ParaHox family genes encode homeodomain transcription factors from the Gsx, Xlox/Pdx, and Cdx classes. The closely related ParaHox and Hox gene families are derived from duplication of an ancestral ProtoHox cluster. Phylogenetic analysis indicates that Gsx genes are most closely related to anterior Hox genes of paralogue groups 1 and 2 (Brooke et al.,1998).
The cephalochordate amphioxus (Branchiostoma floridae) has a single ParaHox gene cluster containing three genes (Brooke et al.,1998). During the evolution of vertebrates from primitive chordates, there have been ParaHox cluster duplication and gene loss events. Thus, in amniotes there are two Gsx genes (Gsh1 and Gsh2), a single Pdx class gene (Xlox/Pdx), and three Cdx genes (Cdx1, Cdx2, and Cdx4) but only a single intact ParaHox cluster consisting of Gsh1, Pdx, and Cdx2. The remaining genes exist in degenerate clusters containing single ParaHox genes (Ferrier et al.,2005).
In the present study, we report the characterization of two Gsx genes (Gsh1 and Gsh2) from the amphibian Xenopus tropicalis. We demonstrate the existence of one intact and three degenerate ParaHox clusters in X. tropicalis. The single cluster, containing Gsh1, Pdx1, and Cdx2, shares gene order and transcriptional orientation with both amphioxus and mammalian ParaHox clusters. Furthermore, the spatial expression of X. tropicalis ParaHox cluster genes is co-linear with regard to their physical position within the intact cluster.
We show that Gsh2 is expressed in the anterior endoderm, demonstrating that the endoderm is a shared site of expression for genes belonging to all three ParaHox gene classes. The most striking feature of Xenopus Gsh1 and Gsh2 is that they each have distinct and highly dynamic expression patterns within the developing nervous system from early neural plate stages. During later development, they are expressed in complex patterns in the forebrain, midbrain, hindbrain, and spinal cord. Our data provide further evidence that Gsx, Nkx, and Msx class homeobox genes are components of a conserved regulatory network involved in patterning the columnar organization of neuronal precursors in both protostome and deuterostome lineages.
Identification and Characterization of Xenopus tropicalis Gsh1 and Gsh2
Searching the Xenopus tropicalis genome database revealed partial genomic sequences for Gsh1 and Gsh2. Single exon probes were generated and used to isolate full-length cDNAs for Gsh1 and Gsh2 (accession numbers DQ195530 and DQ195531). The conceptual Gsh1 and Gsh2 proteins have 77 and 69% identity to the respective murine orthologues (Clustal W alignment; see Supp. Fig. S1, which is available online). A phylogenetic tree reveals that Xenopus Gsh1 and Gsh2 cluster with their putative fish and mammalian orthologues. (Supp. Fig. S2).
Identification of a Complete ParaHox Gene Cluster in Xenopus tropicalis
Analysis of the Xenopus tropicalis genome sequence revealed the presence of a single intact ParaHox gene cluster containing the Gsh1, Pdx1, and Cdx2 genes. The structures of this 117 kb cluster and the ParaHox clusters of amphioxus, mouse, and human are shown in Figure 1A (Brooke et al.,1998; Ferrier et al.,2005). The order and transcriptional orientation of the genes is conserved between all four species, although the size of the clusters varies from species to species.
Spatial Co-Linearity of Expression From Xenopus tropicalis ParaHox Cluster A
It has been previously shown that the expression of amphioxus ParaHox cluster genes is co-linear with respect to their physical position within the cluster (Brooke et al.,1998). Here we show that genes of the intact X. tropicalis ParaHox cluster also exhibit spatial co-linearity of expression (Fig. 1B). At the tailbud stage, Gsh1 is expressed anteriorly in the brain, Pdx1 is intermediate in the endoderm, and Cdx2 is most posterior in the spinal cord and posterior endoderm.
