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Fig. 4 Single-cell transcriptome reveals that a subcluster of glial progenitor and stathmin family members are associated with betaPix mutation. (A) Experimental strategy for single-cell RNA sequencing of embryonic heads from wild-type siblings and betaPix CRISPR mutants at 1 dpf and 2 dpf. (B) UMAP visualization and clustering of cells labeled by cell type. Four samples were aggregated and analyzed together. (C) Proportions of 24 cell clusters were differentially distributed among four sample groups. ctrl_1d, PBS-injected siblings at 1 dpf; ko_1d, betaPix CRISPR mutants at 1 dpf; ctrl_2d, PBS-injected siblings at 2 dpf; ko_2d, betaPix CRISPR mutants at 2 dpf. (D) Enriched GO terms for differentially expressed genes for progenitor sub-cluster comparing ko_2d to ctrl_2d groups. (E) Violin plots showing the expression of the stathmin family genes by all cells among four sample groups (left panel) or by progenitor sub-cluster among four sample groups (right panel). (F) qRT-PCR analysis showing expression of betaPix, stmn1a, stmn1b, stmn4l, and ppfia3 in glia of FACS-sorted betaPixct/ct siblings and betaPixm/m mutants at 48 hpf. Each dot represents cells sorted from one embryo. Data are presented in mean ± SEM. ***p<0.005; ****p<0.001; unpaired Student’s t-test. (G) qRT-PCR analysis showing expression of betaPix, stmn1a, stmn1b, stmn4l, and ppfia3 in glia of FACS-sorted betaPixct/ct siblings and gfap:GFP-Cre; betaPixct/ct mutants at 48 hpf. Each dot represents cells sorted from one embryo. Data are presented in mean ± SEM. ***p<0.005; ****p<0.001; unpaired Student’s t-test. (H) Whole-mount RNA in situ hybridization revealing downregulation of stmn1a, stmn1b, and stmn4l in betaPixct/ct siblings and gfap:GFP-Cre; betaPixct/ct mutant embryos at 48 hpf. Individual scale bars are indicated in the figure.