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. 2021 Apr 26;10:e61974. doi: 10.7554/eLife.61974

Figure 3. Downregulation of SPAG5 phenocopies DIAPH3 deficiency.

Figure 3.

(A–D) Representative images of mitotic cells transfected with scrambled shRNA (A) or SPAG5 shRNA (sh-SPAG5) (B–D) and immunostained with anti-DIAPH3 (green) and anti-α tubulin (magenta) antibodies. Chromosomes were counterstained with DAPI (blue). SPAG5 deficiency downregulated diaphanous three (DIAPH3) and caused mitotic errors. Note the mis-localization of DIAPH3 and spindle abnormalities in sh-SPAG5-transfected cells. Scale bar, 5 µm. n = 100 cells per condition from five distinct experiments. (E, F) Western blot analysis of SPAG5 levels upon shRNA-mediated downregulation in NIH3T3 cells. Transfection of shRNA against SPAG5 (sh-SPAG5) significantly reduced the protein levels (−82.8%, p=0.00001, n = 3 independent experiments, Student’s t-test; error bars represent s.e.m.). Beta-catenin, a protein downregulated by knockdown of SPAG5 (Liu et al., 2019), was used as control of knockdown efficiency. (G) Schematic representation of in utero electroporation of sh-SPAG5 in cortical progenitors at e13.5 and immunohistochemistry (IHC) analysis at e15.5. (H–K) Ventricular zone of telencephalic sections of e15.5 embryos electroporated in utero with scrambled shRNA and GFP (H) or SPAG5 shRNA and GFP (I–K) at e13.5, and immunostained with anti-γ tubulin (magenta) at e15.5. White arrowheads depict normal centrosomes in (H) and numerical abnormalities of the centrosome in (I–K). (K) Illustration of a neural progenitor undergoing a non-planar division. The horizontal and oblique dashed lines delineate the ventricular surface and mitotic spindle orientation, respectively. When the mitotic spindle is parallel to the ventricular surface (0°<angle<30°), the cell division is planar. When it is oblique (30°<angle<60°) or perpendicular (60°<angle<90°) to the ventricular surface, the division is non-planar. Scale bar = 2 µm. n = 5 embryos for both scrambled shRNA and sh-SPAG5. (L, M) Quantification of cell division modalities in electroporated apical neural progenitor cells (aNPCs). There is a bias toward non-planar divisions (larger angles) in sh-SPAG5-electroporated aNPCs when compared with scrambled shRNA, p=0.0009. Student’s t-test; error bars represent s.e.m. (L). 49% (67/137 cells) of sh-SPAG5 progenitors undergo non-planar division compared to 31% (45/147 cells) in scrambled shRNA (M).

Figure 3—source data 1. Quantification of cell division modalities.