Remnants of Four Xenopus tropicalis ParaHox Clusters
The genomic context of Cdx4, Gsh2, and Cdx1 reveals the remnants of three other ParaHox clusters. Figure 1C shows the four X. tropicalis ParaHox clusters, which we have named A–D, following the assignments given to the human ParaHox clusters (Minguillon and Garcia-Fernandez,2003). Each cluster is located on a separate scaffold in the v 4.1 genome assembly. Cluster A (scaffold 80) represents the single intact cluster containing Gsh1, Pdx1, and Cdx1. All four clusters are linked at one end to genes coding for tyrosine kinases closely related to the PDGF receptor. Clusters B (scaffold 10) and C (scaffold 107) both have a Chic family gene at the opposite end. A solute carrier family gene is closely associated with ParaHox cluster D (scaffold 559), which is not present in any of the other clusters.
Developmental Expression of Gsh1
The earliest expression of Gsh1 detected by whole-mount in situ hybridization (WISH) is at early neurula stage 14 in the anterior neural plate (Fig. 2A). Given the later expression of Gsh1, and after referring to relevant fate maps, we suggest that these expression domains represent the anlagen of the pretectum and anterior hindbrain (Neary and Northcutt,1983; Eagleson and Harris,1990; Rubenstein et al.,1998). The level of expression in the presumptive hindbrain increases relative to the presumptive pretectum through the neurula stages (Fig. 2B). At late neurula stage 19, new expression is visible as scattered spots in posterior regions of the hindbrain (Fig. 2C,D). By early tailbud stage 25, there is intense expression in the future pretectum and two parallel domains of expression through the hindbrain, as well as paired expression domains in the forebrain (Fig. 2E–G). By tailbud stage 31, the intensity of expression in the forebrain has increased and there is a definite gap in the hindbrain expression (Fig. 2H–J). Based on the position of the otic vesicle at rhombomere 4 and WISH on sibling embryos with Krox20 (which marks rhombomeres 3 and 5) and Gsh1, this gap in expression is at the level of rhombomere 6 (Fig. 2H, data not shown).
The expression of Gsh1 was examined relative to a number of previously characterized regional markers in the developing CNS of tailbud stage embryos. Expression of the homeobox gene En2 marks the isthmic organizer territory at the midbrain-hindbrain boundary. Figure 2K shows that Gsh1 expression extends into the isthmus and overlaps somewhat with En2.
Lhx9 is a LIM-homeobox gene expressed in the thalamus, anterior tectum, and anterior hindbrain in X. laevis at stage 32 (Bachy et al.,2001). Figure 2L is a double WISH with Lhx9 and Gsh1 in X. tropicalis, showing that both genes are expressed in the pretectum, anterior tectum, and rhombomere 1. In a section through the anterior hindbrain, we see that Gsh1 and Lhx9 are both expressed in rhombomere 1, but their domains are adjacent, not overlapping, with Lhx9 being expressed in a superficial domain and Gsh1 in the ventricular and sub-ventricular regions (Fig. 2M). The medial expression along the dorsoventral axis within the hindbrain is consistent with Gsh1 being expressed in a cell population giving rise to interneurons, as has been previously shown for zebrafish Gsh1 (Cheesman and Eisen,2004).
Dll3 (Distal-less 3; mouse homologue Dlx5) is a homeodomain transcription factor involved in specifying GABAergic neurons in the murine telencephalon (Stuhmer et al.,2002). A recent detailed analysis of the developing Xenopus forebrain shows that in the diencephalon Dll3 is expressed in the hypothalamus. In the ventral telencephalon, Dll3 is expressed in divisions of the sub-pallial region, which are proposed to be homologous to the medial ganglionic eminence (MGE) and lateral ganglionic eminence (LGE) of higher vertebrates (Bachy et al.,2001,2002). Double WISH with Dll3 and Gsh1 revealed co-expression in the prethalamus, anterior hypothalamus, and the ventral regions of the telencephalon corresponding to the proposed MGE and LGE of Xenopus (Fig. 2N, also see S and T). The very anterior forebrain section in Figure 2O shows that Gsh1 expression is more ventricular than Dll3 in the LGE, and that Gsh1, unlike Dll3, is also expressed in the olfactory bulb (Bachy et al.,2002). A slightly more posterior section reveals strong expression in the MGE (Fig. 2P). The section in Figure 2Q shows two domains of expression passing up though the anterior hypothalamus to what may be the zona limitans intrathalamica (ZLI). Intense expression is detected in the thalamus and pretectum, together with a domain of expression in the posterior hypothalamus (Fig. 2R). A cleared tailbud embryo is shown in Figure 2S demonstrating expression in the hypothalamus, MGE, ZLI, thalamus, pretectum, anterior tectum, hindbrain, and weak expression in the LGE. The inset in Figure 2S reveals that, although Gsh1 is expressed in the olfactory bulb, it is absent from the septum (Bachy et al.,2002).
