Fig 2: The endfoot proteome is enriched for ECM and cytoskeletal regulators, including NM IIA and B.(A) STRING analysis of the 47 endfoot-enriched proteins. Dashed lines denote prominent protein classes. Cytoskeletal regulators indicated as blue circles. (B) GO analysis of the endfoot-enriched proteome showing all enriched categories with p = 0.05. (C) Venn diagram comparing synapse proteome [36] (red) and endfoot proteome (green). (D) Venn diagram comparing synapse transcriptomes (yellow [37] and red [36]) and endfoot proteome (green). (E) Scatter plot depicting fold change vs. peptide count in the endfoot fraction for 47 endfoot-enriched proteins. Cytoskeletal regulators indicated as blue circles. (F) Bar graph showing protein fold change of endfeet vs. RGC for non-muscle myosin heavy and light chains, compared to average fold change of all proteins in endfeet (dashed line) and all endfoot enriched proteins (dotted line). (G) Cartoon representation of NM II structure, annotated with heavy and light chain isoforms identified in endfoot proteome. ELC, essential light chain, HC, heavy chain; RLC, regulatory light chain. (H) Cartoon representation of RGC and pial niche with box outlining the location of images in I and J. (I, J) Colocalization of MYH9 (red) (I) or MYH10 (red) (J) with endfeet (green) including orthogonal views. Endfeet marked by white dotted lines. Scale bar (I, J): 5 µm. n = 3 brains, 3 sections per brain (I, J). Data underlying graphs included in S1 Data. ECM, extracellular matrix; GO, Gene Ontology; NM II, non-muscle myosin II; RGC, radial glial cell.

Fig 3: Apical junction defects in Gipc3-knockout mice. (A,B) TJP1 immunoreactivity highlights apical junctions in Gipc3KO IHCs. X-Y (top) and X-Z (middle) slices. Yellow lines in upper panels indicate transects used for X-Z reslices in middle panels; magnified boxed regions of reslices are below. immunolocalization experiments were performed more than three times. IPhC, inner phalangeal cell. (C,D) Tracings of TJP1 labeling from four Gipc3KO/+ (C) and six Gipc3KO/KO (D) cochleas. (E,F) MYH9 immunoreactivity highlights apical junctions in Gipc3KO cochleas. (G) Apical circumference area for the indicated genotypes. Number of cochleas and cells per cochlea analyzed: Gipc3+/+, 2 and 4; Gipc3KO/+, 4 and 4; Gipc3KO/KO, 6 and 2–4. P-values determined from nested one-way ANOVA with Tukey correction. (H,I) SEM of P21.5 Gipc3KO/+ and Gipc3KO/KO IHC region. Insets show the junction region between an IHC, an IPhC, and the next IHC. The yellow dashed lines outline the region in between the two IHCs that is occupied by IPhC microvilli. P21.5 SEM was carried out once, with similar results seen in more than 25 IHCs in each genotype. Panel widths: A,B upper and middle panels, 80 µm; A,B lower panels, 20 µm; E,F, 35 µm; G,H, 30 µm (insets, 2 µm).

Fig 4: Myh9 and Myh10 mRNAs localize to endfeet in complementary patterns that change across development.(A, B) Colocalization of Myh9 smiFISH (white, A) or Myh10 smiFISH (white, B) with electroporated endfeet (green) at E15.5. (Left) Yellow arrows denote endfeet colocalized with smiFISH probes. Yellow box outlines area used for orthogonal views. (Right) Orthogonal views of colocalization. (C) Cartoon representation of RGC with boxes indicating cell body imaged in E and G and endfeet imaged in D and F. (D) Localization of Myh9 smiFISH puncta (white) in endfeet from E12.5 to E16.5. E14.5 to E16.5 endfeet (green) labeled using Nestin-EGFP mouse [46]. Note, EGFP does not clearly label endfeet at E12.5 and E13.5. Yellow dotted lines denote the border between the endfeet (below) and the pia (above). (E) Localization of Myh9 smiFISH puncta (white) in cell bodies from E12.5 to E16.5. Yellow dotted lines denote ventricular border. (F) Localization of Myh10 smiFISH puncta (white) in endfeet from E12.5 to E16.5. E14.5 to E16.5 endfeet (green) labeled using Nestin-EGFP mouse. Yellow dotted lines denote the border between the endfeet (below) and the pia (above). (G) Localization of Myh10 smiFISH puncta (white) in cell bodies from E12.5 to E16.5. Yellow dotted lines denote ventricular border. Samples observed for selection of representative images: n = 3 brains, 3 sections per brain (A, B). n = 2 to 4 brains, 3 sections per brain (D, E, F, G). Scale bars: 10 µm (A, B, D, E, F, G). Black asterisks denote background signal from the meninges (A, B, D, F). RGC, radial glial cell; smiFISH, single-molecule inexpensive fluorescent in situ hybridization.

Fig 5: RGC morphology, regulated by Myh9 and Myh10, influences interneuron number and organization in the MZ.(A) Staining with LHX6 (red) to label interneurons and DAPI (blue) to mark all nuclei. Myh10 Ctrl and cKO brains at E16.5. Asterisks denote vasculature. White dotted line marks boundary between pia and MZ. (B) Quantification of LHX6+ cells in the MZ. n = 9 per genotype from 4 litters. (C) Staining with LHX6+ (red) and DAPI (blue) in the MZ of Ctrl and Myh9 cKO E16.5 brains. (D) Quantification of the number of LHX6+ cells in the MZ of Ctrl and Myh9 cKO brains. n = 10 per genotype from 6 litters. (E) Comparison of LHX6+ (red) cells touching the BM in Ctrl and Myh9 cKO brains. BM labeled with laminin (white) and all nuclei with DAPI (blue). Yellow arrows indicate LHX6+ cells touching the BM. (F) Quantification of the fraction of LHX6+ cells in the MZ touching the BM compared to total LHX6+ cells in the MZ. n = 10 per genotype from 6 litters. (G) Comparison of LHX6+ (red) cells touching the BM in Ctrl and Myh10 cKO brains. BM labeled with COL1 (white) and all nuclei with DAPI (blue). Yellow arrows indicated LHX6+ cells touching the BM. (H) Quantification of the fraction of LHX6+ cells in the MZ touching the BM compared to total LHX6+ cells in the MZ. n = 3 per genotype from 1 litter. Error bars: SD. (B, D, F, H). Student unpaired, two-tailed t test (B, D, F, H). Data points color-coded by litter (B, D, F, H). ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Scale bar: (A, C) 50 μm; (E, G) 25 μm. Data underlying graphs included in S5 Data. BM, basement membrane; COL1, Collagen 1; MZ, marginal zone; RGC, radial glial cell.