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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.11.032 Byline: Eri Mizuhara (a), Yasuko Minaki (a), Tomoya Nakatani (a), Minoru Kumai (b), Takeshi Inoue (a), Keiko Muguruma (c), Yoshiki Sasai (c), Yuichi Ono (a)(b) Keywords: Neph3; E-cadherin; Ptf1a; Purkinje cell; Origin; Neuronal subtype; Cell sorting; Cell surface marker; Fate mapping Abstract: GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3.sup.+ GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3.sup.+ populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3.sup.+ cells were progenitors for Pax2.sup.+ interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3.sup.+ cells. Sorting experiments revealed that the Neph3.sup.+ E-cadherin.sup.high population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2.sup.+ interneurons were enriched in the Neph3.sup.+ E-cadherin.sup.low population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium. Author Affiliation: (a) Group for Neuronal Differentiation, KAN Research Institute Inc., Kobe, Hyogo 650-0047, Japan (b) Group for Transgenic Technology, KAN Research Institute Inc., Kobe, Hyogo 650-0047, Japan (c) Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe, Hyogo 650-0047, Japan Article History: Received 30 July 2009; Revised 12 November 2009; Accepted 30 November 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.010 Byline: Hong-Lin Su (a), Keiko Muguruma (a), Mami Matsuo-Takasaki (a), Mineko Kengaku (b), Kiichi Watanabe (a), Yoshiki Sasai (a) Keywords: Math1; Embryonic stem cells; Neural development; Purkinje cells; Rhombic lip; Cerebellum; Granule cells Abstract: Here, we report in vitro generation of Math1.sup.+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1.sup.+. The induced Math1.sup.+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1.sup.+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7.sup.+/Calbindin-D28K.sup.+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system. Author Affiliation: (a) Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan (b) Laboratory for Neural Cell Polarity, Brain Science Institute, RIKEN, Wako 351-0198, Japan Article History: Received 26 July 2005; Revised 24 October 2005; Accepted 4 November 2005