By mid-tailbud stage 35, the expression in the tectum has expanded posteriorly, a second small region of expression has appeared in the posterior hypothalamus, and expression in the LGE has intensified (Fig. 2T). At late tailbud stage 40, faint expression is visible in the dorsal spinal cord (Fig. 2U). At larval stage 47 (Fig. 2V), Gsh1 expression extends all the way through the tectum ventricular zone, and the hindbrain expression remains. There is no longer any expression in the telencephalon, but high levels are present in the hypothalamus (Fig. 2W).
Developmental Expression of Gsh2
Gsh2 expression is first detectable in the open neural plate at early neurula stage 13 (Fig. 3A). Reference to relevant fate maps indicates that this expression is at the level of the presumptive hindbrain, which is in keeping with the later observed expression of Gsh2 following neural tube closure (Neary and Northcutt,1983; Eagleson and Harris,1990; Rubenstein et al.,1998). Gsh2 expression continues in the presumptive hindbrain region throughout neurula stages (Fig. 3B). At the end of neurula stages, new expression domains are apparent in what we suggest is the prospective prethalamus (Fig. 3C) and the anterior endoderm (Fig. 3D, E).
By the early tailbud stage 25, the hindbrain expression is restricted to the posterior two rhombomeres and weak expression is detected in the posterior spinal cord (Fig. 3F, H). We note that expression in the spinal cord is dynamic, and at a given level in the spinal cord expression is frequently not bilaterally symmetrical across the mid-line (Fig. 3F and data not shown). A section through the posterior spinal cord of a tailbud stage-27 embryo shows that expression extends to the dorsal midline, indicating that Gsh2 is likely to be expressed in cell populations giving rise to both interneurons and sensory neurons (Fig. 3L). Endodermal expression is still detectable at stages 24–25 (Fig. 3H, K). There are two pairs of domains in the forebrain: one in the LGE and one in the prethalamus. Strong expression is detected in the dorsal part of the retinal pigment epithelium (RPE) (Fig. 3G, I, J). By tailbud stages 30–31, the rhombomeres 7–8, LGE, prethalamus, and RPE expression is still visible, but no spinal cord or endodermal expression is detected (Fig. 3M–O).
Using double WISH, the overlap of expression between Gsh1/Gsh2 was examined in the nervous system of tailbud stage embryos. Figure 3P shows that the two Gsx genes are both expressed in the LGE and posterior hindbrain, but that Gsh2 is not expressed in the thalamus or MGE. Gsh2 expression is seen in the LGE (Fig. 3Q–S), ZLI, and prethalamus (Fig. 3Q, T), but not the olfactory bulb (Fig. 3R). A transverse section of the posterior hindbrain shows that expression of Gsh1 and Gsh2 overlaps. However, Gsh1 has a more ventral limit of expression and Gsh2 has a more dorsal limit of expression (Fig. 3U). Supplementary Figure 3 summarizes the results of our in situ hybridization analysis of Gsh1 and Gsh2 expression domains in the brain of a X. tropicalis tailbud stage 31–32 embryo.
