Conditional knockout of protein O-mannosyltransferase 2 reveals tissue-specific roles of O-mannosyl glycosylation in brain development

Abstract

The meninges produce essential signaling molecules and major protein components of the pial basement membrane during normal brain development. Disruptions in the pial basement membrane underlie neural ectopia seen in those congenital muscular dystrophies (CMDs) caused by mutations in genes involved in O-mannosyl glycosylation. In mammals, biosynthesis of O-mannosyl glycans is initiated by a complex of mutually indispensable protein O-mannosyltransferases 1 and 2 (POMT1 and 2). To study the roles of O-mannosylation in brain development we generated a conditional allele of POMT2. POMT2 nulllizygosity resulted in embryonic lethality because of a defective Reichert's membrane. Brain-specific deletion of POMT2 resulted in hypoglycosylation of α-dystroglycan (DG) and abolished laminin binding activity. The effect of POMT2 deletion on brain development was dependent on timing, as earlier deletion resulted in more severe phenotypes. Multiple brain malformations including overmigration of neocortical neurons and migration failure of granule cells in the cerebellum were observed. Immunofluorescence staining and transmission electron microscopy revealed that these migration defects were closely associated with disruptions in the pial basement membrane. Interestingly, POMT2 deletion in the meninges (and blood vessels) did not disrupt the development of the neocortex. Thus, normal brain development requires protein O-mannosylation activity in neural tissue but not the meninges. These results suggest that gene therapy should be directed to the neural tissue instead of the meninges.

Figure 1

Targeting construct and the diagram for generation of floxed POMT2 allele. A: Targeting construct: a cassette containing loxP-Frt-Neo-Frt was inserted into intron 1. A second loxP site was inserted into intron 4. Thus, the targeting vector is designed to have exons 2, 3, and 4 flanked by two loxP sequences. Flanking the two loxP sites are homologous sequences of the POMT2 locus. After homologous recombination in ES cells, one of the two POMT2 alleles will become the targeted allele (shown in C). B: Wildtype POMT2 allele. (C) Targeted POMT2 allele. This allele is present in ES cells that underwent homologous recombination with the targeting vector after electroporation of the targeting vector. D: Floxed POMT2 allele. This allele is obtained by crossing chimeric mice with ROSA26-Flp transgenic mice to remove neomycin resistance cassette. E: Mutant POMT2 allele. This allele is obtained after Cre-mediated conditional removal of the floxed sequences (exons 2–4) of the floxed POMT2 allele. F: RT-PCR showed that the knockout embryos expressed only truncated POMT2 mRNA. The RT-PCR product of truncated POMT2 mRNA is 113 bp. G: E8.5 POMT2 null embryos were runted. H,I: Transmission EM analysis of the Reichert's membrane of control and POMT2 null embryos revealed that the POMT2 null Reichert's membrane was less dense with multiple breaks at E6.5 (arrowheads in I). Scale bar = 2 μm in I (applies to H). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Hypoglycosylation of α-DG in POMT2-deficient cells. Glycoproteins from the neocortex (A) and cloned wildtype and POMT2 knockout neural progenitor cells (B) were used for western blot with an antibody specific for glycosylated form of α-DG (IIH6C4) and laminin overlay experiments. Note dramatically reduced laminin binding and reduced IIH6C4 immunoreactivity in POMT2 conditional knockout brain and undetectable IIH6C4 immunoreactivity in POMT2 knockout neural progenitor cells. β-DG, β-dystroglycan; f/f, floxed/floxed.

Figure 3

Lamination defects in the neocortex and hippocampus of POMT2^{f/f;Emx1-Cre+} mice. Frozen sections were stained with cresyl violet (A–E,G,K–N) or immunostained with antibodies against Ctip2 (F,H) and Cux1 (I,J). A,C,E,F,I,K,M: Cre-negative controls. B,D,G,H,J,L,N: POMT2^{f/f;GFAP-Cre+}. A–D,K–N: Adult. E–J: P0. The knockout forebrain exhibited multiple defects. Cortical layering could not be identified in the knockout (B). Two cerebral hemispheres were fused in the knockout (D). The knockout hippocampus showed abnormal dentate gyrus morphology (arrow in L) and laminination defects of CA neurons. Also note that location of Ctip2- and Cux1-positive neurons was disrupted in newborn knockout (compare H with F and J with I). KO, knockout. Scale bars = 200 μm in H (applies to A–D,K–L); 100 μm for E–H; 25 μm for M–N; 50 μm in J (applies to I).

