Identification of a novel transcriptional corepressor, Corl2, as a cerebellar Purkinje cell-selective marker

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Abstract

The developmental origin of cerebellar Purkinje cells (PCs) has not been precisely mapped and the genetic program of the specification of this neuronal subtype is largely unknown. Here, we report the isolation of a novel mouse gene encoding a transcriptional corepressor, Corl2, and its expression pattern. Corl2 expression was restricted to the central nervous system in both adult and embryonic stages. In situ hybridization and immunohistochemistry using a polyclonal antibody against Corl2 revealed that Corl2 is selectively expressed at high levels in the developing cerebellum, ventral metencephalon and myelencephalon at E12.5. In these brain regions, neural progenitors did not express Corl2 during the proliferative state but started to express it shortly after exit from the cell cycle. In the cerebellum, Corl2 was specifically expressed in PCs at the adult stage, and consistently, most Corl2+ cells expressed PC markers, such as RORα and calbindin, at the late embryonic stage. At E12.5, when PCs are emerging, GABAergic neurons generated from the dorsal part of the Ptf1a+ progenitor domain selectively expressed Corl2. Importantly, Corl2+ cells in the cerebellum did not express the GABAergic interneuron marker Pax2 at any of the developmental stages. Collectively, these results strongly suggest that Corl2 is a specific marker for PCs in the cerebellum from their emergence until the adult stage. Furthermore, this marker was useful for unmasking the precise origin of PCs and delineating the domain map within the ventricular zone that generates cerebellar GABAergic neurons.

Section snippets

Results and discussion

The cerebellum controls movement and posture, and is mainly composed of one glutamatergic neuronal subtype (granule cells) and four GABAergic subtypes (Purkinje, Golgi, stellate and basket cells) (Wang and Zoghbi, 2001). Purkinje cells (PCs) provide the primary output from the cerebellar cortex, and loss of PCs causes severe cerebellar dysfunction (Sidman, 1983, Sotelo, 2004). Although the mechanism of PC differentiation during later developmental stages has been well studied (Gold et al., 2003

Isolation of Corl2

A full-length cDNA for Corl2 was screened from an E12.5 mouse brain cDNA library and sequenced (Mizuhara et al., 2005). The nucleotide sequence of the Corl2 cDNA was deposited in the DDBJ/EMBL/GenBank database under Accession No. AB358976.

RT-PCR

RT-PCR was performed essentially as described previously (Mizuhara et al., 2005). ExTaq polymerase (TAKARA) was used for amplification, which was carried out by denaturation at 94 °C for 30 s (2 min in the first cycle), annealing at 65 °C for 30 s and extension at

Acknowledgements

We are grateful to Dr. T. Imai (KAN Research Institute Inc.) for helpful comments and encouragement. The monoclonal anti-Lhx1/5 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained at the Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242.

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    It is during this stage that the first evidence of PC subtype specification is seen in Early B-cell factor2 (Ebf2) expression. Furthermore, outside the ventricular neuroepithelium, differentiating PCs express Lhx1/Lhx5 (Early B-cell factor2, Morales and Hatten, 2006) and (transcriptional corepressor for the homeodomain transcription factor Lbx1, Minaki et al., 2008) genes that allowed Muguruma et al., (2010) to assess if cells derived from stem cells have acquired the PCs’ lineage. Because there is a direct correlation between the PC’s date of birth and its location in the adult stripe (Hashimoto and Mikoshiba, 2003), both the PC subtype and the position information should be fated at a very early stage of development, just at the end of the proliferation of PC progenitors.

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These authors contributed equally to this work.

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