DEV013847 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Detection of gene products in the E14 rat dorsal telencephalon. (A) Detection of MALS-1, MALS-2, MALS-3, CASK, Mint and PALS1 by RT-PCR from rat E14 dorsal telencephalon (Telen, +RT) and P3 cerebellum (Cerebellum, +RT). Control reactions were without reverse transcriptase (−RT). (B) In situ hybridization to detect MALS-1, MALS-2 and MALS-3 mRNA in sections of E14 rat dorsal telencephalon (Scale bar: 100 µm). MALS-3 appears to be the predominant MALS isoform expressed in VZ cells. (C) Western blot detects MALS-3, MALS-2A and MALS-2B, and MALS-1 at the appropriate molecular weight in E14 rat telencephalon. P, pial surface; PP, preplate; VZ, ventricular zone; V, cerebral ventricle.

• Supplemental Figure S2 -

  Fig. S2. Localization of MALS-3 immunoreactivity in rat tissues at E14. Sections are stained to reveal MALS-3 (green), F-actin (red) and nuclei (blue). (A-C) MALS-3 immunoreactivity at the apical surface of VZ cells is shown to facilitate comparison with other organs from the same embryo. (D-F) No distinct polarization of MALS-3 is seen in the developing eye. (G-O) MALS-3 localizes to the lumenal surface in the oral cavity (G-I), embryonic lung (J-L) and embryonic gut (M-O). Scale bar: 100 µm. P, pial surface; VZ, ventricular zone; V, cerebral ventricle; L, lens; R, retina; RPE, retinal pigmented epithelium; E, epithelia of the gut and oral cavity; L, lumen of the gut and oral cavity. Carets indicate the lumen of the bronchial buds.

• Supplemental Figure S3 -

  Fig. S3. Distribution of proteins between membrane and cytosol in telencephalon postnuclear supernatants. (A) Triton soluble membranes (TX100), triton insoluble membranes (SDS) and cytosol (S100) from E14 rat telencephalon homogenate postnuclear supernatant (PNS) with either magnesium ions (Mg²⁺ group) or a chelating agent (EDTA group) were separated by SDS-PAGE and visualized by western blot. The presence of either Mg²⁺ or EDTA resulted in no difference in the molecular distribution of a variety of proteins between cytosol, Triton soluble membranes and Triton insoluble membranes. (B) Bar graphs compare the separation of membrane-associated from cytosolic proteins using high-speed centrifugation (Mg²⁺ group) versus density-gradient centrifugation (original data not shown). Black and white bars represent relative amounts of cytosolic and membrane protein (based on integrated intensity), respectively. (C) Immunoprecipitation from density gradient fractions. Fractions 5 (light membranes), 14 (dense membranes) and 17 (cytosol) were each pooled from three iodixanol gradients then split into three aliquots that contained beads coated with antibodies directed against MALS-3 (MALS-IP), control rabbit IgG, or no antibody (Start). Western blots revealed that DLG co-immunoprecipitated with MALS in the cytosolic and dense membrane-associated fractions. PALS1 also co-precipitated with MALS; however, the PALS1 antibody detected three bands with slightly different molecular weights, and only the lower molecular weight doublet precipitated with MALS, whereas the higher molecular weight single band remained in the supernatant. Antibody raised against CRB3 also detected a protein that co-precipitated with MALS-3, although the molecular weight of this protein exceeded the published molecular weight of CRB3 (17 kDa) and may instead recognize CRB2 (∼200 kDa). No association could be detected between MALS and β-catenin or p38γ/SAPK3. As expected, naive rabbit IgG failed to precipitate any of these proteins.

• Supplemental Figure S4 -

  Fig. S4. Iodixanol density-gradient separation of buoyant membranes from cytosol in E14 rat telencephalon homogenates. Density-gradient centrifugation of homogenates from the embryonic rat telencephalon followed by a quantitative analysis of proteins present in each fraction following western blot analysis (Vogelmann and Nelson, 2007). The integrated intensity of each band was measured, normalized to the highest value in the set, and plotted on the graph (A; the z-axis scale in each graph has been omitted for clarity). Protein concentration (Protein) of each fraction was also determined and graphed in the same manner. Particles migrated in the gradient to a position that matched their density, with cytosolic proteins remaining in the densest bottom third of the gradient (fractions 16-21) and buoyant particles in the lighter upper two-thirds of the gradient (fractions 1-15). Images of the western blots (B) are also shown, along with bars representing the distribution of cytosolic protein (actin and the majority of cellular protein, fractions 16-21), buoyant material in fractions 1 through 15, plasma membrane proteins (cadherin and the Na⁺/K⁺ ATPase, peaks at fractions 5 and 14), and membranes of the trans-Golgi network (TGN38, peak centered at fraction 12). (C) Bars representing the distributions of the cytosolic, plasma membrane and Golgi proteins in the iodixanol density gradients.

• Supplemental Figure S5 -

  Fig. S5. MALS^TKO cortices do not show lamination defects. (A-F) E18 control and MALS^TKO brains immunostained with layer-specific markers TBR1 (layers 6-5; A,B), CTIP2 (layer 5-6; C,D) and SATB2 (layers 2-4; E,F) do not reveal any obvious changes in expression pattern. Scale bar: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Electroporation of full-length MALS-3 (FL_MALS-3) does not disrupt apicobasal polarity during corticogenesis. (A,B) Analyses of brains electroporated with FL_MALS-3 (A) or empty vector (B) at E13.5 reveal no change in the integrity of the VZ or the positioning of GFP⁺ cells at E18.5. Note that the ventricles appear normal and there are no delaminated cells present. GFP⁺ cells (green) appear to have migrated normally, and immunostaining for MALS (red, A) reveals normal apical staining of the VZ. (C) Breaks in β-catenin immunostaining at the apical surface of the VZ is observed in brains electroporated with the CA-myr_MALS-3 construct. Scale bar: 10 µm.