At late tailbud stage 40, Gsh2 expression continues in the LGE, prethalamus, ZLI, and posterior hindbrain (Fig. 3V). A frontal section shows that Gsh2 expression is restricted to the ventricular zone of the LGE (Fig. 3W). At larval stage 47, there is no expression in the olfactory bulb, but there is expression in the adjacent LGE (Fig. 3X, Y). Neurons connecting the LGE to the prethalamus also express Gsh2, as does the prethalamus itself (Fig. 3X, Z). In addition, there is highly localised expression in some cells of the velar plate, which is derived from the pharynx floor, separating the left and right sides of the branchial cavity (Fig. 3A2).
Gsh2 Expression in the Primary Interneuron Territory of the Neural Plate
Differentiation of neurons in the primary nervous system of Xenopus is initiated during open neural plate stages in three columns on either side of the dorsal midline. Figure 4A is an in situ hybridization for the neuronal differentiation marker N-tubulin showing the positions of the primary neurons in an early neurula stage embryo. The most medial column of differentiating neurons gives rise to motor neurons, the intermediate column gives rise to interneurons, and the lateral column gives rise to Rohon-Beard sensory neurons. During neurula stages, proliferative, non-differentiated progenitor domains are present between the three columns. The exact position of Gsh2 expression relative to the domains of differentiating primary neurons is of considerable interest. We have determined the position of Gsh2 expression by comparing the expression of Gsh2 to other markers of regional identity in the neural plate.
Lbx1 is a marker of interneuron populations in the hindbrain and spinal cord of amniotes (Schubert et al.,2001). Furthermore, Lbx1 has previously been shown to be expressed in the open neural plate and later in development is expressed in a dorso-intermediate position in the X. tropicalis closed neural tube, which is consistent with it being a marker of interneurons (Martin and Harland,2006). Comparison of the Gsh2 expression domain in the neural plate with that of N-tubulin and Lbx1 indicates that Gsh2 is expressed in the region of the differentiating primary interneurons (Fig. 4A–C). Further support for this conclusion is provided by carrying out double in situ hybridization for a number of other markers.
The HMG-box gene Sox3 is expressed during primary neurogenesis in a confluent domain containing the motor neurons, interneurons, and intervening proliferative neuronal precursors (Bellefroid et al.,1998). Thus interneurons are found at the lateral boundary of the Sox3 domain. Double WISH with Gsh2 and Sox3 reveals that Gsh2 is co-expressed with Sox3 at the most lateral part of the Sox3 domain (Fig. 4D–F).
The homeobox gene Iro3 is initially expressed in a similar pattern to Sox3 during open neural plate stages. As development proceeds, its expression becomes increasingly restricted to the domain of non-differentiating neuronal precursors between the motor neuron and interneuron-forming regions (Bellefroid et al.,1998). Double WISH with Gsh2 and Iro3 confirms that Gsh2 is expressed at the edge of this domain in the interneuron region (Fig. 4G and I).
Isl1 encodes a LIM homeodomain transcription factor that marks the primary motor and sensory neurons in zebrafish (Inoue et al.,1994) and Xenopus. Figure 4H and J are double WISH for Isl1 and Gsh2. Absence of expression is observed between the lateral Isl1 domain and the intermediate Gsh2 domain. We interpret this to mean that the domain lacking both Isl1 and Gsh2 expression represents the column of undifferentiated neuronal precursor cells between the sensory neuron and interneuron populations. We conclude that during open neural plate stages, Gsh2 is expressed in the primary interneuron domain in the region fated to give rise to the hindbrain. However, in later development, after closure of the neural tube, it is expressed in a dorsal domain giving rise to both sensory and interneurons.
Conserved Gene Expression During Primary Neurogenesis in Xenopus and Drosophila
The Gsx class gene ind, together with the Nkx class gene vnd and the Msx class gene msh, are expressed in and required for the development of the intermediate, medial, and lateral columns of primary neurons in Drosophila. There has been considerable speculation that genes of these classes are components of a conserved regulatory network required for dorso-ventral patterning of the nervous system in both invertebrate and vertebrate lineages (Arendt and Nubler-Jung,1999; Cornell and Ohlen,2000). With this in mind, we investigated the expression of Gsh2 relative to Nkx and Msx class genes in the open neural plate of Xenopus.
