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First published online 9 April 2008
doi: 10.1242/dev.013847

Development 135, 1781-1790 (2008)
Published by The Company of Biologists 2008
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MALS-3 regulates polarity and early neurogenesis in the developing cerebral cortex

Karpagam Srinivasan1, Jason Roosa1, Olav Olsen2, Soung-Hun Lee1, David S. Bredt3 and Susan K. McConnell1,*

1 Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
2 Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.
3 Lilly Research Laboratories, Indianapolis, IN 46285, USA.


Figure 1
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  		Fig. 1. MALS-3 is expressed by and localizes to the apical surface of NPCs. (A-C) Immunostaining of E14 coronal sections of rat telencephalon with antibodies against MALS-3 (green), ZO2, F-actin or β-catenin (red) reveal the supra-apical localization of MALS-3 relative to ZO2 (A), β-catenin (B) and F-actin (C). Nuclei are stained with SYTO-11 (blue). The en face view in C shows the lumen of the ventricle at the bottom, then (from bottom to top) passes through the apical-most ends of VZ cells and into the domain occupied by adherens junctions, which appear as circles of F-actin staining. (D-L) MALS-3 is initially localized diffusely in VZ cells (D-F), but appears to adopt an apical localization at the onset of neurogenesis (~E11.5, G-I), which is maintained throughout the rest of embryonic life (J-L). Scale bar: 5 µm.

 

Figure 2
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  		Fig. 2. Localization of MALS immunoreactivity compared with other proteins in the VZ. (A-C) Immunostaining of E14 coronal sections of rat telencephalon reveals that MALS is localized supra-apical to F-actin. (D-F) CASK antibody did not reveal specific immunostaining in the VZ. (G-I) PALS1 is also localized supra-apical to F-actin, in a pattern similar to that of MALS. (J-L) CRB3 localization is similar to that of PALS1 and MALS. (M-O) Mint shows a diffuse yet apically biased localization in the VZ. (P-R) DLG, a protein localized basolaterally in many cell types, does not localize to the apical surface in NPCs. (S-U) F-actin colocalized with staining for pan-cadherin in the apical domains of VZ cells. Scale bar: 5 µm.

 

Figure 3
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  		Fig. 3. Distribution of proteins between membrane and cytosol in the postnuclear supernatant. (A) Triton soluble membranes (TX100), triton insoluble membranes (SDS) and cytosol (S100) from E14 rat telencephalon homogenate postnuclear supernatant (PNS) were prepared with either magnesium ions (Mg2+ group) or a chelating agent (EDTA group), separated by SDS-PAGE, and visualized by western blot. The presence of either Mg2+ or EDTA resulted in no reproducible differences in the molecular distributions between cytosol, Triton soluble membranes and Triton insoluble membranes. (B,C) Integrated density plots of the distribution of MALS, CASK and Mint proteins in an iodixanol density gradient (B), with the corresponding western blots of the different fractions (C). The distributions of MALS and CASK showed a strong similarity. (D,E) Integrated density plots of the distribution of known cell polarity proteins (D), together with corresponding western blots (E), reveal the distribution of several cell polarity proteins found in NPCs. Taken together, the data suggest that MALS might interact with CASK and PALS1 in NPCs based on a similar cellular localization. (F) Bars represent the cellular distribution of proteins known to associate with different subcellular compartments (based on data shown in Fig. S4 in the supplementary material).

 

Figure 4
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  		Fig. 4. MALS is required for the maintenance but not establishment of the apical localization of PALS1 and PATJ during corticogenesis. (A-H) Coronal sections of control and MALSTKO mutant brains at E13.5 immunostained for MALS (A,B), PALS1 (C,D), CRB (E,F) and PATJ (G,H) reveal loss of MALS (B) and apical PATJ (H) relative to control. Coronal sections of control and MALSTKO mutant brains at E18.5 immunostained for aPKC{zeta} (I,J), PALS1 (K,L), CRB (M,N) and PATJ (O,P) reveal loss of apical PALS1 (L) and PATJ (P) in MALSTKO mutants. Scale bar: 10 µm. (Q) No changes were observed in total protein levels for any of the above proteins in MALSTKO mutant brains relative to controls.

 

Figure 5
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  		Fig. 5. Adherens junction integrity is not compromised in MALSTKO embryos. (A,B) Coronal sections of E18.5 embryos immunostained for ZO1 to identify adherens junctions reveal normal staining in MALSTKO. (C-F) Antibodies against β-catenin (C,D) and pan-cadherin (E,F), both of which localize to the apical surface at the adherens junctions, reveal no change in MALSTKO mice relative to controls. (G-L) Coronal sections of E17.5 embryos stained with antibodies against nestin (G,H), to identify neural progenitors, and GFAP (K,L), to reveal glial cells, reveal no difference between MALSTKO mice and controls. (I,J) However, antibodies against the neuronal marker TuJ1 reveal a broader TuJ1 staining pattern in the cortex of MALSTKO embryos, suggesting that MALSTKO progenitors quit cycling prematurely and differentiate into neurons. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. MALSTKO embryos display significant differences in labeling index and quit fraction during early neurogenesis. (A-H) Coronal sections of E11.5 (A,B), E12.5 (D,E) and E13.5 (G,H) MALSTKO mutants and matching littermate control brains injected with BrdU 2 hours (A,B,G,H) or 24 hours (D,E) before sacrifice. Sections were immunostained with antibodies to BrdU (A,B,D,E,G,H) and Ki67 (D,E). (C) At E11.5, MALSTKO progenitors have a lower labeling index (total number of BrdU+ cells/total number of cells) than do controls. (F) A larger number of MALSTKO mutant progenitors at E12.5 have quit the cell cycle relative to control littermates (BrdU+ Ki67-/total number of BrdU+ cells). (I) MALSTKO mutant progenitor cells recover normal cell-cycle dynamics just a day later at E13.5, at which time no differences in the labeling index are apparent. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. The mislocalization of MALS-3 disrupts polarity. (A-R) Analyses of brains electroporated with FL_MALS-3/EGFP (A-C), Myr_MALS-3 (E,F,H,I,K,L,N-R) or Myr_CRB3 (D,G,J,M) at E13.5 and sacrificed at E15.5 (D-O) or E18.5 (P-R). Brains electroporated with FL-MALS-3 show no changes in the apical localization of CRB3 (A), PALS1 (B) or aPKC{zeta} (C). Brains electroporated with Myr_CRB3 show no change in the apical localization of ZO1 (red, D), PATJ (red, G), or PALS1 (red, J) in GFP-positive cells (green). (M) CRB protein (red) is apparent in the basolateral regions of GFP-positive cells (green) following electroporation with Myr_CRB3, as expected. Myr_MALS-3 electroporated, GFP-positive cells (green) show a loss of apical localization of ZO1 (E,F), PATJ (H,I) and PALS1 (K,L). CRB staining (red) appears reduced in some GFP-positive cells (N,O). At E18.5, Myr_MALS-3 electroporated brains reveal misplaced cells in the lateral ventricles and breaks in the VZ, and MALS immunostaining is lost at those breaks (P). Larger breaks and loss of PALS1 staining (red) are also observed (Q). The ventricles are populated by delaminated cells that are TuJ1-positive (R). Arrows point to GFP-positive cells in the VZ that are not positive for ZO1 (E), PATJ (H), PALS1 (K), CRB (N), MALS (P) and PALS1 (Q). Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2008