Fig. 3: The diversity of cell type, spatial location and gene regulation of hypothalamic projection neurons.

a, Joint t-SNE of epi-retro-seq (n = 1,572), unbiased snmC-seq (n = 11,554) and scRNA-seq (n = 148,840) data for hypothalamic neurons coloured by cell cluster (top) or projection target (bottom, same colour palette as left row colours in Fig. 1b). Seventeen clusters enriched for the profiled projection neurons are outlined. b, The proportion of each of the 10 projections in each of the 17 projection-enriched clusters, z-score normalized across targets. The asterisk denotes enrichment with FDR < 0.01 (one-sided Fisher exact test, Benjamini–Hochberg procedure; Methods). c,d, t-SNE of cells in the 17 projection-enriched clusters coloured by co-cluster. All cells are colored in c, whereas only cells projecting to one target are colored in each of the subplots in d. e,f, Projection-enriched HY clusters mapped to MERFISH data for six coronal slices (e; two biological replicates (R1 and R2) of slices from anterior to posterior (C6, C8 and C10)) and two sagittal slices (f; S1 and S2 from lateral to medial). Examples of clusters with specific spatial locations are labelled in the enlarged insets of each slice. Scale bars, 15 mm. The colour palette for clusters is the same in b–f. g–i, Normalized gene expression (norm expr, g) and gene-body mCH (h) levels of DEGs (n = 1,163) between the 17 projection-enriched clusters, and mCG levels of DEG-associated DMRs (i; n = 148,897), in each cluster. The values are z-score normalized across clusters. The DEGs and cell clusters are arranged in the same orders in g–i. Only the DMRs with the highest anticorrelation with each DEG are shown in i to make the column orders consistent for g–i. Examples of DEGs with GO annotations related to neuronal function and connectivity are labelled on the x axis. j, Examples of TFs whose binding motifs were enriched in hypo-CG-methylated DMRs of each cluster are shown in the bubble plot. The size of each dot represents the enrichment level (area under the curve (AUC)). The colour of the dot indicates the expression (expr.) level of the TF. The clusters are arranged in the same order as in g–i. Cluster 76 was excluded in i,j as the number of cells in the cluster is too small (<30) to be included in the DMR analysis. k, Example triplet combination of TF–CRE–target gene in GRN of HY. Pearson correlation coefficients (PCCs) are shown on edges. The t-SNE plots share the same coordinates as c. Single cells are greyed and shown in the background. Large dots represent clusters, plotted at the mean coordinates of cells in the cluster. The size of each dot is proportional to the number of cells in the cluster. The dots are coloured by Zic1 expression (left), mCG level of an example DMR (middle) and Zic4 expression (right). Colour scales for gene expression are the same as in g, and the colour scale for mCG is the same as in i.