Aim 1 (“Catalogue cell types, regulatory regions and gene regulatory networks across placozoans”) was fully fulfilled, albeit with less species than initially planned (four instead of six) due to sampling constraints, selecting four species from three genera (Trichoplax, Hoilungia and Cladtertia) instead. This updated sampling covers the global same spectrum of phylogenetic diversity.
Then, we produced complete whole-organism atlases of cell behaviour using single-cell transcriptomic approaches (~15,000 cells sampled in each of the four species), covering all known cell types and even revealing novel ones. In parallel, we produced a complete catalogue of active regulatory regions using bulk ATAC-seq, which we then linked to cell type-specific genes (obtained from the expression data) to obtain cell type-specific sets of regulatory regions. Finally, gene coexpression patterns at the cellular level were leveraged to group genes into coordinated modules. Using this information, I sought to establish the degree of evolutionary conservation and divergence between the four sampled species (“Aim 2 – A cross-species comparative analysis of the determinants of cell type identity”). All four species possessed the same basic cell types (epidermal, gland-secretory, lipophilic cells, fibre contractile cells, and peptidergic-secretory cells), which could be further subdivided in each of the species into functionally distinct subtypes (particularly the peptidergic ones). Based on patterns of expression similarity of orthologous genes across species, I was able to build a phenetic cell type tree that approximated evolutionary relationships between these cell type expression programmes across hundred of millions of years of evolution. However, attempts to obtain similar results based on regulatory region activity were largely unsuccessful, which indicated that this trait was faster-evolving and thus less likely to reflect the underlying phylogenetic signal (“Aim 3 – Build models of cell type and regulatory evolution in placozoans”). This unexpected result prompted me to quantify the rate of conservation decay of various determinants of cell type identity over evolutionary time, revealing consistent patterns whereby, within each homologous cell type, gene expression was more conserved than regulatory element usage (and, within genes, master transcriptional regulators were more conserved than downstream effector genes). Crucially, I was also able to contrast these patterns with those of genome-level sequence evolution.
