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Floor plate (FP) cells, the ventral midline cells of the developing neural tube, have long been thought to be non-neurogenic organizer cells that control neuronal patterning and axonal guidance. Recent studies have revealed that mesencephalic FP (mesFP) cells have neurogenic activity and generate dopaminergic neurons. However, the mechanisms underlying the control of neurogenic potential in FP cells are not yet fully understood. Here we identified the bHLH factor Nato3 as an FP-specific transcription factor. In Nato3-null mutant mice, FP cells in the spinal cord were correctly specified, but could not properly mature. By contrast, in the developing mesencephalon, loss of Nato3 did not affect FP differentiation, but led to loss of neurogenic activity in the medial subpopulation of mesFP cells by suppressing proneural gene expression and inducing cell cycle arrest. As a consequence, the number of midbrain dopaminergic neurons generated was decreased in mutants. We also found that Hes1, which is known to be required for non-dividing organizer cell development in the neural tube, was aberrantly upregulated in the mesFP cells of Nato3 mutants. Consistently, forced expression of Nato3 repressed Hes1 expression and consequently induced premature neurogenesis. Finally, we showed that forced expression of Hes1 in mesFP cells induced cell cycle arrest and downregulation of proneural factors. Taken together, these results suggest that Nato3 confers neurogenic potential on mesFP cells by suppressing classical non-neurogenic FP cell differentiation, at least in part, through repressing Hes1.

Mesencephalic dopaminergic (mesDA) neurons control movement and behavior, and their loss causes severe neurological disorders, such as Parkinson's disease. Recent studies have revealed that mesDA neurons originate from mesencephalic floor plate (FP) cells, which had been thought of as non-neurogenic organizer cells regulating regional patterning and axonal projections. Otx2 and its FP-specific downstream factor Lmx1a have been shown to be sufficient to confer neurogenic activity on FP cells and determine a mesDA fate. However, the mechanism underlying how these factors control mesDA development and how FP cells and mesDA neurons are coordinately specified are still largely unknown. In the present study, we obtained evidence that Lmx1a and Lmx1b cooperate with Foxa2 to specify mesDA neuron identity by gain-of-function approaches using transgenic mice. Lmx1a/b appeared to select a mesDA fate by suppressing red nucleus fate in the context of Foxa2-positive progenitors, at least in part, through repressing the Sim1-Lhx1 and Ngn1 pathways that inhibit proper mesDA differentiation. We also found that, in the mesencephalon, FP cell fate is primarily determined by Foxa2 with a supportive action of Lmx1a/b through repressing Nkx6.1, which inhibits FP cell differentiation. Thus, FP and mesDA identities are determined by distinct specification pathways, both of which are controlled by the same combination of transcription factors, Lmx1a/b and Foxa2, and, as a consequence, mesDA neurons are generated from mesencephalic FP cells., (Copyright 2010 Elsevier Inc. All rights reserved.)

Neuronal differentiation is regulated by many basic-helix-loop-helix (bHLH) family transcriptional activators and repressors, and the balance of activity between these factors is important for the differentiation process. Here, we report the identification of a novel transcriptional repressor, designated Helt. Helt encoded a Hey-related bHLH protein containing the bHLH and Orange domains. Helt could homodimerize, and heterodimerize with Hes5 or Hey2. Both the bHLH and Orange domains were involved in the homodimerization. In contrast, only the bHLH domain was required for the heterodimerization with Hey2, whereas only the Orange domain mediated the interaction between Helt and Hes5. Thus, Helt has two dimerization domains, and these domains independently select a partner. Identification of preferred recognition sequences by CASTing experiments revealed that Helt bound to the E box, which was distinct from the Hes1 optimal sequence around the E box core. Not only the core sequence but also sequences flanking the E box were essential for the recognition by Helt and Hes1. Furthermore, Helt repressed transcription from an artificial promoter through binding to the optimal E box elements, as well as transcription from its own promoter. Using in situ hybridization and immunohistochemistry, Helt expression in embryos was investigated. Helt was mainly expressed in undifferentiated neural progenitors in some of the developing brain regions, including the mesencephalon and diencephalon, at the neurogenesis stage. These results suggest that Helt acts as a transcriptional repressor to regulate neuronal differentiation and/or identity.

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. [Copyright &y& Elsevier]

During development, neuronal identity is determined by a combination of numerous transcription factors. However, the mechanisms of synergistic action of these factors in transcriptional regulation and subsequent cell fate specification are largely unknown. In this study, we identified a novel gene, Corl1, encoding a nuclear protein with homology to the Ski oncoprotein. Corl1 was highly selectively expressed in the central nervous system (CNS). In the embryonic CNS, Corl1 was expressed in a certain subset of postmitotic neurons generated posterior to the midbrain-hindbrain border. In the developing spinal cord, Corl1 was selectively expressed in the dorsal horn interneurons where a homeodomain transcription factor, Lbx1, is required for proper specification. Corl1 was localized in a nuclear dot-like structure and interacted with general transcriptional corepressors. In addition, Corl1 showed transcriptional repression activity in the GAL4-fusion system, indicating its involvement in the regulation of transcriptional repression. Furthermore, Corl1 interacted with Lbx1 and cooperatively repressed transcription, suggesting that it acts as a transcriptional corepressor for Lbx1 in regulating cell fate determination in the dorsal spinal cord. Corl1 corepressor activity did not depend on Gro/TLE activity, and Gro/TLE also functioned as a corepressor for Lbx1. Thus, Lbx1 can select two independent partners, Corl1 and Gro/TLE, as corepressors. Identification of a novel transcriptional corepressor with neuronal subtype-restricted expression might provide insights into the mechanisms of cell fate determination in neurons. [ABSTRACT FROM AUTHOR]