Differential gene regulation in DAPT-treated Hydra reveals molecular pathways dependent on Notch signalling during interstitial cell differentiation and formation of the oral-aboral axis inHydra

The Notch pathway is highly conserved and essential for animal development. We investigated the function of Notch-signalling inHydraby using the presenilin inhibitor DAPT, which efficiently blocks propagation of Notch-signals. InHydra, DAPT treatment prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. Moreover, it causes defects in theHydrahead by compromising the head organizer. In order to understand the molecular mechanisms by which the Notch pathway regulates these processes we performed RNAseq to identify genes that are differentially regulated in response to 48 hours of DAPT-exposure. This revealed downregulation of 624 genes and upregulation of 207 genes. To identify candidate direct regulators of Notch-signalling, we also profiled gene expression changes that occur during restoration of Notch-activity 3 and 6 hours after DAPT-removal. We then analysed gene expression patterns of these Notch-responsive genes in untreated animals by interrogating the available single cell sequencing data set for untreated animals and found that almost half of the Notch responsive genes were specifically expressed in nematocytes and nematocyte progenitors. This confirms the critical role for Notch-signalling in nematocyte development. Promoter analyses and gene expression profiling after DAPT-removal suggested an indirect role for Notch in regulating aPOU-transcription factor, which is critical for nematogenesis. In support of a role for Notch-signalling in head organizer formation, we identified several head organizer genes in the Notch regulated gene data set, includingCngsc, a homologue ofgoosecoid,a gene associated with the Spemann organizer, and the Wnt pathway genesSp5, TcfandWnt-7.Finally, the expression levels of the tentacle patterning genesHyAlxandSp5rapidly recovered after DAPT removal. Given that these genes possess Notch-responsive RBPJ transcription factor binding sites in their regulatory regions, these genes are likely directly targeted by Notch signalling. In summary, our data provide a comprehensive picture of the molecular pathways regulated by Notch signalling in interstitial cell differentiation and formation of the oral-aboral axis inHydra.