Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development Xi Huanga, Jiang Liua, Tatiana Ketovaa, Jonathan T. Fleminga, Vandana K. Groverb, Michael K. Cooperb, Ying Litingtunga, and Chin Chianga,1 Departments of aCell and Developmental Biology and bNeurology, Center for Molecular Neuroscience, Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232 Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved March 18, 2010 (received for review October 13, 2009) Cerebellar neurons are generated from two germinal neuroepithe- mentioned cerebellar cell types (10, 11). Although the cellular lia: the ventricular zone (VZ) and rhombic lip. Signaling mechanisms origin and final fate of many cerebellar neurons are known, the that maintain the proliferative capacity of VZ resident progenitors signaling pathways that maintain the proliferative capacity of remain elusive. We reveal that Sonic hedgehog (Shh) signaling is resident progenitors in the ventricular germinal neuroepithe- active in the cerebellar VZ and essential to radial glial cell proli- lium remain elusive. feration and expansion of GABAergic interneurons. We demon- The role of Shh signaling in regulating adult forebrain sub- strate that the cerebellum is not the source of Shh that signals ventricular zone neural stem cells has been studied extensively to the early VZ, and suggest a transventricular path for Shh ligand (reviewed in ref. 12). Although these studies focused primarily on delivery. In agreement, we detected the presence of Shh protein in the role of Shh signaling in postnatal and adult stages, its definitive the circulating embryonic cerebrospinal fluid. This study identifies function during embryonic forebrain radial glia cell development Shh as an essential proliferative signal for the cerebellar ventricular has not been demonstrated. Moreover, prototypic midline signal- germinal zone, underscoring the potential contribution of VZ pro- ing centers, such as the prechordal plate and floor plate, that are genitors in the pathogenesis of cerebellar diseases associated with continuous with the embryonic neural epithelium and normally deregulated Shh signaling, and reveals a transventricular source associated with neural progenitor specification/maintenance are of Shh in regulating neural development. absent during cerebellar VZ development. Here, we reveal an essential and previously unappreciated function of Shh signaling in NEUROSCIENCE fl cerebellum | cerebrospinal uid | choroid plexus | radial glia | Shh embryonic cerebellar VZ development, providing evidence that Shh signaling regulates proliferation and expansion of cerebellar he cerebellum plays important roles in sensory perception and radial glia and neural progenitors derived from the VZ. We further Tmotor coordination. These functions are critically dependent determined that the cerebellum itself is not a source of Shh that on diverse neurons and glia originating from two distinct germinal targets the early cerebellar VZ and strongly implicate a trans- neuroepithelia: the rhombic lip (RL) and ventricular zone (VZ). ventricular route for delivery of Shh ligand to the cerebellar VZ. As in the cerebrum, neurons of the cerebellum are either Because current pathologic studies associated with deregulation of inhibitory or excitatory, depending on the expression of neuro- Shh signaling are mainly focused on external granule neurons, our transmitter GABA or glutamate, respectively (1). The principal findings may provide additional insights into the diverse cellular glutamatergic neurons residing in the cerebellar granule layer are origin of cerebellar diseases. granule cells. During development, granule precursor cells gen- erated from the RL migrate tangentially to the external granule Results layer (2), where they proliferate in response to Purkinje-derived Cerebellar Ventricular Zone Progenitors Display Shh Signaling. We Sonic hedgehog (Shh) during late embryogenesis and the early have recently reported that the developing hindbrain choroid postnatal period (3–5). Aberrant activation of the Shh pathway in plexus epithelium (hChPe) robustly expresses Shh, which targets a Patched1 heterozygous mice results in medulloblastoma (MB), a discrete epithelial tissue situated between the hChP and lower malignant cerebellar tumor that is thought to stem from massive RL (13). This distinct epithelial domain functions as the hChPe deregulated granule neuron precursor proliferation (6). Similarly, progenitor domain, which directly contributes to hChP epithelial mutations in PATCHED1 significantly increase the occurrence of growth by a proliferative process that depends on active Shh sig- sporadic MB in humans (7). naling activity (13). To our surprise, during our examination of In contrast to glutamatergic neurons, the GABAergic neurons the expression of Gli1 and Ptch1 by RNA in situ hybridization for are generated from the VZ and consist of at least five different Shh pathway activation in the