The cerebellar phenotype of Gpr56 −/− mice provides new insights into the cellular roles of this orphan GPCR and potential mechanisms involved in the pathogenesis of BFPP. The pattern of GPR56 expression together with the nature of the defects in Gpr56 knockouts indicate that GPR56 plays a critical role in the morphogenesis of the rostral cerebellum. Furthermore, GPR56 appears to perform this role by regulating adhesion of developing granule cells to ECM components of the pial BM. Loss of GPR56 in cerebellar granule cells results in fragmentation of the BM and disruption of Bergmann glial endfeet. Since the cerebellum and its BM appear normal prior to E18.5, GPR56 seems to be dispensable for the initial formation of the rostral cerebellum but essential for its integrity during the perinatal period, when the rostral EGL undergoes rapid expansion.
Previous studies haves shown that integrity of the pial BM and glia limitans is critical for the morphogenesis of the cerebral and cerebellar cortices (Graus-Porta et al., 2001
; Halfter et al., 2002
; Moore et al., 2002
; Beggs et al., 2003
; Niewmierzycka et al., 2005
; Belvindrah et al., 2007
; Hu et al., 2007
; Voss et al., 2008
). Our results provide new appreciation of the potential role of neurons in the preservation of the cerebellar BM, with several lines of evidence suggesting that granule cells contribute to BM integrity through GPR56. First, at the critical time-period when the BM defects arise in Gpr56
knockouts, the only cells expressing GPR56 are developing granule cells (Bergmann glial expression appears later). Second, granule cells (but not glia) from the affected lobules in knockouts show a specific loss of adhesion to BM molecules, but no defects in proliferation, migration and neurite outgrowth. Third, the loss of adhesion can be mimicked by knockdown of GPR56 in granule cells.
Electron microscopy of the EGL has shown that during perinatal age, the glia limitans overlying the cerebellum is not yet continuous, and that developing granule cells directly contact a substantial portion of the pial BM in between the glial endfeet (Sievers et al., 1981
). These direct contacts remain for some time postnatally even as granule cells begin their inward migration (Sievers et al., 1981
; Hausmann and Sievers, 1985
). The timing of these events raises the intriguing possibility that these granule cell-BM contacts may involve GPR56-mediated interactions. The possibility that granule cell-BM interactions are mediated by GPR56 is also supported by the observation that fluorescently tagged soluble GPR56 N-terminal peptides bind to the cerebellar BM and meninges (Li et al., 2008
and data not shown), suggesting that these structures contain a GPR56 ligand.
Our findings establish that GPR56 plays a role in regulating adhesion of cerebellar granule cells of the perinatal rostral cerebellum. Furthermore, based on our observations that exogenous GPR56 peptides and GPR56 expression in heterologous cells do not alter adhesion, GPR56 appears to contribute to cell adhesion indirectly rather than by directly binding to laminin-1 or fibronectin. The molecular interactors of GPR56 that may mediate its role in adhesion remain unknown. However, it is noteworthy that GPR56 associates with tetraspanins (Little et al., 2004
), which in turn interact with integrins and other ECM binding proteins (Berditchevski and Odintsova, 1999
; Yunta and Lazo, 2003
) - molecules known to contribute to the assembly, remodeling, and maintenance of BMs (Henry and Campbell, 1998
; Schwarzbauer, 1999
). Thus, GPR56 may play a role in BM integrity by interacting with some of these surface receptors. Although we have not found conclusive alterations in Gpr56
−/− cerebella in the expression patterns of several key integrin subunits involved in laminin-1 and fibronectin binding, we cannot rule out other changes in integrins or the involvement of other adhesion molecules in the observed phenotypes.
−/− phenotypes in the developing forebrain (Li et al., 2008
) and the rostral cerebellum share significant features, including rupture of the pial BM, disorganization of the glial scaffold and abnormal neuronal positioning. Together, these observations indicate that a key role of GPR56 in the brain is to maintain the integrity of the pial BM during expansion of the underlying cell layers. However, the cell types expressing GPR56 in the perinatal rostral cerebellum and embryonic cortex are different: developing neurons and radial glia, respectively. The distinctive role of granule cells in BM integrity may reflect the uniquely close association of these cells with the pial BM in the cerebellum.
