Fig. 4: Cell type-specific depletion of eRegulon TFs in the adult testis reveals functional roles in stem cell regulation.
From: Dissecting the enhancer gene regulatory network in early Drosophila spermatogenesis
a Schematic of the experimental strategy for TF validation. Lineage-specific GAL4 drivers (carrying UAS-Cas9) were crossed to U6:gRNA lines targeting selected TFs. Crosses were maintained at 18 °C until eclosion, then shifted to 29 °C to induce GAL4-driven expression. b Left: UMAP embedding of the dataset in transcriptional space, with cells colored by lineage-specific latent time. Right: Expression of indicated genes projected onto the UMAP; expression levels are shown as increasing intensity of purple. c Left: Representative smFISH images of Mad transcripts in control and germline-specific Mad knockout (KO) testes. Right: Quantification of Mad smFISH signal across genotypes. d Left: Representative images of control and germline-specific Mad CRISPR mutagenesis testes. Right: Quantification of GSC number, GSC diameter, and total germ cell number. e Left: Representative smFISH images of crc in control and germline-specific crc CRISPR mutagenesis testes. Right: Quantification of crcsmFISH signal. f Left: Control and crc CRISPR mutagenesis testes. Right: Quantification of GSC number, GSC diameter, and total germ cell number. g Left: Representative smFISH images of ovo in control and germline-specific ovo CRISPR mutagenesis testes. Right: Quantification of ovo smFISH signal. h Left: Control and ovo CRISPR mutagenesis testes. Right: Quantification of GSC number, GSC diameter, and total germ cell number. i Left: Representative smFISH images of klu in control and germline-specific klu CRISPR mutagenesis testes. Right: Quantification of klu smFISH signal. j Left: Control and klu CRISPR mutagenesis testes. Right: Quantification of GSC number, GSC diameter, and total germ cell number. In all images, Vasa (green) labels the germline, Fas3 (magenta) marks hub cells, and smFISH signals are shown in white. Scale bars, 20 µm. (c, e, g, i: n = 8), (d, f, h, j: n = 5). Boxplots represent median values within the Q1–Q3 range, while whiskers include the 0–100 percentiles. p values are calculated with two-sided independent t-tests. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Exact p values can be found in the Source data file. See also Supplementary Fig. 3, Supplementary Tables 1, 2. Source data is provided as a Source Data file (Fig. 4).