hChP and surrounding tissue, the neuronal subtypes, including Purkinje cells and GABAergic in- adjacent cerebellum, we also detected Shh signaling pathway terneurons (8). Birthdating studies have revealed that neurons of activity in the cerebellar VZ along the medial–lateral axis at E14.5 the cerebellar VZ are generated in three sequential but over- (Fig. 1). We observed similar expression pattern in sections from fi lapping waves (8, 9). The rst-born are small DCN neurons Ptch1-LacZ mice containing a LacZ reporter knock-in at the [embryonic day (E)10 to E12 in mice], which eventually settle in endogenous Ptch1 locus (Fig. 1). Shh signaling continued to be the white matter beneath internal granule neurons. Purkinje cell detected at E16.5 (Fig. 1) in the cerebellar medial VZ and vermal progenitors are second to be generated from the VZ, and they region. Although Gli1 expression was relatively higher in the become postmitotic from E11 to E13 (8). Purkinje cells then migrate dorsally along guiding radial glial processes to their final destination beneath the external granule layer. A third population Author contributions: X.H., Y.L., and C.C. designed research; X.H., J.L., T.K., J.T.F., and of neurons, which consists of GABAergic interneurons of the V.K.G. performed research; V.K.G. and M.K.C. contributed new reagents/analytic tools; DCN, stellate, basket, Lugaro, and Golgi cells, is generated X.H., J.L., J.T.F., Y.L., and C.C. analyzed data; and X.H., Y.L., and C.C. wrote the paper. during late embryonic (from E14) and postnatal development. The authors declare no conflict of interest. In addition to GABAergic neurons, astrocytes including Berg- This article is a PNAS Direct Submission. mann glia and oligodendrocytes are also derived from the VZ 1To whom correspondence should be addressed. E-mail: [email protected]. (9). By fate-mapping studies, radial glial cells are recognized as This article contains supporting information online at www.pnas.org/cgi/content/full/ VZ multipotent progenitors that give rise to all of the afore- 0911838107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0911838107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 f/- Gli1 Ptch1 Ptch1-LacZ Nestin-cre;Smo compared with WT (52% ± 3.8% vs. 100% ± 7.5% in vermal region, P < 0.001; 48% ± 4.9% vs. 100% ± 6% in CB CB < 5.21E lateral VZ, P 0.001, n = 3; Fig. 2 B and C), demonstrating a hChP dramatic reduction of GABAergic interneuron population. Similarly, the expression of homeodomain protein Lhx1/5, which marks the immature Purkinje cell (8) and Pax2+ GABAergic interneurons (Fig. S1A), were also significantly reduced in Nes- f/- 5.41E CB tin-cre;Smo mutants (66% ± 2.9% vs. 100% ± 1.6% in vermal < ± ± < hChP region, P 0.001; 68% 2.9% vs. 100% 5% in lateral VZ, P 0.001, n =3;Fig.2B and C). Together, we conclude that lack of sufficient Shh signaling in the embryonic cerebellar VZ leads to fi 5.61E signi cant reduction in VZ proliferation and GABAergic inter- CB neuron progenitor cell expansion. hChP To further support the critical role of Shh in promoting em- bryonic cerebellar VZ proliferation, we crossed the Nestin-cre BLBP gal Merge driver strain with SmoM2 conditional mutant, which harbors a constitutively activated form of Smo (17), to generate mice with ectopic Shh signaling. At E14.5, we observed enhanced Gli1 Zc expression in Nestin-cre;SmoM2 cerebella (Fig. 2A). Interestingly, aL fi -1 ectopic Shh signaling was largely con ned to the VZ, where Shh h signaling normally occurs, despite widespread Nestin-cre;R26R c tP reporter expression in almost all neural cells (Fig. 2A). Nestin-cre; SmoM2 VZ displayed augmented proliferation and enhanced 4th Ventricle BrdU incorporation compared with WT (231% ± 8% vs. 100% ± 3.4% in the vermal region, P < 0.001; 345% ± 17.5% vs. 100% ± Fig. 1. Shh signaling in the embryonic cerebellum. Cerebellar VZ displays Shh 7.2% in lateral VZ, P < 0.001, n = 3; Fig. 2 B and C). CyclinD1 signaling activity by E14.5. At E16.5, Shh signaling continues to be apparent was robustly expressed in a larger percentage of cells in Nestin-cre; along cerebellar VZ, with higher expression in the medial portion. Note that SmoM2 compared with WT (221% ± 3.5% vs. 100% ± 8.8% in at this stage, there are many positive cells beyond VZ within the cerebellar LacZ/+ the vermal region, P < 0.001; 158% ± 6% vs. 100% ± 6.1% in tissue. β-gal+ cells overlap with BLBP+ cells in E13.5 Ptch1 embryos. lateral VZ, P < 0.001, n = 3; Fig. 2 B and C). In agreement, a larger number of GABAergic neuronal progenitors was present in medial VZ, Ptch1 and Ptch1-lacZ staining seemed to span the VZ the ventral cerebellum of Nestin-cre;SmoM2 compared with WT, medial–lateral axis. Shh signaling activity was not detectable in such as Pax2+ (237% ± 2.2% vs. 100% ± 7.4%, in vermal region, the cerebellum at E12.5, although evident in the developing hChP P < 0.001; 276% ± 18.1% vs. 100% ± 6% in lateral VZ, P < 0.001, at all stages presented (Fig.