Gene regulatory networks of mesodermal transcription factors in Cnidaria

One of the fundamental questions in Biology is how complex body plans evolved during animal evolution. Until recently, it was widely assumed that increasing morphological complexity went hand in hand with a corresponding increase in genetic complexity. However, recent genome and transcriptome analyses from a variety of "simple" or basal organisms revealed that the gene repertoire is astonishingly similar in all animals examined. If complexity of the gene repertoire itself is not causal for the emergence of new structures and body plans, then the regulatory networks that link these genes may be the critical factor. One crucial step in animal evolution was the emergence of the third germ layer, the mesoderm. While most animals are composed of three germ layers (endoderm, ectoderm and mesoderm), which are formed during early embryogenesis, mostly at gastrulation, a few basal phyla, including the Cnidaria, consist of only two germ layers, ectoderm and endoderm. We propose to analyse the gene regulatory networks (GRNs) of a few well-selected transcription factors, Brachyury, FoxA, Twist and Mef2, in the sea anemone Nematostella vectensis on a genome- wide level. These proteins play crucial roles in the formation and differentiation of the mesoderm in Bilateria. In Cnidaria, they are either involved in gastrulation or in endoderm formation, but obviously not in mesoderm differentiation. How the corresponding genes became mesodermal master regulators will be addressed by revealing all target genes of these transcription factors on a genome-wide level. The method of choice is Chromatin- Immunoprecipitation followed by massive sequencing (ChIP-seq). Mapping of these reads will reveal all binding sites in the genome in wildtype or gene knockdown conditions. To unravel the relevance of the binding events, we will combine the ChIP-seq with RNA-seq from wildtype and perturbed embryos of defined developmental stages. These data will then be compared with existing or currently emerging data from Bilateria in order to reconstruct how GRNs in development can evolve and contribute to the formation of new morphological structures.

How did complex body plans evolve and is this correlated with changes in the genome? This is one of the fundamental questions in evolutionary biology. In this respect cnidarians (sea anemones, corals and jellyfish) are particularly instructive as they are morpholigically simple and split about 600 Mio years from the sister group, the Bilateria. One of the key differences between Bilateria and Cnidaria is that Bilateria are built from three germ layers, while Cnidaria are composed of two cell layers, and seem to lack the mesoderm, which gives rise to muscles and bones in vertebrates. Genome analyses revealed a surprising complexity of cnidarian genomes and in fact cnidarians possess most of the "mesodermal" developmental determinants. In order to trace the origin of the mesoderm, we identified in genome-wide analyses all target genes regulated by the conserved mesodermal determinants in the sea anemone Nematostella vectensis and compared them to those in sea urchin and diverse vertebrates. We identified some very conserved parts of the network that date back 600 Mio years to the common ancestor, but also those, which are specific to individual lineages like vertebrates. Future validations of these candidate genes may finally explain the evolution of the mesoderm. To test, whether there is already some sort of segregation of mesodermal and endodermal functions in the diploblastic cnidarians, we analysed the spatial expression of over 50 mesodermal and endodermal marker genes in the sea aneomen. Surprisingly, we found that all endodermal function genes (e.g. digestive enzymens, Insulin) are expressed in the most inner tip of the internal septae, called septal filament, while all "mesodermal" functions (muscle, gonad, nutrient storage) are localized in the rest of the endoderm. Thus, despite the diploblastic organisation, there is already a topological separation of functions. To address how the decision to generate ectoderm and endoderm is made we analysed body axis and germ layer formation in aggregates of dissociated embryonic cells. We found that this decision is governed by a small number of organizer cells at the margin of the blastopore, marking the boundary between ectoderm and endoderm. These cells exert this function by secreting specific signaling factors of the Wnt family, which are sufficient - if applied ectopically - to induce the differentiation of endoderm in cells normally fated to become ectoderm. These experiments reveal the plasticity of cell fate decision making in a self-organising developmental system. Taken together, our work contributed to the understanding how a major step in evolution, the formation of the third germ layer, may have occured by changes in the gene regulatory network of conserved transcription factors.