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Evolution of gene regulatory networks in animal development

Final Report Summary - EVONET (Evolution of gene regulatory networks in animal development)

Initial training network (ITN) EVONET

Final publishable summary report

A major challenge for the next generation of researchers in biology will be to integrate knowledge of developmental biology, bioinformatics, functional genomics and evolutionary biology. This requires a new combination of interdisciplinary training to bridge the gap between what are currently quite separate disciplines. The EVONET initial training network (see http://www.itn-evonet.com) brought together eight leading European groups to apply systems biology approaches to the understanding of the evolution of gene regulatory networks (GRNs). We sought to integrate information on GRNs from diverse animal systems, representing all major animal lineages, with particular emphases on the mesoderm specification network and the head regionalisation network. To this end, this network provided early researchers with the skills necessary to apply state of the art systems biology, genomics and bioinformatics tools to emerging model organisms. To reach this goal, we organised two major complementary courses, one focusing on the technology of chromatin-immunoprecipation followed by high throughput sequencing (ChIP-seq) and the subsequent bioinformatic analysis (at the EMBL/Heidelberg) and one focusing on the knowledge of and the molecular approach to diverse marine organisms (in a marine station in Sweden). Yearly graduate schools at all locations of the EVONET partners as well as site-visits at innovative small and medium sized enterprises (SMEs) provided a platform for interaction and soft-skill training for the fellows.

On the scientific level we aimed at generating comprehensive and robust data on the GRN in each of the diverse animal organisms studied among the network members, including vertebrates, sea urchins, sea anemones, fruit flies, polychaete worms, centipedes, urochordates and flatworms. The ultimate and future goal of this project was to be able to make comparisons between organisms spanning the whole animal kingdom in order to identify conserved and divergent nodes and components in the GRNs of shared developmental regulators. As most groups pioneered in the establishment of new model organisms, our milestones were first to establish the reagents and tools in these systems, for instance, by cloning a large number of genes to be studied, by generating antibodies against proteins of interest, by establishing new protocols for chromatin immunoprecipitation (ChIP) followed by high throughput sequencing in organisms where this has never been done and by working out new protocols of isolating and sequencing pluripotent stem cells involved in regeneration.

In both work packages (WPs), i.e. in 'A. Mesoderm network' and 'B. Head regionalisation network', we successfully reached these milestones and even went one step further to generate the corresponding data of the GRN. Newly established collaborations among the EVONET partners have turned out to be most fruitful and will continue to be instrumental in common experimental approaches, meta-analyses and exchanges of lab members.

In the mesoderm network WP, the group of Jim Smith (NIMR London) studied the role of the transcription factor Ladybird in the frog xenopus laevis and - in collaboration with the group of Ulrich Technau (Uni Vienna) - in the sea anemone nematostella vectensis. The early stage researcher (ESR) Anna Strobl performed morpholino knockdown experiments in both organisms and generated a list of potential direct or indirect target genes affected by the perturbation of the ladybird gene. In order to discriminate between direct and indirect target genes and to also identify further target genes, she generated an antibody against xenopus ladybird protein and performed ChIP-seq. In a parallel experiment, both groups also investigated on a genome-wide level all target genes of the transcription factor Brachyury in both the sea anemone and the frog. The results revealed a large set of conserved target genes between these organisms, which are separated for about 600 million years.

Brachyury has also been studied in the sea urchin by the group of Ina Arnone (Stazione Zoologica Naples), besides a number of other mesodermal and myogenic genes. The group successfully generated an antibody against Brachyury and established immunohistochemistry and ChIP-seq protocol in collaboration with the Technau group (Vienna). The mesodermal transcription factors Tinman, twist, Mef2 and bagpipe and their GRN were the focus of the group of Eileen Furlong (EMBL Heidelberg) who compared two drosophila species with remarkably different developmental timing. Obviously, drosophila is technically much more advanced compared to the other animal systems of the ITN, but this project provided equal challenge and training for the fellows as it involved new Drosophila species. Another group contributing to the understanding of the evolution of the mesoderm differentiation was the one of Michael Akam (Cambridge University), who studied a basal arthropod, a centipede. Akam's group has been leading the sequencing of the centipede genome and preliminary analysis has corroborated the ancestral nature of this organism. It also allowed them to isolate many genes of interest, which have been studied by in situ hybridisation in order to get a spatio-temporal network of gene expression during mesoderm formation. Additional studies also provide surprising and important insights into the evolution of segmentation in arthropods.

In the head regionalisation network WP, the groups involved studied in detail the spatio-temporal expression pattern of a number of genes which play a conserved role in at least some model species. The group of Michael Akam (Cambridge) isolated numerous new genes from the centipede and studied their expression pattern during development. They could show by comparative gene expression analysis that most genes show a pattern that is conserved between the centipede and drosophila, arguing for an ancient origin of the head patterning GRN in arthropods. Since part of the network is also conserved to vertebrates, we conclude that the common ancestor of Bilaterians, some 540 million years ago, must have had a head which was patterned by the same set of transcription factors. One of the most well known conserved developmental principles of axial development are the hox genes, which specify the anterior-posterior axis in most animals in a conserved manner. Daniel Chourrout's group (Sars Centre Bergen) studied their role in the closest relatives of the vertebrates, the urochordate Oikopleura dioca, among the most abundant zooplancton in the ocean. Notably, unlike in vertebrates and many other animals, the hox cluster is completely disintegrated in Oikopleura. As yet it is unclear how this impacts on the function of hox genes in axis specification. The Chourrout group generated antibodies against specific hox proteins for ChIP-seq experiments and was able to establish a protocol to study gene function by gene knockdown. Hox genes play an important role in specifying the nervous system along the anteroposterior (AP) axis. Another source of neurons and other cell types is the neural crest, which originates from the margin of the neural fold and the epidermis. The neural crest is considered one of the key innovations of the vertebrate and its evolutionary origin is unclear. The Arendt group (EMBL Heidelberg) addressed this question, by interrogating the expression pattern of a number of conserved neural crest markers in the polychaete worm. On the basis of the concept of the molecular fingerprint they were able to identify a cell population at a corresponding position in the body which expresses the same set of neural crest markers, indicating an ancient origin of these cell types which gave rise to neural crest cells in the vertebrate lineage. Only few organisms have broad capacities of regeneration and even fewer can regenerate a full head with a complete nervous system. An example for such an organism is the planarian flatworm. Regeneration in these animals is governed by pluripotent stem cells, which can give rise to all differentiated cell types of the animal. The group of Nikolaus Rajewsky (MDC Berlin) established a method to dissociate and isolate the stem cells (termed neoblasts) by FACS. They then studied the transcriptome of this purified population of neoblasts by high throughput sequencing. This work revealed hundreds of genes specific to pluripotent stem cells and as such candidates for the involvement of regeneration capacity. Notably, the stem cells in the planarians have a highly similar gene signature as human pluripotent stem cells, suggesting that they could be used as a model for stem cell biology in the future.

Socioeconomic impact of EVONET

This project is predominantly promoting basic research, supported by the non-beneficiary industrial partner AGILENT. The major impact for society lies in the training of the ITN fellows in state of the art techniques and conceptual knowledge from the interdisciplinary and innovative fields of functional genomics, developmental biology, evolutionary biology and systems biology. The successful achievements of the major milestones and objectives opened the field to address new questions in a number of emerging model systems. The results obtained in this ITN revealed patterns of surprisingly deep conservation among distantly related organisms, as well as cases of divergence giving insight into how evolution works on development in order to give rise to diverse body plans. This may contribute to our understanding of fundamental processes in development and disease.