Genomic and transcriptomic survey of bryozoan Hox and ParaHox genes with emphasis on phylactolaemate bryozoans.

BACKGROUND: Bryozoans are mostly sessile aquatic colonial invertebrates belonging to the clade Lophotrochozoa, which unites many protostome bilaterian phyla such as molluscs, annelids and brachiopods. While Hox and ParaHox genes have been extensively studied in various lophotrochozoan lineages, investigations on Hox and ParaHox gene complements in bryozoans are scarce. RESULTS: Herein, we present the most comprehensive survey of Hox and ParaHox gene complements in bryozoans using four genomes and 35 transcriptomes representing all bryozoan clades: Cheilostomata, Ctenostomata, Cyclostomata and Phylactolaemata. Using similarity searches, phylogenetic analyses and detailed manual curation, we have identified five Hox genes in bryozoans (pb, Dfd, Lox5, Lox4 and Post2) and one ParaHox gene (Cdx). Interestingly, we observed lineage-specific duplication of certain Hox and ParaHox genes (Dfd, Lox5 and Cdx) in some bryozoan lineages. CONCLUSIONS: The bryozoan Hox cluster does not retain the ancestral lophotrochozoan condition but appears relatively simple (includes only five genes) and broken into two genomic regions, characterized by the loss and duplication of serval genes. Importantly, bryozoans share the lack of two Hox genes (Post1 and Scr) with their proposed sister-taxon, Phoronida, which suggests that those genes were missing in the most common ancestor of bryozoans and phoronids.

Transcriptional regulation of the Ciona Gsx gene in the neural plate.

The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.

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 ß-clusters) bearing five ParaHox genes (Gsxα, Pdxα, Cdxα, Gsxß, and Cdxß). 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 Gsxß in the ß-cluster is inverted. Interestingly, Gsxß 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.

Amphioxus SYCP1: a case of retrogene replacement and co-option of regulatory elements adjacent to the ParaHox cluster.

Retrogenes are formed when an mRNA is reverse-transcribed and reinserted into the genome in a location unrelated to the original locus. If this retrocopy inserts into a transcriptionally favourable locus and is able to carry out its original function, it can, in rare cases, lead to retrogene replacement. This involves the original, often multi-exonic, parental copy being lost whilst the newer single-exon retrogene copy 'replaces' the role of the ancestral parent gene. One example of this is amphioxus SYCP1, a gene that encodes a protein used in synaptonemal complex formation during meiosis and which offers the opportunity to examine how a retrogene evolves after the retrogene replacement event. SYCP1 genes exist as large multi-exonic genes in most animals. AmphiSYCP1, however, contains a single coding exon of ~ 3200 bp and has inserted next to the ParaHox cluster of amphioxus, whilst the multi-exonic ancestral parental copy has been lost. Here, we show that AmphiSYCP1 has not only replaced its parental copy, but also has evolved additional regulatory function by co-opting a bidirectional promoter from the nearby AmphiCHIC gene. AmphiSYCP1 has also evolved a de novo, multi-exonic 5'untranslated region that displays distinct regulatory states, in the form of two different isoforms, and has evolved novel expression patterns during amphioxus embryogenesis in addition to its ancestral role in meiosis. The absence of ParaHox-like expression of AmphiSYCP1, despite its proximity to the ParaHox cluster, also suggests that this gene is not influenced by any potential pan-cluster regulatory mechanisms, which are seemingly restricted to only the ParaHox genes themselves.

Ordered expression pattern of Hox and ParaHox genes along the alimentary canal in the ascidian juvenile.

The Hox and ParaHox genes of bilateria share a similar expression pattern along the body axis and are known to be associated with anterior-posterior patterning. In vertebrates, the Hox genes are also expressed in presomitic mesoderm and gut endoderm and the ParaHox genes show a restricted expression pattern in the gut-related derivatives. Regional expression patterns in the embryonic central nervous system of the basal chordates amphioxus and ascidian have been reported; however, little is known about their endodermal expression in the alimentary canal. We focus on the Hox and ParaHox genes in the ascidian Ciona intestinalis and investigate the gene expression patterns in the juvenile, which shows morphological regionality in the alimentary canal. Gene expression analyses by using whole-mount in situ hybridization reveal that all Hox genes have a regional expression pattern along the alimentary canal. Expression of Hox1 to Hox4 is restricted to the posterior region of pharyngeal derivatives. Hox5 to Hox13 show an ordered expression pattern correlated with each Hox gene number along the postpharyngeal digestive tract. This expression pattern along the anterior-posterior axis has also been observed in Ciona ParaHox genes. Our observations suggest that ascidian Hox and ParaHox clusters are dispersed; however, the ordered expression patterns along the alimentary canal appear to be conserved among chordates.

