ParaHox Gene Cluster Research Articles

Mutation of amphioxus Pdx and Cdx demonstrates conserved roles for ParaHox genes in gut, anus and tail patterning

BackgroundThe homeobox genes Pdx and Cdx are widespread across the animal kingdom and part of the small ParaHox gene cluster. Gene expression patterns suggest ancient roles for Pdx and Cdx in patterning the through-gut of bilaterian animals although functional data are available for few lineages. To examine evolutionary conservation of Pdx and Cdx gene functions, we focus on amphioxus, small marine animals that occupy a pivotal position in chordate evolution and in which ParaHox gene clustering was first reported.ResultsUsing transcription activator-like effector nucleases (TALENs), we engineer frameshift mutations in the Pdx and Cdx genes of the amphioxus Branchiostoma floridae and establish mutant lines. Homozygous Pdx mutants have a defect in amphioxus endoderm, manifest as loss of a midgut region expressing endogenous GFP. The anus fails to open in homozygous Cdx mutants, which also have defects in posterior body extension and epidermal tail fin development. Treatment with an inverse agonist of retinoic acid (RA) signalling partially rescues the axial and tail fin phenotypes indicating they are caused by increased RA signalling. Gene expression analyses and luciferase assays suggest that posterior RA levels are kept low in wild type animals by a likely direct transcriptional regulation of a Cyp26 gene by Cdx. Transcriptome analysis reveals extensive gene expression changes in mutants, with a disproportionate effect of Pdx and Cdx on gut-enriched genes and a colinear-like effect of Cdx on Hox genes.ConclusionsThese data reveal that amphioxus Pdx and Cdx have roles in specifying middle and posterior cell fates in the endoderm of the gut, roles that likely date to the origin of Bilateria. This conclusion is consistent with these two ParaHox genes playing a role in the origin of the bilaterian through-gut with a distinct anus, morphological innovations that contributed to ecological change in the Cambrian. In addition, we find that amphioxus Cdx promotes body axis extension through a molecular mechanism conserved with vertebrates. The axial extension role for Cdx dates back at least to the origin of Chordata and may have facilitated the evolution of the post-anal tail and active locomotion in chordates.

Lampreys, the jawless vertebrates, contain only two ParaHox gene clusters

ParaHox genes (Gsx, Pdx, and Cdx) are an ancient family of developmental genes closely related to the Hox genes. They play critical roles in the patterning of brain and gut. The basal chordate, amphioxus, contains a single ParaHox cluster comprising one member of each family, whereas nonteleost jawed vertebrates contain four ParaHox genomic loci with six or seven ParaHox genes. Teleosts, which have experienced an additional whole-genome duplication, contain six ParaHox genomic loci with six ParaHox genes. Jawless vertebrates, represented by lampreys and hagfish, are the most ancient group of vertebrates and are crucial for understanding the origin and evolution of vertebrate gene families. We have previously shown that lampreys contain six Hox gene loci. Here we report that lampreys contain only two ParaHox gene clusters (designated as α- and β-clusters) bearing five ParaHox genes (Gsxα, Pdxα, Cdxα, Gsxβ, and Cdxβ). The order and orientation of the three genes in the α-cluster are identical to that of the single cluster in amphioxus. However, the orientation of Gsxβ in the β-cluster is inverted. Interestingly, Gsxβ is expressed in the eye, unlike its homologs in jawed vertebrates, which are expressed mainly in the brain. The lamprey Pdxα is expressed in the pancreas similar to jawed vertebrate Pdx genes, indicating that the pancreatic expression of Pdx was acquired before the divergence of jawless and jawed vertebrate lineages. It is likely that the lamprey Pdxα plays a crucial role in pancreas specification and insulin production similar to the Pdx of jawed vertebrates.

