The evolution of Runx genes II. The C-terminal Groucho recruitment motif is present in both eumetazoans and homoscleromorphs but absent in a haplosclerid demosponge

Background. The Runt DNA binding domain (Runx) defines a metazoan family of sequence-specific transcription factors with essential roles in animal ontogeny and stem cell based development. Depending on cis-regulatory context, Runx proteins mediate either transcriptional activation or repression. In many contexts Runx-mediated repression is carried out by Groucho/TLE, recruited to the transcriptional complex via a C-terminal WRPY sequence motif that is found encoded in all heretofore known Runx genes. Findings. Full-length Runx genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans, including placozoans and sponges, the most basally branching members of that clade. No sequences with significant similarity to the Runt domain were found in the genome of the choanoflagellate Monosiga brevicollis, confirming that Runx is a metazoan apomorphy. A contig assembled from genomic sequences of the haplosclerid demosponge Amphimedon queenslandica was used to construct a model of the single Runx gene from that species, AmqRunx, the veracity of which was confirmed by expressed sequences. The encoded sequence of the Runx protein OscRunx from the homoscleromorph sponge Oscarella carmella was also obtained from assembled ESTs. Remarkably, a syntenic linkage between Runx and Supt3h, previously reported in vertebrates, is conserved in A. queenslandica. Whereas OscRunx encodes a C-terminal Groucho-recruitment motif, AmqRunx does not, although a Groucho homologue is found in the A. queenslandica genome. Conclusion. Our results are consistent with the hypothesis that sponges are paraphyletic, and suggest that Runx-WRPY mediated recruitment of Groucho to cis-regulatory sequences originated in the ancestors of eumetazoans following their divergence from demosponges

Early evolution of the LIM homeobox gene family

Background: LIM homeobox (Lhx) transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template

GENOME CONTENT AND DEVELOPMENTAL EXPRESSION OF TRANSCRIPTION FACTOR GENES IN THE DEMOSPONGE AMPHIMEDON QUEENSLANDICA: INSIGHTS INTO THE FIRST MULTICELLULAR ANIMAL

Recent phylogenetic insights suggest that the body plan and life cycle of the oldest metazoans, sponges, may resemble the last common ancestor to all animals. While their adult body plan differs drastically from other animals, sponge development displays many metazoan hallmarks. Enabling a framework for the spatial organisation of a variety of cell types, the invention of a basic developmental program may have been the key step allowing the transition to multicellularity in animals. Novel transcription factors were likely to have been crucial to the increase in regulatory complexity that must have accompanied this step. Many developmental regulatory pathways are conserved throughout animal evolution and investigations into the molecular basis of eumetazoan development have led to reconstruction of ancestral developmental states. In this study, I investigate the genomic and developmental innovations that occurred at the base of Metazoa in view of reconstructing the first metazoan developmental program and identifying fundamental characters that enabled the transition to multicellularity in animals. As in other metazoans, in the haplosclerid demosponge Amphimedon queenslandica, fertilisation, cleavage, and gastrulation yield a larva with various cell types patterned along an anteriorposterior axis and into germ layers. Non-neural sensory structures enable the larva to respond to environmental conditions. Drawing on the recently sequenced genome of A. queenslandica, I show that this demosponge has a limited set of homeobox, Sox, Fox, T-box, and nuclear receptor transcription factors, which have important roles during eumetazoan development. Using in situ hybridisation on sponge developmental stages for the first time, I also analyse the expression of a subset of these genes during development. Remarkably, A. queenslandica possesses a clear NK cluster of ANTP genes but lacks Hox, ParaHox, or EHGBox genes. This suggests that an NK cluster was present in the metazoan last common ancestor and that a protoHox gene - progenitor to Hox, ParaHox, and EHGBox genes - arose from within this cluster after demosponges had branched off the main metazoan lineage. Importantly, this implies that Hox genes were co-opted into an existing system for anteriorposterior axial patterning. Using the completed genomes of A. queenslandica and the cnidarian Nematostella vectensis as well as preliminary genome traces of the choanoflagellate Monosiga brevicollis, I have analysed the origin and early evolution of transcription factors through phylogenetic analyses. Metazoanspecific transcription factor classes seem to have evolved before demosponges departed from the main metazoan lineage. It appears that these classes gradually diversified by duplication in different ways, particularly in the periods preceding and following the demosponge/metazoan split. Largely unlike cnidarians, sponges seem to constitute an intermediate stage in the evolution of the metazoan genome and, as such, the transcription factor genes present in their genome may have been part of a core developmental program sufficient for the transition to multicellularity. Many transcription factor genes are expressed during A. queenslandica development and a subset of these genes seems to display some conservation in their developmental expression between sponges and other metazoans. AmqHNF4 expression suggests that epithelium evolution went through a stage without a basement membrane. Based on AmqLim3 and AmqSoxB expression, neurons may be related to pre-neural cells associated with photo- and chemosensory structures. AmqBsh and AmqSoxF were probably involved in regulation of cell adhesion and extracellular matrix molecules during the development of the metazoan ancestor. AmqProx2, AmqPaxB, and AmqPoul expression is only detected in early embryogenesis while AmqSoxC, like most other genes in this study, may be involved in fate determination and differentiation of a specific migrating cell lineage throughout development. Expression data paint the picture of an ancestral metazoan developmental program where asymmetric cleavage participates in early cell fate determination and differentiation and where specified cells respond in different ways to signalling cues by following complex migratory trajectories throughout embryogenesis. From a genomic and developmental perspective, sponges appear to be an intermediate in metazoan evolution. The developmental program seems to have gradually increased in complexity in pre-Cambrian animals. However, the last common ancestor of all extant metazoans may have already utilised many of the same molecular tools during development in the same way as most contemporary animals. The evolutionary steps that occurred at the base of Metazoa and led to a relatively complex core developmental program do not seem to have been recorded until present day

