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

Congenital muscular dystrophies (CMDs) that are associated with central nervous system (CNS) malformations, Walker–Warburg syndrome (WWS), muscle–eye–brain disease (MEB), Fukuyama congenital muscular dystrophy (FCMD), and congenital muscular dystrophy 1D (MDC1D), can be caused by mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) including POMT1, POMT2, POMGnT1, FKTN, FKRP, and LARGE (Kobayashi et al., 1998; Brockington et al., 2001a,b; Yoshida et al., 2001; Beltran-Valero de et al., 2002; de Bernabe et al., 2003; Longman et al., 2003; van Reeuwijk et al., 2005; Currier et al., 2005). At least some of these genes are involved in the synthesis of O-linked mannosyl glycans such as Siaα2,3Galβ1,4GlcNAcβ1,2-Man-Ser/Thr and Galβ1,4(Fucα1,3)GlcNAcβ1,2Man-Ser/Thr (Chiba et al., 1997; Sasaki et al., 1998; Smalheiser et al., 1998). POMT1 and 2 (protein O-mannosyltransferases 1 and 2) are an enzyme complex that transfers mannose to serine or threonine residues; this is the first step in biosynthesis of this glycosylation (Manya et al., 2004; Akasaka-Manya et al., 2006). POMGnT1 (protein O-mannose N-acetylglucosaminyltransferase 1) then transfers N-acetylglucosamine to O-linked mannose (Yoshida et al., 2001; Zhang et al., 2002). The functions of the protein product of FKTN and LARGE, fukutin and LARGE, are not yet fully elucidated. Recent data indicate that LARGE is involved in extension of an unidentified phosphoryl glycosylation branch on O-linked mannose (Yoshida-Moriguchi et al., 2010) and complex N- and mucin O-glycosylations of α-dystroglycan (DG) (Patnaik and Stanley, 2005; Aguilan et al., 2009). O-linked mannosyl glycans account for 1/3 of O-linked glycans in the brain (Finne et al., 1979; Krusius et al., 1986; Chai et al., 1999; Kogelberg et al., 2001). The bestknown O-mannosylated glycoprotein is α-DG, a cell surface glycoprotein that binds to the transmembrane β-DG (Ervasti and Campbell, 1991; Ibraghimov-Beskrovnaya et al., 1992). Both DG subunits are widely expressed and function as a transmembrane linker between the extracellular matrix and cytoskeleton (Ervasti and Campbell, 1991; Winder, 2001). α-DG binds with high affinity to laminin as well as several other extracellular matrix components including agrin, perlecan, neurexin, and pikachurin (Ervasti and Campbell, 1993; Gee et al., 1993; Yamada et al., 1994; Smalheiser and Kim, 1995; Montanaro et al., 1999; Sato et al., 2008). Mutations in POMT1, POMT2, and POMGnT1, as well as mutations in LARGE and fukutin, lead to hypoglycosylation of α-DG with markedly reduced laminin binding activity (Grewal et al., 2001; Michele et al., 2002; Kano et al., 2002; Takeda et al., 2003; Kim et al., 2004; Liu et al., 2006). Mouse knockout of dystroglycan recapitulates CMD phenotypes (Satz et al., 2008), supporting that defective glycosylation of α-DG is the key molecular cause of these CMDs. CMDs with CNS manifestations are characterized by congenital muscular dystrophy, retinal atrophy, and type II lissencephaly in the brain. Overmigration of some neurons beyond the limits of the cerebral cortical boundary (Dobyns et al., 1985; Parano et al., 1995; Haltia et al., 1997; van der Knaap et al., 1997; Ross and Walsh, 2001; Jimenez-Mallebrera et al., 2005; Lian and Sheen, 2006) is a hallmark of these brain malformations. Analysis of animal models has provided insights into the mechanisms of type II lissencephaly (Holzfeind et al., 2002; Chiyonobu et al., 2005; Liu et al., 2006). Disruptions in the pial basement membrane are the key initial events leading to overmigration of neurons during development (Hu et al., 2007). Major components of the pial basement membrane are produced by the meninges, a tissue of mesenchymal origin essential for brain development (Sievers et al., 1994). Destruction of meninges in the cerebellum of newborn rodents by 6-hydroxydopamine causes disruptions of the pial basement membrane with ectopia of granule cells, suggesting continued production of the extracellular matrix proteins by the meninges is required to maintain integrity of the pial basement membrane (Sievers et al., 1983, 1994). Interestingly, a meninges-specific deletion of focal adhesion kinase (FAK) disrupts the pial basement membrane and leads to overmigration of neurons through the disruptions. Thus, FAK is required in the meninges to maintain the integrity of the pial basement membrane, suggesting that the meninges function as more than just a source of extracellular matrix molecules (Beggs et al., 2003). The meninges have also been shown to be a source of retinoic acid that regulates proliferation of neural progenitor cells in the forebrain (Siegenthaler et al., 2009). Analysis of POMGnT1 knockout mice shows a requirement for O-mannosyl glycosylation in brain development. However, knockout of POMGnT1 does not affect the first step of O-mannosyl glycosylation, which apparently provides residual laminin binding activity to α-DG (Kanagawa et al., 2009). In contrast, POMT1 nulllizygosity causes embryonic lethality and precludes the analysis of fetal development (Willer et al., 2004). Thus, a detailed examination of O-mannosyl glycosylation in brain development has not been performed nor is it known if O-mannosyl glycosylation is required in the meninges for normal brain development. In this study we generated POMT2-floxed allele to study the effect of deleting O-mannosyl glycosylation in developing brain in a tissue-specific manner. POMT2-floxed mice were crossed with GFAP-Cre (Zhuo et al., 2001), Emx1-Cre (Gorski et al., 2002), and Wnt1-Cre (Danielian et al., 1998) to specifically knockout POMT2 in the forebrain and the forebrain meninges. Our results indicated that O-mannosyl glycosylation is required in the brain for normal neocortical development in a temporal-specific manner. Surprisingly, POMT2 deletion in the meninges does not affect forebrain development.