Workpackage 1: Description of the development of target species using advanced imaging technologies and molecular approaches.
During the whole Action we focussed on the description of the development of multiple target species and here with a focus on molecular pathways that are related to mesoderm development. With this project we delivered new insights into the developmental process in all the target species. Further, we included new species which showed mesodermal organ development specifics (e.g. phoronids). One of the major insights came from the description of the processes of gastrulation, the time in embryogenesis, during which the germ layers including the mesoderm are specified. Here we discovered that the behaviour of the site of gastrulation (blastopore) is depending on the processes of axial elongation and mesoderm internalization and does not recapitulate evolutionary ancient processes, as it was proposed by Ernst Haeckel (Martin-Duran et al 2015, Nature Ecology and Evolution). Regarding the late development of mesodermal organs, we studied the nephridiogenesis from a comparative aspect and provided the molecular basis for the homology of all excretory organs that are nephridia like (Gasiorowski et al 2022).
Workpackage 2: Bioinformatic approaches to study genes involved in mesoderm development
During the whole project we studied the gene orthology of mesodermal transcription factors and signalling pathways throughout the animal kingdom. We applied modern phylogenetic analysis and expanded our studies also to structural genes e.g. in Andrikou et al 2019 PLOS Biology). This challenge we learned to overcome by using advanced orthology assessment, which was successful for most genes. With the emergence of single cell sequencing technologies, we started to early implement these methods. We show that is is essential to incorporate phylogenetic methods in comparative molecular studies (Dunn et al 2018, PNAS) and that care should be taken when homologizing structures based on their molecular underpinnings.
Workpackage 3: Functional studies to test the developmental role of genes using gene knockout and knockdown technologies
Functional studies on non-model organisms are very difficult and this has been proven again in this project. It was one of the high/medium risk elements of EVOMESODERM. However, we performed the inference of signalling pathways using inhibitors in numerous of our studies and were able to dissect the function of e.g. the FGF pathway during mesoderm development in spiralian species. Injection of snRNA, morpholinos were hampered by the fact that some embryos are extremely difficult to inject, even by injection experts.
Workpackage 4. Transgenic animal production
Although we switched to CRISPr Cas9 genome editing from the proposed TALEN techniques, throughout the project it was difficult to establish one species as the major research organism, where the effort to establish the method would have been fruitful. The discoveries made in the other work packages were the main drivers of the studies. In addition, it turned out that in many species mesoderm formation is a very late process from an embryonic perspective, and the transgenesis would infere with earlier processes. In summary, it turned out to be more successful to 1. expand the study to more species and 2. include more pathways for understanding the evolutionary process than to focus on the establishment of transgenesis in 1-2 species.
