Precocious cerebellum development and improved motor functions in mice lacking the astrocyte cilium-, patched 1-associated Gpr37l1 receptor Daniela Marazziti1, Chiara Di Pietro1, Elisabetta Golini, Silvia Mandillo, Gina La Sala, Rafaele Matteoni, and Glauco P. Tocchini-Valentini2 Consiglio Nazionale delle Ricerche, European Mouse Mutant Archive-Infrafrontier-International Mouse Phenotyping Consortium, Istituto di Biologia Cellulare e Neurobiologia, I-00015 Monterotondo Scalo (Rome), Italy Contributed by Glauco P. Tocchini-Valentini, August 12, 2013 (sent for review July 17, 2013) In the developing cerebellum, the proliferation and differentiation high levels of the Shh pathway components, including the trans- of glial and neuronal cell types depend on the modulation of membrane transporter-like patched 1 (Ptch1) and the seven- the sonic hedgehog (Shh) signaling pathway. The vertebrate G- transmembrane span smoothened (Smo) proteins (10, 11). protein-coupled receptor 37-like 1 (GPR37L1) gene encodes a puta- Shh signaling is regulated by several ligand-binding factors, tive G-protein–coupled receptor that is expressed in newborn and including Ptch1, its primary receptor, and associated coreceptors adult cerebellar Bergmann glia astrocytes. This study shows that (12, 13). The binding of Shh to the Ptch1 complex leads to the the ablation of the murine Gpr37l1 gene results in premature derepression of Smo. This in turn triggers an intracellular signal down-regulation of proliferation of granule neuron precursors transduction cascade, with transactivation of several target genes (14). Many different proteins are involved in the regulation of and precocious maturation of Bergmann glia and Purkinje neurons. the Shh mitogenic signal, but their specific roles and mechanisms These alterations are accompanied by improved adult motor learn- −/− of action are only partially established (10). ing and coordination. Gpr37l1 mice also exhibit specificmodifi- fi fi Mammalian genome-wide expression studies have identi ed cations of the Shh signaling cascade. Speci c assays show that in gene sets whose transcripts and protein products are enriched in Bergmann glia cells Gpr37l1 is associated with primary cilium mem- developing and adult cerebellar neurons and glial cells (15, 16). branes and it specifically interacts and colocalizes with the Shh Among them, the GPR37L1 gene was found highly expressed primary receptor, patched 1. These findings indicate that the in developing (Cerebellar Development Transcriptome Da- patched 1–associated Gpr37l1 receptor participates in the regula- tabase, accession no. CD06446) and adult Bergmann glia tion of postnatal cerebellum development by modulating the Shh astrocytes (17). It was also identified as a modulator of the pathway. genesis and assembly of the primary cilium, a cellular organelle required for Shh–Smo signaling (18, 19). The vertebrate G- mutant mouse model | mitogenic signaling protein–coupled receptor 37 and G-protein–coupled receptor 37-like 1 (GPR37 and GPR37L1) genes encode seven-trans- membrane span proteins with amino acid sequence homology to he cytoarchitecture of the cerebellum includes only a few cell types, thus offering a convenient system to study the regu- T fi latory pathways that coordinate brain cell proliferation, survival, Signi cance and differentiation (1). Granule and Purkinje cell neurons and Bergmann glia astrocytes concomitantly differentiate in the de- A new mouse mutant strain carrying a genetic deletion of the veloping mouse cerebellum. In the earliest postnatal period, the G-protein–coupled receptor 37l1 (Gpr37l1) was established and external granular layer (EGL) contains granule cell precursors characterized. Null mutant animals exhibit specific alterations (GCPs). Their proliferation is sustained by interneuronal con- of postnatal cerebellar development, with premature down- tacts, whereas interactions with astrocytes inhibit the mitotic regulation of granule neuron proliferation, precocious Berg- cycle and induce their differentiation (2). Postmitotic granule mann glia, and Purkinje neuron maturation and layer forma- cells migrate along glial radial fibers, through the molecular layer tion. The postnatal expression of several components of the (ML) and Purkinje cell layer (PCL), thus forming the internal sonic hedgehog protein mitogenic pathway is consistently granular layer (IGL) (3). Before the migratory process, in the changed in cerebellar samples from Gpr37l1 knock-out mice. inner EGL, each granule neuron generates a single axonal out- These alterations are associated with precocious juvenile motor growth, which extends during neuron migration along the glial abilities and improved adult motor learning and coordination. processes, while producing bifurcate axonal fibers. These run The Gpr37l1 and patched 1 proteins are found to colocalize and parallel to the cerebellar pial surface and make synaptic contacts interact in Bergmann glia cells during cerebellar development. with the developing Purkinje cell arbors (3). At about 2 wk after The reported experimental data indicate that Gpr37l1 partic- birth, the EGL structure is not distinguishable anymore, and ipates in the regulation of sonic hedgehog signaling during postnatal cerebellar development. postmitotic granule cells have all completed their migration into the IGL (4). Author contributions: D.M. and G.P.T.