While ataxia is consistently observed in BFPP patients, little is known about the characteristics of their cerebellar defects and the contribution of these to their motor symptoms. Our findings suggest that, in mice, the motor deficits are caused primarily by cerebellar abnormalities rather than defects in sensory or motor cortex or the pons, and that the anterior cerebellum is preferentially affected. Our findings may have relevance to other human cerebellar malformations as well. For example, the defects we describe are reminiscent of cerebellar polymicrogyria, including the presence of ectopic granule cells, fusion of adjacent folia, misdirected Bergmann glial processes, and region-specific differences in severity (Aida et al., 1994
; Demaerel et al., 1998
; Patel and Barkovich, 2002
; Soto-Ares et al., 2002
). It would be interesting to examine if GPR56
mutations or dysregulation play a role in some forms of cerebellar polymicrogyria.
The phenotype of Gpr56
−/− mice sheds light on several key processes during cerebellar development. First, in these mice many granule cells can be found in ectopic locations in the adult, supporting other evidence that these neurons can survive and mature without association with their normal neighboring cells (Blaess et al., 2004
; Kerjan et al., 2005
). Second, the outward migration of granule cells through breaches in the BM suggests that chemoattractive signals that may exist in the cerebellar anlage to guide granule cell migration inward (Borghesani et al., 2002
) are in themselves insufficient to attract these cells in the absence of a viable pial barrier, or that GPR56 is required for granule cells to respond to these signals. Third, our findings add to evidence that despite the relatively uniform cellular architecture of the cerebellar cortex, there are developmentally critical molecular differences between regions, with some of these differences existing only transiently (Millen et al., 1995
; Herrup and Kuemerle, 1997
While a number of mutant mice with disrupted cerebellar lamination and presence of ectopic neurons have been described, the phenotype produced by GPR56 loss-of-function has distinctive features. For example, the cerebella of mice lacking the genes for β1-integrin (Graus-Porta et al., 2001
; Blaess et al., 2004
), dystroglycan (Moore et al., 2002
), semaphorin-6A (Kerjan et al., 2005
), or integrin-linked kinase (Belvindrah et al., 2006
) share some key features in common with Gpr56
−/− cerebella, including ruptures in the pial BM, disruptions of the glial scaffold, and presence of ectopic granule cells. However, in these mice the defects occur throughout the cerebellum while in Gpr56
−/− mice the defects are rostral-specific. Whether the expression of any of these genes is affected only rostrally in the Gpr56
−/− cerebellum, and could play a role in the Gpr56
−/− phenotype, merits further study. There is a handful of mouse mutants in which the affected region is primarily rostral, for example, rostral cerebellar mutant (rcm
) (Ackerman et al., 1997
; Eisenman and Brothers, 1998
) and meander tail (mea
) (Ross et al., 1990
; Hawkes and Eisenman, 1997
). However, the loss of Gpr56
leads to a cerebellar phenotype different from that of these mice. In the rcm
mutant, defects in guidance cause some granule cells to be misrouted to the midbrain, whereas in Gpr56
−/− mice the mislocalization of granule cells is restricted to the cerebellum. Similarly, although the affected region in mea
knockout mice is similar, the major defect in mea
is the loss of rostrally-located granule cells whereas granule cell proliferation and overall numbers are unaffected in Gpr56
The cerebellar phenotype in Gpr56
−/− mice is strikingly similar to those observed when the meninges overlying the cerebellum are ablated at birth using 6-hydroxydopamine (Sievers et al., 1985
; von Knebel Doeberitz et al., 1986
; Sievers et al., 1994b
). This raises the intriguing possibility that the meninges are necessary for GPR56 function in rostral cerebellar development, potentially as a source of the GPR56 ligand. The finding that a putative GPR56 ligand is present in the meninges (Li et al., 2008
and data not shown) is consistent with this hypothesis.
In summary, we show that the orphan GPCR, GPR56, plays a novel role in regulating adhesion of developing neurons to BM components, and is essential for pial integrity and proper development of the rostral cerebellum. These observations point to defects in cell adhesion as a potential mechanism underlying the anatomical abnormalities in BFPP patients. Given the critical role of GPR56 described here and its broad expression in neuronal precursors in other brain regions, it is possible that GPR56 mutations or dysregulation could underlie other congenital brain defects in addition to BFPP.