The vertebrate Nkx2 genes are orthologous to Drosophila vnd, but in Xenopus, the Nkx2 genes are not expressed in the midline region of the open neural plate (Saha et al.,1993; Hollemann and Pieler,2000; Small et al.,2000). However, we find that the related Nkx6.1 gene is expressed in the medial region that will give rise to motor neurons (Fig. 4K). Gsh2 is expressed in the intermediate neurogenic region (Fig. 4B). Similarly, Figure 4L shows that Msx1, an orthologue of msh, is expressed in a domain at the lateral margin of the neural plate that has been previously been demonstrated to give rise to both primary sensory neurons and neural crest derivatives (Bang et al.,1999). Thus, we conclude that relative positions of the Nkx, Gsx, and Msx expression domains in the early neurogenic regions have been conserved between Drosophila and Xenopus (summarised in Fig. 4M).
The Amphibian ParaHox Clusters
We report that a single intact ParaHox gene cluster (ParaHox cluster A) containing Gsh1, Pdx1, and Cdx2 orthologues, is present in the genome of the amphibian Xenopus tropicalis, together with three degenerate ParaHox clusters (clusters B, C, and D) in Xenopus. While this organization of ParaHox genes has been reported for some amniotes (Ferrier et al.,2005), not all vertebrate genomes exhibit the same organization. Interestingly, an intact ParaHox cluster is absent in teleost fish, including zebrafish and pufferfish. It has been speculated that genome duplication during evolution of the teleost lineage led to relaxation of the constraints that have maintained the ParaHox cluster in amniotes and presumably amphibians (Mulley et al.,2006). It is interesting to note that, although the size of the ParaHox cluster varies considerably between chordate groups, the relative spacing of the genes within the cluster is very similar. It has been speculated that this constraint on gene spacing might reflect an underlying regulatory mechanism required for correct expression of genes within the cluster (Ferrier et al.,2005). Comparison of ParaHox cluster organization and evolution in basal vertebrate lineages, amphibians, teleosts, and amniotes is likely to provide insights into the evolutionary pressures that have maintained the organization of these clusters during evolution.
We report that the spatial expression along the developing body axis from genes of the amphibian ParaHox cluster is co-linear with regard to position within the cluster. This feature has also been noted for genes of the amphioxus ParaHox gene cluster (Brooke et al.,1998). Interestingly, unlike genes of the closely related Hox gene clusters, genes from the amphibian ParaHox cluster do not exhibit temporal co-linearity of expression. Typically, anterior (3′) Hox genes are expressed earlier in development than more posterior (5′) genes. In contrast, in Xenopus the posterior ParaHox cluster gene Cdx2 is expressed from the early gastrula stage (Reece-Hoyes et al.,2002), several hours before expression of the anterior Gsh1 gene, indicating that mechanisms underlying temporal and spatial co-linearity are separable.
Our detailed analysis of amphibian Gsx expression reveals that Gsh2 is expressed in the anterior gut endoderm. This indicates that genes from all three ParaHox genes classes are expressed in the developing gut endoderm (Guo et al.,2004; Rosanas-Urgell et al.,2005). We find no expression of Gsx genes in mesodermal derivatives. However, the most striking feature of amphibian Gsx gene expression is their complex expression within the developing nervous system.
Gsx Class Genes and the Anterior Nervous System
The previously reported expression patterns of chordate Gsx class genes reveal that expression in the most anterior regions of the nervous system is a common feature. The amphioxus Gsx gene is expressed in a single domain, the cerebral vesicle, which is the anatomical homologue of the vertebrate forebrain/midbrain (Brooke et al.,1998). A putative Gsx gene has been identified in Ciona; this gene is also expressed in the anterior nervous system, specifically in the photosensitive sensory vesicle (Hudson and Lemaire,2001).
