Periodic Reporting for period 4 - EVOMESODERM (The evolution of mesoderm and its differentiation into cell types and organ systems)
Reporting period: 2019-12-01 to 2021-05-31
Objective 1: Determining the evolutionary origin of the mesoderm and characterizing the
nature of the first mesodermal tissues.
Objective 2: Identifying the changes in developmental programs underlying mesoderm
formation that have caused its diversification in different animal lineages.
Objective 3: Tracing the evolutionary differentiation of mesodermal cell types and their
organization into novel organ systems.
We have described and published the development of two target species with gene expression and morphological approaches, the bryozoan M. membranacea (Vellutini et al 2017, BMC Biol) and Lineus ruber (Martin-Duran et al 2015, EvoDevo). Furthermore, we have established a safer in situ hybridization protocol in which formamide is replaced by Urea (Sinigaglia et al 2017 Dev Biol). The study of the target species T. transversa and P. caudatus have been described in several different publications, each focussing on a specific aspect of the development and often in the context and comparison with other species. For example, data from the study of the brachiopod T. transversa is part of the publication about neural elements (Martin-Duran et al 2017, Nature) and the publication about the fate of cells of the blastopore is published in Martin-Duran et al 2016, Nature Ecol Evol. In both manuscripts it was necessary to include mesodermal candidate genes to better discriminate the early cell fates in the embryo. Especially the results of the latter manuscript are highly relevant for this project, since we discovered that the location of the early mesoderm specification in the gastrula influences the future fate of the blastopore (protostomic or deuterostomic).
Workpackage 2: Bioinformatic approaches to study genes involved in mesoderm development
First we developed an advanced orthology assessment pipeline called “Leap-frog” (Martin-Duran et al 2017, Genome Research) in line with the proposed use of larger taxon sampling across animal taxa. This pipeline allows the improved detection of orthologs and the detection of ‘hidden-orthologs’, fast evolving orthologs that cannot be detected with other methods. We also tested the effect of comparative methods versus very commonly used pairwise comparisons of transcriptomic data. To our surprise we found that pair-wise comparisons can lead to false conclusions about evolutionary processes and that it is necessary to use comparative methods when analysing data on an evolutionary background (published in Dunn et al 2018 PNAS). Aware of this issue and having novel tools in our hands we are currently analysing the gene complement in major animal evolutionary lineages (our and others published genomes and transcriptomes) which will be published as a reference compendium.
Workpackage 3: Functional studies to test the developmental role of genes using gene knockout and knockdown technologies
Some of our recent publications of the descriptive approach contain already the in the proposal outlined experiments using pharmaceutical inhibitors against crucial developmental pathways (see Vellutini et al 2017; Martin-Duran et al 2016; 2017). The application of these inhibitors is informative to a specific degree and can be easily applied to the embryos. We combine these experiment with live-recordings (Vellutini et al 2017). During the course of the project we have also applied inhibitors against the ammonium secretion pathway to also characterize the role of nephridia in this process better (Andrikou et al. BioRxiv doi:10.1101/136788). This is a promising approach that will also be applied in the other species. The improvement of this publication also includes successfully RNAi as gene specific knockdown tool in I. pulchra.
Workpackage 4. Transgenic animal production
We switched to CRISPr Cas9 genome editing and are currently establishing the method in one of the target species (Dinophilus gyrociliatus).
Objective 1: “Origins” - Determining the evolutionary origin of the mesoderm and characterizing the nature of the first mesodermal tissues of animals.
Our research reveals so far that the first mesodermal tissue is musculature. Our approach to detect discrete excretory organs in xenacoelomorphs led to the novel insight that excretion before the evolution of specialized excretory organs likely happened through the digestive tissue (Andrikou et al 2017 biorXiv: 10.1101/136788). This raises the question how new mesodermal tissues can originate from musculature (if mesoderm is homologous at all) and are right in the question about how new cell types evolve. We currently follow up on this fascinating topic and test the hypothesis about a possible multiple origin of mesoderm.
Objective 2: “Deviations” - Identifying the changes in developmental programs underlying mesoderm formation that have led to diversification in different animal lineages.
The most significant finding so far is that the place and time of mesoderm formation plays a key-role in determining the fate of the blastopore (Martin-Duran et al 2017, Nature Ecology & Evolution). Equal distribution of mesodermal precursors around the blastopore causes a deuterostomic embryo, while asymmetric shifts of the cells during gastrulation to different locations can cause protostomic embryos. This fundamental difference in animal embryogenesis can be deducted from a simple early embryonic change in the way mesodermal precursors are internalized into the embryo.
Objective 3: “Novelties” - Tracing the evolutionary diversification of mesodermal cell types and their organization into novel and specialized organ systems.
Our bioinformatics approach to the cell type sequencing delivered evidence that nephridia, although originating partly from ectoderm, express many mesodermal genes. Here we cannot exclude a re-programming of ectodermal cells to mesodermal ones during development, which questions the germ layer consistency. Further experiments are currently conducted to test this hypothesis.