-V. designed research; D.M., C.D.P., E.G., S.M., and The molecular regulation of this complex set of cellular G.L.S. performed research; D.M., C.D.P., E.G., S.M., G.L.S., and R.M. analyzed data; and interactions and differentiation events is only partially un- D.M., S.M., R.M., and G.P.T.-V. wrote the paper. derstood (5). The duration and intensity of the GCP pro- The authors declare no conflict of interest. liferation phase determine the size of the mature granule cell Data deposition: The mouse Gpr37l1-floxed and Gpr37l1-knock-out allele sequences pool and therefore influence the final morphology and physiol- reported in this paper have been deposited in the Mouse Genome Informatics, www. ogy of the cerebellum (6). In the earliest postnatal stage, Pur- informatics.jax.org [MGI accession nos. 5439168 (Gpr37l1<tm1.1Gtva>)and5439169 kinje neurons secrete the cholesterol-modified sonic hedgehog (Gpr37l1<tm1.2Gtva>)]. protein (Shh), which crucially stimulates postnatal GCP pro- 1D.M. and C.D.P. contributed equally to this work. liferation (7–9). Shh also affects the Bergmann glia population, 2To whom correspondence should be addressed. E-mail: [email protected]. promoting its maturation/differentiation (7). During postnatal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cerebellar development, both glial astrocytes and GCPs express 1073/pnas.1314819110/-/DCSupplemental. 16486–16491 | PNAS | October 8, 2013 | vol. 110 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1314819110 Downloaded by guest on October 2, 2021 G-protein–coupled receptors for the endothelin and bombesin S2) and no alteration of adult cerebellar layer cytoanatomy and peptides (17, 20, 21). Recently, the secreted neuro- and glio- organization (Fig. S2A; see Fig. S5 C and E). protective glycoprotein prosaposin and derived peptides have − − been shown in vitro to interact with and activate both putative Premature Down-Regulation of EGL Proliferation in Gpr37l1 / Mice. Cerebellar EGL neurons were stained with the Hoechst nuclear receptors (22). −/− To test a functional role of Gpr37l1 in cerebellar Shh signal- dye in midsagittal sections from wild-type or Gpr37l1 litter- ing, a unique mouse strain with a targeted deletion of the mates at distinct postnatal stages. Both genotype groups showed Gpr37l1 gene was produced and characterized. Homozygous null similar folia anatomy at all stages studied (Fig. 1D and Fig. S2) mutant mice exhibited specific alterations of postnatal cerebellar but the average thickness of individual cerebellar layers was al- development, with premature down-regulation of GCP pro- tered in P10 and, less markedly, in P15 null mutant animals [Fig. liferation, precocious Bergmann glia, and Purkinje neuron mat- 1D and Fig. S2B). At P10, they exhibited a decrease in the EGL −/− t t = P < t uration and cerebellar layer formation. Gpr37l1 mice also ( test, (1,4) 3.43, 0.05], a marked increase in the ML [ test, t = − P < E showed consistent changes of the postnatal expression levels of (1,4) 4.14, 0.05] and no variation in the IGL (Fig. 1 ). No Shh and various components of its signaling pathway. Specific significant difference was detected in P0, P3, and P5 pups assays indicated that in Bergmann glia cells, Gpr37l1 is associ- (Fig. S2). ated with primary cilium structures and interacts and colocalizes EGL neuron proliferation was studied in P3, P5, and P7 cer- Gpr37l1−/− with Ptch1, during postnatal cerebellar development. Further- ebellar samples of wild-type or littermates upon in ′ more, premature cerebellar differentiation in the mutant mice is vivo 5-bromo-2 -deoxyuridine (BrdU) incorporation followed by accompanied by specific changes in motor function development, immunostaining 2 h postinjection. The overall localization of including precocious juvenile motor abilities and improved adult BrdU incorporation was similar, but in the null mutant pups the motor learning and coordination. Taken together, the reported average number of BrdU-positive cells was increased at P3 and reduced at P7, whereas it did not vary at P5, in comparison with data indicate that Gpr37l1 participates in the cerebellar