Stepwise emergence of the neuronal gene expression program in early animal evolution

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.We thank Alex de Mendoza, Manuel Irimia, and Marta Iglesias for critical comments on the manuscript. We also thank Nikolaus Leisch for advice on placozoan fixation for in situ hybridization; Vikas Trivedi, Krisztina Arato, Kerim Anlas, and Maayan Schwarzkopf for help with in situ HCR; and Jase Gehring, Tara Chari, and Lior Pachter for counsel about the Clicktag (CT) protocol. Finally, we thank Niccolò Arecco for help with ChimeraX and Òscar Fornas and the CRG flow cytometry staff for help with FACS sorting and flow cytometry data analysis. Research in A.S.-P. group was supported by the European Research Council (ERC-StG 851647), the Spanish Ministry of Science and Innovation (PID2021-124757NB-I00), and AGAUR (2021-SGR2021-01219). We also acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme (Generalitat de Catalunya). Research in B.S. group was supported by the Alexander von Humboldt Foundation and a TiHo President’s grant (ITZ-20-2). H.G.-V. was funded by the Max Planck Society through N. Dubilier and by the DFG (495424697). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech). E.S. and C.C. acknowledge support from the Spanish Ministry of Science and Innovation (PID2020-115092GB-I00) and AGAUR (2021SGR01225). D.C and L.S acknowledge funding from the FWO (S000722N). X.G.-B. is supported by the European Union’s H2020 research and innovation program under Marie Skłodowska-Curie grant agreement 101031767. A.E. and C.N. are supported by FPI PhD fellowships from the Spanish Ministry of Science and Innovation