Genome sequence of a diabetes-prone rodent reveals a mutation hotspot around the ParaHox gene cluster

The sand rat Psammomys obesus is a gerbil species native to deserts of North Africa and the Middle East, and is constrained in its ecology because high carbohydrate diets induce obesity and type II diabetes that, in extreme cases, can lead to pancreatic failure and death. We report the sequencing of the sand rat genome and discovery of an unusual, extensive, and mutationally biased GC-rich genomic domain. This highly divergent genomic region encompasses several functionally essential genes, and spans the ParaHox cluster which includes the insulin-regulating homeobox gene Pdx1. The sequence of sand rat Pdx1 has been grossly affected by GC-biased mutation, leading to the highest divergence observed for this gene across the Bilateria. In addition to genomic insights into restricted caloric intake in a desert species, the discovery of a localized chromosomal region subject to elevated mutation suggests that mutational heterogeneity within genomes could influence the course of evolution.

Cdx ParaHox genes acquired distinct developmental roles after gene duplication in vertebrate evolution.

BackgroundThe functional consequences of whole genome duplications in vertebrate evolution are not fully understood. It remains unclear, for instance, why paralogues were retained in some gene families but extensively lost in others. Cdx homeobox genes encode conserved transcription factors controlling posterior development across diverse bilaterians. These genes are part of the ParaHox gene cluster. Multiple Cdx copies were retained after genome duplication, raising questions about how functional divergence, overlap, and redundancy respectively contributed to their retention and evolutionary fate.ResultsWe examined the degree of regulatory and functional overlap between the three vertebrate Cdx genes using single and triple morpholino knock-down in Xenopus tropicalis followed by RNA-seq. We found that one paralogue, Cdx4, has a much stronger effect on gene expression than the others, including a strong regulatory effect on FGF and Wnt genes. Functional annotation revealed distinct and overlapping roles and subtly different temporal windows of action for each gene. The data also reveal a colinear-like effect of Cdx genes on Hox genes, with repression of Hox paralogy groups 1 and 2, and activation increasing from Hox group 5 to 11. We also highlight cases in which duplicated genes regulate distinct paralogous targets revealing pathway elaboration after whole genome duplication.ConclusionsDespite shared core pathways, Cdx paralogues have acquired distinct regulatory roles during development. This implies that the degree of functional overlap between paralogues is relatively low and that gene expression pattern alone should be used with caution when investigating the functional evolution of duplicated genes. We therefore suggest that developmental programmes were extensively rewired after whole genome duplication in the early evolution of vertebrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0165-x) contains supplementary material, which is available to authorized users.

Reinforcing the egg-timer: recruitment of novel lophotrochozoa homeobox genes to early and late development in the pacific oyster.

The metazoan superclade Lophotrochozoa includes mollusks, annelids, and several other animal phyla. It is reasonable to assume that this organismal diversity may be traced, in part, to changes in developmentally important genes, such as the homeobox genes. Although most comparative studies have focussed on ancient homeobox gene families conserved across bilaterians, there are also “novel” homeobox genes that have arisen more recently in evolution, presumably by duplication followed by radical divergence and functional change. We classify 136 homeobox genes in the genome sequence of the Pacific oyster, Crassostrea gigas. The genome shows an unusually low degree of homeobox gene clustering, with disruption of the NK, Hox, and ParaHox gene clusters. Among the oyster genes, 31 do not fall into ancient metazoan or bilaterian homeobox gene families; we deduce that they originated in the lophotrochozoan clade. We compared eight lophotrochozoan genomes to trace the pattern of homeobox gene evolution across this clade, allowing us to define 19 new lophotrochozoan-specific clades within the ANTP, PRD, TALE, ZF, SIX, and CUT classes. Using transcriptome data, we compared temporal expression of each homeobox gene in oyster development, and discovered that the lophotrochozoan-specific homeobox genes have peak expression either in early development (egg to gastrula) or in late development (after the trochophore larval stage), but rarely in between. This finding is consistent with the egg-timer, hourglass or phylotypic stage model of developmental evolution, in which there is a conserved central phase of development, but more evolutionarily labile early and late phases.

