s004270050306.View ArticlePubMedGoogle Scholar
- Hudson C, Lemaire P: Induction of anterior neural fates in the ascidian Ciona intestinalis. Mechanisms of Development. 2001, 100: 189-203. 10.1016/S0925-4773(00)00528-1.View ArticlePubMedGoogle Scholar
- Corrado M, Aniello F, Fucci L, Branno M: Ci-IPF1, the pancreatic homeodomain transcription factor, is expressed in neural cells of Ciona intestinalis larva. Mech Dev. 2001, 102: 271-274. 10.1016/S0925-4773(01)00311-2.View ArticlePubMedGoogle Scholar
- Bleidorn C, Vogt L, Bartolomaeus T: New insights into polychaete phylogeny (Annelida) inferred from 18S rDNA sequences. Mol Phylogenet Evol. 2003, 29: 279-288. 10.1016/S1055-7903(03)00107-6.View ArticlePubMedGoogle Scholar
- Hall KA, Hutchings PA, Colgan DJ: Further phylogenetic studies of the Polychaeta using I8S rDNA sequence data. Journal of the Marine Biological Association of the United Kingdom. 2004, 84: 949-960. 10.1017/S0025315404010240h.View ArticleGoogle Scholar
- Arendt D: Spiralians in the limelight. Genome Biol. 2003, 5: 303-10.1186/gb-2003-5-1-303.View ArticlePubMed CentralPubMedGoogle Scholar
- Ushakov R: Polychaete Worms. 1. Fauna of the USSR. 1972, Leningrad, NaukaGoogle Scholar
- Tessmar-Raible K, Arendt D: Emerging systems: between vertebrates and arthropods, the Lophotrochozoa. Curr Opin Genet Dev. 2003, 13: 331-340. 10.1016/S0959-437X(03)00086-8.View ArticlePubMedGoogle Scholar
- Raible F, Tessmar-Raible K, Osoegawa K, Wincker P, Jubin C, Balavoine G, Ferrier D, Benes V, de Jong P, Weissenbach J, Bork P, Arendt D: Vertebrate-type intron-rich genes in the marine annelid Platynereis dumerilii. Science. 2005, 310: 1325-1326. 10.1126/science.1119089.View ArticlePubMedGoogle Scholar
- Wilson EB: The cell lineage of Nereis. J Morphol. 1892, 6:Google Scholar
- Nielsen C: Trochophora Larvae: Cell-Lineages, Ciliary Bands, and Body Regions. 1. Annelida and Mollusca. Journal of Experimental Zoology Part B Molecular and Developmental Evolution. 2004, 302: 35-68.View ArticleGoogle Scholar
- Ackermann C, Dorresteijn A, Fischer A: Clonal domains in postlarval Platynereis dumerilii (Annelida: Polychaeta). J Morphol. 2005, 266: 258-280. 10.1002/jmor.10375.View ArticlePubMedGoogle Scholar
- Brusca RC, Brusca GJ: Invertebrates. 2003, Sunderland, Sinauer Associates, 2ndGoogle Scholar
- Arendt D, Tessmar K, de Campos-Baptista MI, Dorresteijn A, Wittbrodt J: Development of pigment-cup eyes in the polychaete Platynereis dumerilii and evolutionary conservation of larval eyes in Bilateria. Development. 2002, 129: 1143-1154.PubMedGoogle Scholar
- Denes AS, Jekely G, Steinmetz PR, Raible F, Snyman H, Prud'homme B, Ferrier DE, Balavoine G, Arendt D: Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in bilateria. Cell. 2007, 129: 277-288. 10.1016/j.cell.2007.02.040.View ArticlePubMedGoogle Scholar
- Invanova-Kazas OM: Comparative embryology of invertebrates. 1977, Moskva, NaukaGoogle Scholar
- Kulakova MA, Kostyuchenko RP, Andreeva TF, Dondua AK: The abdominal-B-like gene expression during larval development of Nereis virens (Polychaeta). Mech Dev. 2002, 115: 177-179. 10.1016/S0925-4773(02)00113-2.View ArticlePubMedGoogle Scholar
- Punin MJ: Histological Organization of the intestinal epithelium of priapulans, brachiopods, bivalves, and polychaetes. 1991, St. Petersburg, NaukaGoogle Scholar
- Ferrier DE, Holland PW: Sipunculan ParaHox genes. Evol Dev. 2001, 3: 263-270. 10.1046/j.1525-142x.2001.003004263.x.View ArticlePubMedGoogle Scholar
- Urbach R, Technau GM: Molecular markers for identified neuroblasts in the developing brain of Drosophila. Development. 2003, 130: 3621-3637. 10.1242/dev.00533.View ArticlePubMedGoogle Scholar
- Hsieh-Li HM, Witte DP, Szucsik JC, Weinstein M: Gsh-2, a murine homeobox gene expressed in the developing brain. Mech Dev. 1995, 50 (2–3): 177-86.View ArticlePubMedGoogle Scholar
- Li H, Zeitler PS, Valerius MT, Small K, Potter SS: Gsh-1, an orphan Hox gene, is required for normal pituitary development. Embo Journal. 1996, 15: 714-724.PubMed CentralPubMedGoogle Scholar
- Toresson H, Campbell K: A role for Gsh1 in the developing striatum and olfactory bulb of Gsh2 mutant mice. Development. 2001, 128: 4769-4780.PubMedGoogle Scholar