Figure 4

Wnt1-Cre mediated deletion of POMT2 in the meninges does not affect forebrain development. A–C: Wnt1-Cre positive (ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo} transgenic mice revealed specificity of Cre-recombination event in the forebrain meninges. D: RT-PCR of POMT2 using total RNA isolated from the meningeal cells of POMT2^{f/f;Wnt1-Cre-} (ff/Cre-) and POMT2^{f/f;Wnt1-Cre+} (ff/Cre+) mice. Wildtype POMT2 mRNA was barely detectable in the meninges of the knockout by RT-PCR (413 bp), while mutant mRNA was detected (113 bp). E,F: Cresyl violet stain of Cre-negative control and POMT2^{f/f;Wnt1-Cre+} mice, respectively, showing normal histology in the knockout. G,H: Distribution of Ctip2-positive neurons in POMT2^{f/f;Wnt1-Cre+} animals was indistinguishable from the controls at P0. KO, knockout. Scale bars = 100 μm in C (applies to A,B); 200 μm in F (applies to E); 100 μm in H (applies to G).

Figure 5

Disruptions of the pial basement membrane covering the neocortex of POMT2^{f/f;Emx1-Cre+} mice. Frozen sections of adult forebrain were immunofluorescence stained with anti-laminin (A,B), ER-TR7 (C,D), and anti-GFAP (E,F). A,C,E: Cre-negative control. B,D,,F: POMT2^{f/f;Emx1-Cre+}. Note absence of the pial basement membrane (B) and glia limitans (F) at the neocortical surface in POMT2^{f/f;Emx1-Cre+} animals. Ectopic meningeal fibroblasts were observed in the cortex (arrowheads in D). Excess GFAP-positive astrocytes were observed in the upper half of the knockout neocortex (F). Scale bar = 50 μm.

Figure 6

Disruptions of the pial basement membrane during development underlie the lamination defects in POMT2^{f/f;Emx1-Cre+} mice. Transmission EM (A–D) and immunofluorescence staining for laminin-1 (E,F) were carried out to evaluate the pial basement membrane at E13.5 and 15.5, respectively. A: Control. B–D: POMT2^{f/f;Emx1-Cre+}. E: Control. F: POMT2^{f/f;Emx1-Cre+}. Note intact pial basement membrane in the control (arrows in A) and disrupted pial basement membrane in the knockout (arrowheads in B–D). $ symbol in C indicates the leading process of a cell (its nucleus indicated by # symbol) that is migrating through the disruption of the pial basement membrane. Asterisks in F indicate location of the disrupted pial basement membrane and remnants of the pial basement membrane can clearly be identified at lateral aspect of this section (arrowheads). CP, cortical plate; fib, fibroblast; IZ, intermediate zone; KO, knockout; SVZ, subventricular zone; VZ, ventricular zone. Scale bars = 500 nm in A–D; 100 μm in E (applies to F).

Figure 7

Abnormal radial glial morphology and displacement of Cajal-Retzius cells are associated with neuronal overmigration in POMT2^{f/f;Emx1-Cre+} mice. E15.5 frozen sections were immunostained with RC2 antibody (green fluorescence) (A,B) and double stained with anti-laminin (red fluorescence) and CR-50 (green fluorescence) (C,D). Propidium iodide (magenta) was used as a nuclear counterstain in A and B while DAPI (blue) was used as a nuclear counterstain stain in C and D. A,C: Cre-negative controls. B,D: POMT2^{f/f;Emx1-Cre+} mice. Note protrusion of radial glia fibers into the DCZ and displacement of Cajal-Retzius cells in the DCZ (arrowheads in B,D, respectively). Scale bar = 50 μm.