Evolution of homeobox genes

Many homeobox genes encode transcription factors with regulatory roles in animal and plant development. Homeobox genes are found in almost all eukaryotes, and have diversified into 11 gene classes and over 100 gene families in animal evolution, and 10 to 14 gene classes in plants. The largest group in animals is the ANTP class which includes the well-known Hox genes, plus other genes implicated in development including ParaHox (Cdx, Xlox, Gsx), Evx, Dlx, En, NK4, NK3, Msx, and Nanog. Genomic data suggest that the ANTP class diversified by extensive tandem duplication to generate a large array of genes, including an NK gene cluster and a hypothetical ProtoHox gene cluster that duplicated to generate Hox and ParaHox genes. Expression and functional data suggest that NK, Hox, and ParaHox gene clusters acquired distinct roles in patterning the mesoderm, nervous system, and gut. The PRD class is also diverse and includes Pax2/5/8, Pax3/7, Pax4/6, Gsc, Hesx, Otx, Otp, and Pitx genes. PRD genes are not generally arranged in ancient genomic clusters, although the Dux, Obox, and Rhox gene clusters arose in mammalian evolution as did several non-clustered PRD genes. Tandem duplication and genome duplication expanded the number of homeobox genes, possibly contributing to the evolution of developmental complexity, but homeobox gene loss must not be ignored. Evolutionary changes to homeobox gene expression have also been documented, including Hox gene expression patterns shifting in concert with segmental diversification in vertebrates and crustaceans, and deletion of a Pitx1 gene enhancer in pelvic-reduced sticklebacks. WIREs Dev Biol 2013, 2:31-45. doi: 10.1002/wdev.78 For further resources related to this article, please visit the WIREs website. The author declares that he has no conflicts of interest.

Caudal-related Homeobox (Cdx) Protein-dependent Integration of Canonical Wnt Signaling on Paired-box 3 (Pax3) Neural Crest Enhancer

One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

Parallel retention of Pdx2 genes in cartilaginous fish and coelacanths.

The Pdx1 or Ipf1 gene encodes an important homeodomain-containing protein with key roles in pancreas development and function. Mutations in human PDX1 are implicated in developmental defects and disease of the pancreas. Extensive research, including genome sequencing, has indicated that Pdx1 is the only member of its gene family in mammals, birds, amphibians, and ray-finned fish, and with the exception of teleost fish, this gene forms part of the ParaHox gene cluster along with Gsx1 and Cdx2. The ParaHox cluster, however, is a remnant of a 4-fold genome duplication; the three other ParaHox paralogues lack a Pdx-like gene in all vertebrate genomes examined to date. We have used bacterial artificial chromosome cloning and synteny analysis to show that the ancestor of living jawed vertebrates in fact had more ParaHox genes, including two Pdx genes (Pdx1 and Pdx2). Surprisingly, the two Pdx genes have been retained in parallel in two quite distantly related lineages, the cartilaginous fish (sharks, skates, and chimeras) and the Indonesian coelacanth, Latimeria menadoensis. The Pdx2 gene has been lost independently in ray-finned fish and in tetrapods.

Chordate Hox and ParaHox Gene Clusters Differ Dramatically in Their Repetitive Element Content

The ParaHox and Hox gene clusters control aspects of animal anterior-posterior development and are related as paralogous evolutionary sisters. Despite this relationship, it is not clear if the clusters operate in similar ways, with similar constraints. To compare clusters, we examined the transposable-element (TE) content of amphioxus and mammalian ParaHox and Hox clusters. Chordate Hox clusters are known to be largely devoid of TEs, possibly due to gene regulation and constraints on clustering in these animals. Here, we describe several novel amphioxus TEs and show that the amphioxus ParaHox cluster is a hotspot for TE insertion. TE contents of mammalian ParaHox loci are at background levels, in stark contrast to chordate Hox clusters. This marks a significant difference between Hox and ParaHox clusters. The presence of so many potentially disruptive elements implies selection constrains these ParaHox clusters as they have not dispersed despite 500 My of evolution for each lineage.