- Valerius MT, Li H, Stock JL, Weinstein M, Kaur S, Singh G, Potter SS: Gsh-1: a novel murine homeobox gene expressed in the central nervous system. Dev Dyn. 1995, 203: 337-351.View ArticlePubMedGoogle Scholar
- Deschet K, Bourrat F, Chourrout D, Joly JS: Expression domains of the medaka (Oryzias latipes) Ol-Gsh 1 gene are reminiscent of those of clustered and orphan homeobox genes. Dev Genes Evol. 1998, 208: 235-244. 10.1007/s004270050178.View ArticlePubMedGoogle Scholar
- Finnerty JR, Paulson D, Burton P, Pang K, Martindale MQ: Early evolution of a homeobox gene: the parahox gene Gsx in the Cnidaria and the Bilateria. Evol Dev. 2003, 5: 331-345. 10.1046/j.1525-142X.2003.03041.x.View ArticlePubMedGoogle Scholar
- Hayward DC, Catmull J, Reece-Hoyes JS, Berghammer H, Dodd H, Hann SJ, Miller DJ, Ball EE: Gene structure and larval expression of cnox-2Am from the coral Acropora millepora. Development Genes and Evolution. 2001, 211: 10-19. 10.1007/s004270000112.View ArticlePubMedGoogle Scholar
- Pérez-Villamil B, Schwartz PT, Vallejo M: The pancreatic homeodomain transcription factor IDX1/IPF1 is expressed in neural cells during brain development. Endocrinology. 1999, 140: 3857-3860. 10.1210/en.140.8.3857.View ArticlePubMedGoogle Scholar
- Schwartz PT, Perez-Villamil B, Rivera A, Moratalla R, Vallejo M: Pancreatic homeodomain transcription factor IDX1/IPF1 expressed in developing brain regulates somatostatin gene transcription in embryonic neural cells. J Biol Chem. 2000, 275: 19106-19114. 10.1074/jbc.M000655200.View ArticlePubMedGoogle Scholar
- Ferrier DE, Dewar K, Cook A, Chang JL, Hill-Force A, Amemiya C: The chordate ParaHox cluster. Curr Biol. 2005, 15: R820-R822. 10.1016/j.cub.2005.10.014.View ArticlePubMedGoogle Scholar
- Kriks S, Lanuza GM, Mizuguchi R, Nakafuku M, Goulding M: Gsh2 is required for the repression of Ngn1 and specification of dorsal interneuron fate in the spinal cord. Development. 2005, 132: 2991-3002. 10.1242/dev.01878.View ArticlePubMedGoogle Scholar
- van den Akker E, Forlani S, Chawengsaksophak K, de Graaff W, Beck F, Meyer BI, Deschamps J: Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation. Development. 2002, 129: 2181-2193.PubMedGoogle Scholar
- Gaunt SJ, Drage D, Trubshaw RC: cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse. Int J Dev Biol. 2005, 49: 901-908. 10.1387/ijdb.052021sg.View ArticlePubMedGoogle Scholar
- Skromne I, Thorsen D, Hale M, Prince VE, Ho RK: Repression of the hindbrain developmental program by Cdx factors is required for the specification of the vertebrate spinal cord. Development. 2007, 134: 2147-2158. 10.1242/dev.002980.View ArticlePubMed CentralPubMedGoogle Scholar
- de Rosa R, Prud'homme B, Balavoine G: caudal and even-skipped in the annelid Platynereis dumerilii and the ancestry of posterior growth. Evolution and Development. 2005, 7: 574-587. 10.1111/j.1525-142X.2005.05061.x.View ArticlePubMedGoogle Scholar
- Korchagina NM, Monteiro AS, Nagarajan AJ, Andreeva T, Holland PWH, Ferrier DEK: Hox and ParaHox gene cluster in a polychaete (Annelida): ; Edinburgh. 2005, BBSRCGoogle Scholar
- Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BLM, Wright CVE: PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development. 1996, 122: 983-995.PubMedGoogle Scholar
- Beck F, Chawengsaksophak K, Waring P, Playford RJ, Furness JB: Reprogramming of intestinal differentiation and intercalary regeneration in Cdx2 mutant mice. Proc Natl Acad Sci U S A. 1999, 96: 7318-7323. 10.1073/pnas.96.13.7318.View ArticlePubMed CentralPubMedGoogle Scholar
- Rosanas-Urgell A, Marfany G, Garcia-Fernandez J: Pdx1-related homeodomain transcription factors are distinctly expressed in mouse adult pancreatic islets. Mol Cell Endocrinol. 2005, 237: 59-66. 10.1016/j.mce.2005.03.008.View ArticlePubMedGoogle Scholar
- Cook CE, Smith ML, Telford MJ, Bastianello A, Akam M: Hox genes and the phylogeny of the arthropods. Curr Biol. 2001, 11: 759-763. 10.1016/S0960-9822(01)00222-6.View ArticlePubMedGoogle Scholar
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