Does the high gene density in the sponge NK homeobox gene cluster reflect limited regulatory capacity?

A huge discrepancy in morphological diversity exists between poriferans and eumetazoans. The disparate evolutionary outcomes of these two ancient metazoan lineages may be reflected in the composition, architecture, and regulation of genomes of modern representatives. As a case study, we compare the sizes of upstream intergenic regions of genes found within the NK homeobox cluster of the demosponge Amphimedon queenslandica with eumetazoan orthologs. This analysis includes NK genes as well as five structural genes interspersed in the cluster. The upstream intergenic regions of the homeobox genes are significantly smaller in Amphimedon than in eumetazoan orthologs, suggesting that the sponge genes house less cis-regulatory information. In contrast, the upstream intergenic regions of the structural genes are not significantly different. The simple developmental expression patterns of representative NK genes in Amphimedon lends support to the proposition that their regulatory apparatuses, unlike those of bilaterians, do not encode the information for dynamic, pleiotropic gene expression. On the basis of this example, we suggest that the size of the intergenic regions upstream of the transcription start site may act as a proxy for estimating regulatory complexity and reflect the limitations of the sponge genome to direct complex and varied morphogenetic processes

Whole-mount in situ hybridization in Amphimedon

Developmental gene expression is analyzed predominantly via whole-mount in situ hybridization using digoxigenin-labeled RNA probes. This protocol describes how to perform this procedure in Amphimedon queenslandica, including fixation, hybridization, and sectioning of embryonic, larval, and post-larval juvenile stages

Demosponge and Sea Anemone Fibrillar Collagen Diversity Reveals the Early Emergence of A/C Clades and the Maintenance of the Modular Structure of Type V/XI Collagens from Sponge to Human*S⃞

Collagens are often considered a metazoan hallmark, with the fibril-forming fibrillar collagens present from sponges to human. From evolutionary studies, three fibrillar collagen clades (named A, B, and C) have been defined and shown to be present in mammals, whereas the emergence of the A and B clades predates the protostome/deuterostome split. Moreover, several C clade fibrillar collagen chains are present in some invertebrate deuterostome genomes but not in protostomes whose genomes have been sequenced. The newly sequenced genomes of the choanoflagellate Monosiga brevicollis, the demosponge Amphimedon queenslandica, and the cnidarians Hydra magnipapillata (Hydra) and Nematostella vectensis (sea anemone) allow us to have a better understanding of the origin and evolution of fibrillar collagens. Analysis of these genomes suggests that an ancestral fibrillar collagen gene arose at the dawn of the Metazoa, before the divergence of sponge and eumetazoan lineages. The duplication events leading to the formation of the three fibrillar collagen clades (A, B, and C) occurred before the eumetazoan radiation. Interestingly, only the B clade fibrillar collagens preserved their characteristic modular structure from sponge to human. This observation is compatible with the suggested primordial function of type V/XI fibrillar collagens in the initiation of the formation of the collagen fibrils

Whole-Mount In Situ Hybridization in Amphimedon

INTRODUCTIONDevelopmental gene expression is analyzed predominantly via whole-mount in situ hybridization using digoxigenin-labeled RNA probes. This protocol describes how to perform this procedure in Amphimedon queenslandica, including fixation, hybridization, and sectioning of embryonic, larval, and post-larval juvenile stages