Features of the ancestral bilaterian inferred from Platynereis dumerilii ParaHox genes

BackgroundThe ParaHox gene cluster is the evolutionary sister to the Hox cluster. Whilst the role of the Hox cluster in patterning the anterior-posterior axis of bilaterian animals is well established, and the organisation of vertebrate Hox clusters is intimately linked to gene regulation, much less is known about the more recently discovered ParaHox cluster. ParaHox gene clustering, and its relationship to expression, has only been described in deuterostomes. Conventional protostome models (Drosophila melanogaster and Caenorhabditis elegans) are secondarily derived with respect to ParaHox genes, suffering gene loss and cluster break-up.ResultsWe provide the first evidence for ParaHox gene clustering from a less-derived protostome animal, the annelid Platynereis dumerilii. Clustering of these genes is thus not a sole preserve of the deuterostome lineage within Bilateria. This protostome ParaHox cluster is not entirely intact however, with Pdu-Cdx being on the opposite end of the same chromosome arm from Pdu-Gsx and Pdu-Xlox. From the genomic sequence around the P. dumerilii ParaHox genes the neighbouring genes are identified, compared with other taxa, and the ancestral arrangement deduced.ConclusionWe relate the organisation of the ParaHox genes to their expression, and from comparisons with other taxa hypothesise that a relatively complex pattern of ParaHox gene expression existed in the protostome-deuterostome ancestor, which was secondarily simplified along several invertebrate lineages. Detailed comparisons of the gene content around the ParaHox genes enables the reconstruction of the genome surrounding the ParaHox cluster of the protostome-deuterostome ancestor, which existed over 550 million years ago.

ParaHox Cluster Evolution — Hagfish and Beyond

Abstract The ParaHox genes comprise three Hox-related homeobox gene families, found throughout the animals. They were first discovered in the invertebrate chordate amphioxus, where they are tightly clustered. In this paper we carry out a comparative review of ParaHox gene cluster organization among the deuterostomes, and discuss how the recently published hagfish ParaHox clusters fit into current theories about the evolution of this group of genes.

Comparative genomics of ParaHox clusters of teleost fishes: gene cluster breakup and the retention of gene sets following whole genome duplications

BackgroundThe evolutionary lineage leading to the teleost fish underwent a whole genome duplication termed FSGD or 3R in addition to two prior genome duplications that took place earlier during vertebrate evolution (termed 1R and 2R). Resulting from the FSGD, additional copies of genes are present in fish, compared to tetrapods whose lineage did not experience the 3R genome duplication. Interestingly, we find that ParaHox genes do not differ in number in extant teleost fishes despite their additional genome duplication from the genomic situation in mammals, but they are distributed over twice as many paralogous regions in fish genomes.ResultsWe determined the DNA sequence of the entire ParaHox C1 paralogon in the East African cichlid fish Astatotilapia burtoni, and compared it to orthologous regions in other vertebrate genomes as well as to the paralogous vertebrate ParaHox D paralogons. Evolutionary relationships among genes from these four chromosomal regions were studied with several phylogenetic algorithms. We provide evidence that the genes of the ParaHox C paralogous cluster are duplicated in teleosts, just as it had been shown previously for the D paralogon genes. Overall, however, synteny and cluster integrity seems to be less conserved in ParaHox gene clusters than in Hox gene clusters. Comparative analyses of non-coding sequences uncovered conserved, possibly co-regulatory elements, which are likely to contain promoter motives of the genes belonging to the ParaHox paralogons.ConclusionThere seems to be strong stabilizing selection for gene order as well as gene orientation in the ParaHox C paralogon, since with a few exceptions, only the lengths of the introns and intergenic regions differ between the distantly related species examined. The high degree of evolutionary conservation of this gene cluster's architecture in particular – but possibly clusters of genes more generally – might be linked to the presence of promoter, enhancer or inhibitor motifs that serve to regulate more than just one gene. Therefore, deletions, inversions or relocations of individual genes could destroy the regulation of the clustered genes in this region. The existence of such a regulation network might explain the evolutionary conservation of gene order and orientation over the course of hundreds of millions of years of vertebrate evolution. Another possible explanation for the highly conserved gene order might be the existence of a regulator not located immediately next to its corresponding gene but further away since a relocation or inversion would possibly interrupt this interaction. Different ParaHox clusters were found to have experienced differential gene loss in teleosts. Yet the complete set of these homeobox genes was maintained, albeit distributed over almost twice the number of chromosomes. Selection due to dosage effects and/or stoichiometric disturbance might act more strongly to maintain a modal number of homeobox genes (and possibly transcription factors more generally) per genome, yet permit the accumulation of other (non regulatory) genes associated with these homeobox gene clusters.

