Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.

The mechanism underlying the determination of neurotransmitter phenotype in the developing mesencephalon, particularly GABAergic versus glutamatergic fate, remains largely unknown. Here, we show in mice that the basic helix-loop-helix transcriptional repressor gene Helt (also known as Megane and Heslike) functions as a selector gene that determines GABAergic over glutamatergic fate in the mesencephalon. Helt was coincidently expressed in all the progenitor domains for mesencephalic GABAergic neurons. In the mesencephalon of Helt-deficient embryos, GABAergic neurons were mostly absent and glutamatergic neurons emerged instead. Conversely, ectopically expressed Helt suppressed glutamatergic formation and induced GABAergic neurogenesis. However, the Helt mutants showed normal progenitor domain formation. In consequence, postmitotic expression of the homeodomain factor Nkx2.2, which was specifically expressed by GABAergic populations in wild-type embryos, was maintained despite the transmitter phenotype conversion from GABAergic to glutamatergic in the Helt mutants, suggesting that Helt is not involved in neuronal identity specification. Furthermore, we identified proneural genes Ngn1 and Ngn2, which were selectively expressed in glutamatergic progenitors in the developing mesencephalon and had the ability to confer the glutamatergic fate, as downstream target genes of Helt. These results suggest that Helt determines GABAergic over glutamatergic fate, at least in part, by repressing Ngn (Neurog) genes and that basic helix-loop-helix transcription factor networks involving Helt and Ngns are commonly used in the mesencephalon for determination of the GABAergic versus glutamatergic transmitter phenotype.

In resting T cells, Csk is constitutively localized in lipid rafts by virtue of interaction with a phosphorylated adaptor protein, Csk-binding protein (Cbp)/phosphoprotein associated with glycolipid-enriched microdomains, and sets an activation threshold in TCR signaling. In this study, we examined a kinase responsible for Cbp phosphorylation in T cell membrane rafts. By analyzing T cells from Fyn−/− mice, we clearly demonstrated that Fyn, but not Lck, has its kinase activity in membrane rafts, and plays a critical role in Cbp phosphorylation, Cbp-Csk interaction, and Csk kinase activity. Naive CD44lowCD62 ligandhigh T cells were substantially reduced in Fyn−/− mice, presumably due to the inhibition of Cbp phosphorylation. Thus, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts by phosphorylating Cbp. Csk recruited to rafts would then be activated and inhibit the kinase activity of Lck to keep resting T cells in a quiescent state. Our results elucidate a negative regulatory role for Fyn in proximal TCR signaling in lipid rafts.

In the cerebellum, all GABAergic neurons are generated from the Ptf1a-expressing ventricular zone (Ptf1a domain). However, the machinery to produce different types of GABAergic neurons remains elusive. Here we show temporal regulation of distinct GABAergic neuron progenitors in the cerebellum. Within the Ptf1a domain at early stages, we find two subpopulations; dorsally and ventrally located progenitors that express Olig2 and Gsx1, respectively. Lineage tracing reveals the former are exclusively Purkinje cell progenitors (PCPs) and the latter Pax2-positive interneuron progenitors (PIPs). As development proceeds, PCPs gradually become PIPs starting from ventral to dorsal. In gain- and loss-of-function mutants for Gsx1 and Olig1/2, we observe abnormal transitioning from PCPs to PIPs at inappropriate developmental stages. Our findings suggest that the temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs is negatively regulated by Olig2 and positively by Gsx1, and contributes to understanding temporal control of neuronal progenitor identities.