Hox and ParaHox genes: a review on molluscs.

Hox and ParaHox genes are involved in patterning the anterior-posterior body axis in metazoans during embryo development. Body plan evolution and diversification are affected by variations in the number and sequence of Hox and ParaHox genes, as well as by their expression patterns. For this reason Hox and ParaHox gene investigation in the phylum Mollusca is of great interest, as this is one of the most important taxa of protostomes, characterized by a high morphological diversity. The comparison of the works reviewed here indicates that species of molluscs, belonging to different classes, share a similar composition of Hox and ParaHox genes. Therefore evidence suggests that the wide morphological diversity of this taxon could be ascribed to differences in Hox gene interactions and expressions and changes in the Hox downstream genes rather than to Hox cluster composition. Moreover the data available on Hox and ParaHox genes in molluscs compared with those of other Lophotrochozoa shed light on the complex and controversial evolutionary histories that these genes have undergone within protostomes.

Genomic organization of Hox and ParaHox clusters in the echinoderm, Acanthaster planci.

The organization of echinoderm Hox clusters is of interest due to the role that Hox genes play in deuterostome development and body plan organization, and the unique gene order of the Hox complex in the sea urchin Strongylocentrotus purpuratus, which has been linked to the unique development of the axial region. Here, it has been reported that the Hox and ParaHox clusters of Acanthaster planci, a corallivorous starfish found in the Pacific and Indian oceans, generally resembles the chordate and hemichordate clusters. The A. planci Hox cluster shared with sea urchins the loss of one of the medial Hox genes, even-skipped (Evx) at the anterior of the cluster, as well as organization of the posterior Hox genes.

Spatial Colinear but Broken Temporal Expression of Duplicated ParaHox Genes in Asexually Reproducing Annelids, Nais communis and Pristina longiseta.

ParaHox genes are key developmental regulators involved in the patterning of the digestive tract along the anteroposterior axis and the development of the nervous system. Most studies have focused on the function of these genes in embryogenesis, while their expression patterns in postembryonic development often remain unknown. In this study, we identified for the first time all ParaHox orthologs in two naidid oligochaetes, N. communis and P. longiseta, and described their expression patterns during normal growth and fission in these animals. We showed that Gsx and Cdx are presented by two paralogs, while Xlox is a single copy gene in both species. Using whole-mount in situ hybridization, we also found that orthologs, except for the Xlox gene, have similar activity patterns with minor differences in details, while the expression patterns of paralogs can differ significantly. However, all these genes are involved in axial patterning and/or in tissue remodeling during growth and asexual reproduction in naidids. Moreover, during paratomic fission, these genes are expressed with spatial colinearity but temporal colinearity is broken. The results of this study may be evidence of the functional diversification of duplicated genes and suggest involvement of the ParaHox genes in whole-body patterning during growth and asexual reproduction in annelids.

Sponges Lack ParaHox Genes.

Addressing the origin of axial-patterning machinery is essential for understanding the evolution of animal form. Historically, sponges, a lineage that branched off early in animal evolution, were thought to lack Hox and ParaHox genes, suggesting that these critical axial-patterning genes arose after sponges diverged. However, a recent study has challenged this long-held doctrine by claiming to identify ParaHox genes (Cdx family) in two calcareous sponge species, Sycon ciliatum and Leucosolenia complicata. We reanalyzed the main data sets in this paper and analyzed an additional data set that expanded the number of bilaterians represented and removed outgroup homeodomains. As in the previous study, our Neighbor-Joining analyses of the original data sets recovered a clade that included sponge and Cdx genes, whereas Bayesian analyses placed these sponge genes within the NKL subclass of homeodomains. Unlike the original study, only one of our two maximum-likelihood analyses was congruent with Cdx genes in sponges. Our analyses of our additional data set led to the sponge genes consistently being placed within the NKL subclass of homeodomains regardless of method or model. Our results show more support for these sponge genes belonging to the NKL subclass, and therefore imply that Hox and ParaHox genes arose after Porifera diverged from the rest of animals.

Comparative genomics reveals the distinct evolutionary trajectories of the robust and complex coral lineages.