A Degenerate ParaHox Gene Cluster in a Degenerate Vertebrate

The ParaHox genes consist of 3 homeobox gene families, Gsx, Xlox, and Cdx, all of which have fundamental roles in development. Xlox (known as IPF1 or PDX1 in vertebrates), for example, is crucial for development of the vertebrate pancreas and is also involved in regulation of insulin expression. The invertebrate amphioxus has a gene cluster containing one gene from each of the gene families, whereas in all vertebrates examined to date there are additional copies resultant from ParaHox gene cluster duplications at the base of the vertebrate lineage. Extant vertebrates basal to bony and cartilaginous fish are central to the question of when and how these multiple genes arose in the vertebrate genome. Here, we report the mapping of a ParaHox gene cluster in 2 species of hagfishes. Unexpectedly, these basal vertebrates have lost a functional Xlox gene from this cluster, unlike every other vertebrate examined to date. Furthermore, our phylogenetic analyses suggest that hagfishes may have diverged from the vertebrate lineage before the duplications, which created the multiple ParaHox clusters in jawed vertebrates.

Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements

Bilaterian animals have a Hox gene cluster essential for patterning the main body axis, and a ParaHox gene cluster. Comparison of Hox and ParaHox genes has led workers to postulate that both clusters originated from the duplication of an ancient cluster named ProtoHox, which contained up to four genes with at least the precursors of anterior and posterior Hox/ParaHox genes. However, the way in which genes diversified within the ProtoHox, Hox and ParaHox clusters remains unclear because no systematic study of non-bilaterian animals exists. Here we characterize the full Hox/ParaHox gene complements and genomic organization in two cnidarian species (Nematostella vectensis and Hydra magnipapillata), and suggest a ProtoHox cluster simpler than originally thought on the basis of three arguments. First, both species possess bilaterian-like anterior Hox genes, but their non-anterior genes do not appear as counterparts of either bilaterian central or posterior genes; second, two clustered ParaHox genes, Gsx and a gene related to Xlox and Cdx, are found in Nematostella vectensis; and third, we do not find clear phylogenetic support for a common origin of bilaterian Cdx and posterior genes, which might therefore have appeared after the ProtoHox cluster duplication. Consequently, the ProtoHox cluster might have consisted of only two anterior genes. Non-anterior genes could have appeared independently in the Hox and ParaHox clusters, possibly after the separation of bilaterians and cnidarians.

Breakup of a homeobox cluster after genome duplication in teleosts.

Several families of homeobox genes are arranged in genomic clusters in metazoan genomes, including the Hox, ParaHox, NK, Rhox, and Iroquois gene clusters. The selective pressures responsible for maintenance of these gene clusters are poorly understood. The ParaHox gene cluster is evolutionarily conserved between amphioxus and human but is fragmented in teleost fishes. We show that two basal ray-finned fish, Polypterus and Amia, each possess an intact ParaHox cluster; this implies that the selective pressure maintaining clustering was lost after whole-genome duplication in teleosts. Cluster breakup is because of gene loss, not transposition or inversion, and the total number of ParaHox genes is the same in teleosts, human, mouse, and frog. We propose that this homeobox gene cluster is held together in chordates by the existence of interdigitated control regions that could be separated after locus duplication in the teleost fish.