The expression patterns of vertebrate Gsh1 and Gsh2 are complex and dynamic, being expressed in overlapping domains in the forebrain, hindbrain, and spinal cord. Additionally, Gsh1 is expressed in the anterior midbrain. The expression of Xenopus Gsh1 and Gsh2 within the ventral telencephalon is broadly similar to that reported for Gsx class genes in other vertebrates (Szucsik et al.,1997; Deschet et al.,1998; Yun et al.,2001; Cheesman and Eisen,2004). This provides further evidence to support the proposal that the basic organization of the telencephalon is conserved between amphibians and other vertebrates (Bachy et al.,2002).
Gsx Class Genes and Dorsoventral Patterning
There has been much speculation that the conserved expression domains of Nkx, Gsx, and Msx class genes in the developing nervous systems of Drosophila and mouse provide evidence for a conserved regulatory system patterning the dorsoventral axis of the nervous system of both protostomes and deuterostomes (Cornell and Ohlen,2000; Mizutani et al.,2006). However, it has been noted that expression of these genes in the mouse neural tube is rather late in development compared with the expression in the early Drosophila neuroectoderm, a stage that is perhaps more comparable to the open neural plate stage of vertebrate embryos (Cheesman and Eisen,2004). Our study for the first time demonstrates that Nkx, Gsx, and Msx class genes are expressed in distinct midline to lateral domains in the open neural plate of a vertebrate.
vnd has the greatest sequence identity with the Nkx2 genes, and comparisons have been made with the expression of mouse Nkx2 genes in motor neurons, but no Nkx2 genes have been reported as being expressed in the amphibian embryo at the open neural plate stage. Nkx2.1, Nkx2.2, and Nkx2.4 are all expressed slightly later and at the extreme anterior in the developing Xenopus telencephalon (Saha et al.,1993; Hollemann and Pieler,2000; Small et al.,2000). In contrast, Nkx6.1 is expressed early in the medial neural plate of both higher (Qiu et al.,1998) and lower vertebrates (Cheesman et al.,2004), suggesting that vertebrate Nkx6 genes may have an earlier role in neural patterning than Nkx2 genes.
Both Nkx2.2 and Nkx6 genes are required for motor neuron development (Pattyn et al.,2003). In Drosophila, the nkx6 gene is also expressed in the midline cells and a subset of ventral (medial) and intermediate column neuroblasts (Uhler et al.,2002; Cheesman et al.,2004). These findings indicate that the roles and relationships of Nkx family genes have altered somewhat since the divergence of protostomes and deuterostomes. Thus in Drosophila and vertebrates, Nkx6 genes are closely associated with the development of medial column neurons, and in vertebrate lineages they may perform some of the functions of vnd in Drosophila. For example, Nkx6.1 represses interneuron development from the intermediate column in zebrafish (Cheesman et al.,2004). The parallels to vnd repression of ind during Drosophila neurogenesis are striking (Cowden and Levine,2003). Further studies are planned to investigate if the negative regulatory interactions that have been shown to operate between the Nkx, Gsx, and Msx gene families in Drosophila neurogenesis are conserved during amphibian primary neurogenesis (Cowden and Levine,2003).
Gsx Genes and Neurogenesis
Drosophila null mutants in the Gsx class gene ind have reduced numbers of intermediate column neuroblasts (Weiss et al.,1998). Functional studies of mouse Gsh1 and Gsh2 indicate multiple roles for these genes in the development of the nervous system. Analysis of Gsh1/Gsh2 compound knockout mice indicates that Gsx function is required for the proliferation of subsets of neuronal precursors in the ventricular zone of the ventral telencephalon (Toresson and Campbell,2001). A general role for Gsx function in regulating neuronal proliferation is further suggested by the demonstration that Gsh1 expression is associated with proliferating cells in the optic tectum and hindbrain of medaka (Nguyen et al.,1999). Our data indicate that Gsh1 and Gsh2 are expressed in the proliferative ventricular zones in several areas of the developing central nervous. We also note that Gsh2 is expressed in the deep layer of progenitor cells in the primary interneuron domain of the early neural plate. Together, these observations may indicate a role for Gsx genes in the balance between proliferation and differentiation of neuronal precursor cells from the very earliest stages of neurogenesis in the frog.
