Final Report Summary - MESODERM EVOLUTION (The development and evolution of the mesoderm in basal bilaterian acoel worms) We have investigated the mesoderm development in the representative acoel species Isodiametra pulchra using fate map analysis and gene expression studies. The primary objectives of the proposal were:Objective 1: Description of endoderm and mesoderm formation and identification of candidate genes in an acoel species.We have used 4D-microscopic and fate mapping approaches to characterise the mesoderm development in the acoel Isodiametra pulchra. Our results that the mesoderm is formed from two blastomeres at the vegetal pole of the 16-cell stage embryo. This is consistent with previous descriptions but we have gained a more detailed fatemap using confocal imaging of living fluorescently labeled worms. We found that the acoel stem cells (neoblasts) - are in low numbers in the juvenile worm and will grow to larger numbers during development. We have studies the expression of mesodermal transcription factors in the acoel and found that only a small number of mesodermal cell types are present. The so-called ‘parenchyme’ must be secondarily derived from mesoderm in the lineage to the Acoel a (Chiodin et al 2013). This work is a starting point and also led to the establishment of several methods in the acoel species I. pulchra: I. microinjection of cell tracing dyes into individual blastomeres of the 16-cell stage, II. Live imaging microscopy using a 3D-timelapse microscope II. double fluorescent in situ hybridization in combination with EdU as stem cell marker. Further investigations will be necessary to unravel the differentiation of mesodermal cell types.Objective 2: Quantification and timing of expression of candidate genesWe have successfully established a method for stage specific next generation sequencing to determine the rate of expression during different developmental stages. This replaced the method originally proposed (microarrays). In combination with the sequenced genome of Isodiametra pulchra, we are able to determine the amount of expression during a dozen developmental stages. Currently we are improving the method to single-cell sequencing of blastomeres and tissues to determine the temporary and quality changes during the development. The tissue specific transcriptomes will also help to determine lineage specific genes that are responsible for mesoderm formation.Objective 3: Approach to identify the specific function of the genes and the reconstruction of the gene regulatory networkThe most ambitious objective was only reached partially. We have established RNA inference methods by injecting double stranded RNA into individual blastomeres. We were successfully able to knock down the function of some genes. We also decided to establish ballistic transformations in Isodiametra pulchra.We have furthermore contributed to sequencing the genome and transcriptome of the acoel which is currently being analyzed in comparative manner with that of other acoel, nemertodermatid and Xenoturbella species.