The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus

A ParaHox gene cluster is composed of three genes (Gsx, Xlox, and Cdx). It has been proposed that all three ParaHox genes were present in the last common ancestor to the lophotrochozoan protostomes and the deuterostomes and that gene loss event has occurred in the ecdysozoan lineage. In this paper, we report the existence of all three ParaHox genes in Perionyx excavatus, a clitellate annelid. Although orthologs of each of the three ParaHox genes were previously discovered from other lopotrochozoan taxa, this study constitutes the first reported isolation of all three ParaHox genes in the same clitellate species.

All the three ParaHox genes are present in Nuttallochiton mirandus (Mollusca: polyplacophora): evolutionary considerations

The ParaHox gene cluster contains three homeobox genes, Gsx, Xlox and Cdx and has been demonstrated to be an evolutionary sister of the Hox gene cluster. Among deuterostomes the three genes are found in the majority of taxa, whereas among protostomes they have so far been isolated only in the phylum Sipuncula. We report the partial sequences of all three ParaHox genes in the polyplacophoran Nuttallochiton mirandus, the first species of the phylum Mollusca where all ParaHox genes have been isolated. This finding has phylogenetic implications for the phylum Mollusca and for its relationships with the other lophotrochozoan taxa.

Pdx1-related homeodomain transcription factors are distinctly expressed in mouse adult pancreatic islets

Complex gene networks are responsible for the proper operation of the endocrine pancreas. A central member of such networks, the homeodomain transcription factor Pdx1, belongs to the ParaHox gene cluster, an array of Hox-like homeobox genes. With a combination of mRNA in situ hybridisation and immunodetection, we have found that the rest of ParaHox cluster genes, Cdx1, Cdx2/3, and Cdx4, and Gsh1 and Gsh2, are all expressed in specific islet cell types of the endocrine pancreas. To our knowledge, this is the first report that locates ParaHox genes other than Pdx1 and Cdx2/3 in a place as to be involved in the pancreatic transcriptional regulatory networks, potentially regulating glucagon–insulin homeostasis.

Hox, ParaHox, ProtoHox: facts and guesses

The Hox gene cluster has captivated the imagination of evolutionary and developmental biologists worldwide. In this review, the origin of the Hox and ParaHox gene clusters by duplication of a ProtoHox gene cluster, and the changes in their gene numbers in major Metazoan Transitions are reviewed critically. Re-evaluation of existing data and recent findings in Cnidarians, Acoels, and critical stages of vertebrate evolution suggest alternative scenarios for the origin, structure, and changes in Hox gene numbers in relevant events of Metazoan evolution. I discuss opposing views and propose that (i) the ProtoHox cluster had only two genes, and not four as commonly believed: a corollary is that the origin of Bilaterians was coincident with the invention of new Hox and ParaHox gene classes, which may have facilitated such a transition; (ii) the ProtoHox cluster duplication was a cis duplication event, rather than a trans duplication event, as previously suggested, and (iii) the ancestral vertebrate cluster possessed 14 Hox genes, and not the 13 generally assumed. These hypotheses could be verified or refuted in the near future, but they may help critical discussion of the evolution of the Hox/ParaHox family in the metazoan kingdom.

Evolution of the Hox/ParaHox gene clusters.

The Hox gene cluster is a guiding force within the field of Evolutionary Developmental Biology. In large part our understanding of this gene cluster comes from only a few model organisms in developmental biology. The situation is gradually changing. A comparative review of the organisation of the Hox and ParaHox gene clusters and, in particular, instances of cluster disintegration, leads us to the view that the phenomenon of Temporal Colinearity is the major constraining force in maintaining these gene clusters over such long evolutionary timespans.