BACKGROUND: Despite the biological and economic significance of scleractinian reef-building corals, the lack of large molecular datasets for a representative range of species limits understanding of many aspects of their biology. Within the Scleractinia, based on molecular evidence, it is generally recognised that there are two major clades, Complexa and Robusta, but the genomic bases of significant differences between them remain unclear. RESULTS: Draft genome assemblies and annotations were generated for three coral species: Galaxea fascicularis (Complexa), Fungia sp., and Goniastrea aspera (Robusta). Whilst phylogenetic analyses strongly support a deep split between Complexa and Robusta, synteny analyses reveal a high level of gene order conservation between all corals, but not between corals and sea anemones or between sea anemones. HOX-related gene clusters are, however, well preserved across all of these combinations. Differences between species are apparent in the distribution and numbers of protein domains and an apparent correlation between number of HSP20 proteins and stress tolerance. Uniquely amongst animals, a complete histidine biosynthesis pathway is present in robust corals but not in complex corals or sea anemones. This pathway appears to be ancestral, and its retention in the robust coral lineage has important implications for coral nutrition and symbiosis. CONCLUSIONS: The availability of three new coral genomes enabled recognition of a de novo histidine biosynthesis pathway in robust corals which is only the second identified biosynthetic difference between corals. These datasets provide a platform for understanding many aspects of coral biology, particularly the interactions of corals with their endosymbionts.

Solving Classification Problems for Large Sets of Protein Sequences with the Example of Hox and ParaHox Proteins.

Phylogenetic methods are key to providing models for how a given protein family evolved. However, these methods run into difficulties when sequence divergence is either too low or too high. Here, we provide a case study of Hox and ParaHox proteins so that additional insights can be gained using a new computational approach to help solve old classification problems. For two (Gsx and Cdx) out of three ParaHox proteins the assignments differ between the currently most established view and four alternative scenarios. We use a non-phylogenetic, pairwise-sequence-similarity-based method to assess which of the previous predictions, if any, are best supported by the sequence-similarity relationships between Hox and ParaHox proteins. The overall sequence-similarities show Gsx to be most similar to Hox2-3, and Cdx to be most similar to Hox4-8. The results indicate that a purely pairwise-sequence-similarity-based approach can provide additional information not only when phylogenetic inference methods have insufficient information to provide reliable classifications (as was shown previously for central Hox proteins), but also when the sequence variation is so high that the resulting phylogenetic reconstructions are likely plagued by long-branch-attraction artifacts.

Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle.

INTRODUCTION: Bivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly. RESULTS: We present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome. CONCLUSIONS: Both the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.

Did homeobox gene duplications contribute to the Cambrian explosion?

The Cambrian explosion describes an apparently rapid increase in the diversity of bilaterian animals around 540-515 million years ago. Bilaterian animals explore the world in three-dimensions deploying forward-facing sense organs, a brain, and an anterior mouth; they possess muscle blocks enabling efficient crawling and burrowing in sediments, and they typically have an efficient 'through-gut' with separate mouth and anus to process bulk food and eject waste, even when burrowing in sediment. A variety of ecological, environmental, genetic, and developmental factors have been proposed as possible triggers and correlates of the Cambrian explosion, and it is likely that a combination of factors were involved. Here, I focus on a set of developmental genetic changes and propose these are part of the mix of permissive factors. I describe how ANTP-class homeobox genes, which encode transcription factors involved in body patterning, increased in number in the bilaterian stem lineage and earlier. These gene duplications generated a large array of ANTP class genes, including three distinct gene clusters called NK, Hox, and ParaHox. Comparative data supports the idea that NK genes were deployed primarily to pattern the bilaterian mesoderm, Hox genes coded position along the central nervous system, and ParaHox genes most likely originally specified the mouth, midgut, and anus of the newly evolved through-gut. It is proposed that diversification of ANTP class genes played a role in the Cambrian explosion by contributing to the patterning systems used to build animal bodies capable of high-energy directed locomotion, including active burrowing.

ParaHox genes in pancreatic cell cultures: effects on the insulin promoter regulation.

The gene encoding PDX1 (pancreatic duodenum homeobox 1), the main transcription factor regulating the glucose-dependent transactivation of the insulin promoter in pancreatic beta-cells, clusters with two closely related homeobox genes (Gsh1 and Cdx2/3), all of them belonging to the ParaHox gene family. The ParaHox gene evolutionary history in the vertebrate lineage involved duplications of the cluster and subsequent loss of some members, so that eventually, the human and murine genomes contain only 6 ParaHox genes. The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established. There is some data on CDX2/3 function in alpha-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas. Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters. Therefore, we explored the effects of the Parahox proteins (GSH1, GSH2, CDX1, CDX2/3 and CDX4) on the regulation of the insulin promoter in transfected alpha- and beta- cultured cell lines at different glucose concentrations and compared them to those of PDX1. Noticeably, several ParaHox transcription factors are able to transactivate or inhibit the insulin promoter, depending on the cell type and glucose concentration, thus suggesting their possible participation in the regulation of similar target genes, such as insulin, either by silencing or activating them, in the absence of PDX1.