In Vitro Fertilization and Xenopus tropicalis Embryo Culture
Embryos were generated by in vitro fertilization (Reece-Hoyes et al.,2002). Staging was according to Nieuwkoop and Faber (Nieuwkoop and Faber,1967).
PCR Cloning of Genomic Fragments of Xenopus tropicalis Gsh1, Gsh2, and Pdx1
The X. tropicalis genome sequence database (http://genome.jgi-psf.org/Xentr4/Xentr4.home.html) was searched using the mouse Gsh1, Gsh2, and Xenopus laevis Xlhbox8 (Pdx1) peptide sequences. PCR primers were designed to amplify fragments of exon 2 from Gsh1, and exon 1 from Gsh2 and Pdx1, from genomic DNA. The PCR fragments were ligated into the pGEM®-T Easy vector (Promega).
cDNA Library Screening
An X. tropicalis stage 20–30 head library in pCS107 (kind gift of Enrique Amaya) was screened for Gsh1 and Gsh2 using genomic PCR products as probes. Putative full-length clones of X. tropicalisGsh1 (1,456 bp) and Gsh2 (1,202 bp) were isolated and sequenced. Accession numbers are DQ195530 and DQ195531, respectively.
Genomic Analysis of the Xenopus tropicalis ParaHox Clusters
As part of the JGI BAC sequencing project, one BAC containing X. tropicalis Cdx2 (accession number, AC147904) and another BAC containing X. tropicalis Pdx1 (accession number, AC146803) have been sequenced. Contig 7 from the Cdx2 BAC (containing Gsh1) and contig 12 from the Pdx1 BAC (containing Pdx1 and Cdx2) were compiled to give a super-contig that covers the whole of the X. tropicalis ParaHox cluster. Scaffold 80 of the X. tropicalis genome sequence database, assembly 4.1 (http://genome.jgi-psf.org/Xentr4/Xentr4.home.html), was also shown to contain the X. tropicalis ParaHox cluster, and the two sources are in agreement.
Wholemount In Situ Hybridization (WISH)
Embryos were fixed in MEMFA and in situ hybridizations were carried out as per Harland (1991) with the modifications described in (Pownall et al.,1996). Details of the in situ probe plasmids are shown Table 1; those with accession numbers were obtained from MRC Geneservice (www.hgmp.mrc.ac.uk/geneservice/index.shtml). A few X. laevis probes were used (indicated by X rather than Xt). Where indicated, in situ probes were hydrolysed to 250 bases. Double WISH was carried as per Isaacs et al. (1998). Hybridizing probes were detected using anti-DIG or anti-fluorescein AP-coupled antibodies (Roche). The substrate for single WISH was BM purple (Roche). For double WISH, BCIP (Roche) and magenta-phosphate (Sigma) AP substrates were used. For embryos older than early neurula stages, 1 mM levamisole was added to the substrate reaction. Following colour development, embryos were bleached in 6% H2O2 in PBS with 0.1% Tween. Clearing of embryos was in 1,2,3,4-tetrahydronaphthalene.
After WISH, unbleached embryos were fixed in 4% paraformaldehyde in PBS overnight. Embryos were embedded in paraplast media (Sigma) through an ethanol-Histoclear (National Diagnostics) series and 14 μm microtome sections were counterstained with eosin or picric acid. Some embryos were embedded in 4% agarose and 40–50 μm sections were cut using a Leica VT 1000 S vibratome.
The authors acknowledge Enrique Amaya for the X. tropicalis stage 20–30 head cDNA library, David Ferrier and Alan Roberts for helpful conversations, Heithem El-Hodiri for advice on the double WISH protocol, Ann Bamford for her dedication to the frogs' welfare, and the BBSRC for studentships BBSSC200212885 and BBSSC200513195 to J.C.I. and E.F.W.