Hydra vulgaris AEP and Hydra vulgaris transgenic lines were dissociated into single cells and were prepared for Drop-seq (49 (link)); FACS was used to enrich for neurons. Sequencing reads were mapped to a de novo assembled transcriptome and a Hydra genome reference and clustering was performed. Subclustering was performed on the following subsets of the data: epithelial ectodermal cells, epithelial endodermal cells, interstitial cells, and neurons and neuronal progenitors. The in situ location of neuron subclusters was determined using in situ hybridization and differential gene expression analysis of separated epithelial layers. URD (24 (link)) was used to build differentiation trajectories for the interstitial and male germline lineages and to analyze the spatial expression of genes in the ectodermal, endodermal, and gland lineages. To analyze regulatory regions, co-expression modules were identified using NMF, ATAC-seq was performed to identify regions of open chromatin, and motif enrichment analysis was used to identify candidate regulators of the gene modules. Colorimetric in situ hybridization, fluorescent in situ hybridization, immunohistochemistry, and generation of transgenic lines was performed and used to validate biomarkers and cell states. For complete methods see supplementary material and methods.