Spatiotemporal analysis of cerebellar neurons from the ventricular progenitors revealed by in utero electroporation

Yoshiaki Kita1, Kazuhiko Nishida1, Koichi Kawakami2,Yoshiko Takahashi3, Fujio Murakami3

1)Grad Sch Frontier Biosci, Osaka Univ, Suita, Japan, 2)Natl Inst Genetics, Mishima, Japan,3)Nara Inst Sci and Tech, Ikoma, Japan

 INeurons in the cerebellum are thought to be derived from two progenitor zones, the ventricular zone (VZ) and the rhombic lip. Some transcription factors have recently been shown to be expressed in these progenitor zones. However, since expressions of these transcription factors are not localized to the progenitor zones and occur over extended period of time during development, fate-mapping studies based on expression of these transcription factors do not exactly point to spatiotemporal origin of cerebellar neurons. Birthdating studies, which are usually useful to examine neuronal origin, do not reveal the location of progenitors of cerebellar neurons, particularly because the final division of most cerebellar neurons does not occur in the VZ. To reveal the spatiotemporal origin of cerebellar neurons, we utilized in utero electroporation, which enables labeling of VZ progenitors at specific developmental stages. We performed electroporation at stages between embryonic day 10.5 and 14.5 and observed labeled cells in matured cerebellum. To overcome the problem caused by dilution of plasmid that occurs as a consequence of cell division, we utilized transposon-mediated gene transfer, which enables expression of electroporated DNAs persistently in VZ-derived cells by genomic integration. After electroporation of conventional plasmids, Purkinje cells, Golgi cells, Lugaro cells, unipolar brush cells, and a minor subset of granule cells were labeled. Transposon-mediated gene transfer caused labeling of additional cell types, stellate, basket, and a vast majority of granule cells. Types of labeled neurons were electroporated stage-dependent. Our data suggest that progenitors in the VZ produce major types of cerebellar neurons, although stellate and basket cells are generated after their repeated division. In addition, difference in spatiotemporal origin is important to produce a variety of neuronal types.