Figure 7. Characteristics of the Hub.

(A–E) Expression of the indicated genes (upd1, hh, CadN, Fas3, tup) projected onto the testis snRNA-seq UMAP with the 79 definitive hub nuclei outlined (small red box), with reprojection of hub nuclei (larger red box). Color intensity corresponds to expression level, shown as normalized average logFC. Red dashed circles contain non-hub nuclei of cluster 90 (see text and Figure 5). (F-F’’’) Apical tip of adult testis carrying a Tup::GFP fusion transgene revealing protein expression largely restricted to hub nuclei delimited by Fas3 (magenta). Nuclei (blue). Scale bar: 10 μm. (G–J) Paired Venn diagrams, comparing up-regulated genes in the hub vs. clusters containing either CySCs, spermatogonia, hemocytes, or terminal epithelial cells. Overlap in upregulated genes was greatest between Hub and CySCs. In each pairing, circle size reflects the number of genes compared for each cluster. Genes are listed in Figure 7—source data 1; the specific genes for hub vs. CySCs in Figure 7—source data 2. (K) Dot plot of expression of selected genes comparing hub to CySCs, the early Cyst lineage, Spermatogonia, Hemocytes, and Terminal epithelium (see also Figure 7—figure supplements 1 and 2).

Figure 7—figure supplement 1. Hub justification using markers.

Justification for excluding 41 nuclei originally assigned to cluster 90. (A) Cluster 90 nuclei (blue). Red box: 79 nuclei of the definitive hub. Solid and dashed red circles: 4 other spatially separate domains of original cluster 90. Dashed circles: left wing (14 nuclei), right wing (21 nuclei). (B) Expression of Rbp4, a gene highly expressed in the germline, and in both wings but barely detected in the definitive hub. (C–E) Expression of three genes (C) zpg, (D) p53, (E) vas, all highly expressed in the germ line, detected in the right wing but barely detected in the definitive hub. Scale bar: normalized logFC values.

Figure 7—figure supplement 2. Further support for Hub identity using alternate clustering methodology.

(A) Graph-based subclustering of the 120 nuclei originally assigned to cluster 90. (Top) Nuclei colored according to cluster identity, based on highly expressed and highly variable genes in those 120 nuclei. (Bottom) ‘True Hub’ (i.e. the 79 definitive hub nuclei) are colored in red. ‘True Hub’ annotation almost perfectly overlaps with cluster 0 in the graph-based subclustering. (B) A dendrogram derided from hierarchical clustering of the 120 nuclei originally assigned to cluster 90. The 79 definitive hub nuclei largely cluster together (red text). Nuclei are named by their cell barcode, with the suffix ‘_HUB’ added to the 79 hub nuclei. The bolded lines indicate a single clade composed entirely of definitive hub nuclei.