Unipolar brush cells of the cerebellum are produced in the rhombic lip and migrate through developing white matter.

Unipolar brush cells (UBCs) are glutamatergic interneurons in the cerebellar cortex and dorsal cochlear nucleus. We studied the development of UBCs, using transcription factor Tbr2/Eomes as a marker for UBCs and their progenitors in embryonic and postnatal mouse cerebellum. Tbr2+ UBCs appeared to migrate out of the upper rhombic lip via two cellular streams: a dorsal pathway into developing cerebellar white matter, where the migrating cells dispersed widely before entering the internal granular layer, and a rostral pathway along the cerebellar ventricular zone toward the brainstem. Ablation of the rhombic lip in organotypic slice cultures substantially reduced the production of Tbr2+ UBCs. In coculture experiments, Tbr2+ UBCs migrated from rhombic lip explants directly into the developing white matter of adjacent cerebellar slices. The origin of Tbr2+ UBCs was confirmed by colocalization with beta-galactosidase expressed from the Math1 locus, a molecular marker of rhombic lip lineages. Moreover, the production of Tbr2+ UBCs was Math1 dependent, as Tbr2+ UBCs were severely reduced in Math1-null cerebellum. In reeler mutant mice, Tbr2+ UBCs accumulated near the rhombic lip, consistent with impaired migration through developing white matter. Our results suggest that UBCs arise from the rhombic lip and migrate via novel pathways to their final destinations in the cerebellum and dorsal cochlear nucleus. Our findings support a model of cerebellar neurogenesis, in which glutamatergic and GABAergic neurons are produced from separate progenitor pools located mainly in the rhombic lip and the cerebellar ventricular zone, respectively.

Development of the deep cerebellar nuclei: transcription factors and cell migration from the rhombic lip.

The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.

Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex.

The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 --> Tbr2 --> Tbr1 in the differentiation of radial glia --> intermediate progenitor cell --> postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia --> postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

Dystroglycan on radial glia end feet is required for pial basement membrane integrity and columnar organization of the developing cerebral cortex.

Interactions between the embryonic pial basement membrane (PBM) and radial glia (RG) are essential for morphogenesis of the cerebral cortex because disrupted interactions cause cobblestone malformations. To elucidate the role of dystroglycan (DG) in PBM-RG interactions, we studied the expression of DG protein and Dag1 mRNA (which encodes DG protein) in developing cerebral cortex and analyzed cortical phenotypes in Dag1 CNS conditional mutant mice. In normal embryonic cortex, Dag1 mRNA was expressed in the ventricular zone, which contains RG nuclei, whereas DG protein was expressed at the cortical surface on RG end feet. Breaches of PBM continuity appeared during early neurogenesis in Dag1 mutants. Diverse cellular elements streamed through the breaches to form leptomeningeal heterotopia that were confluent with the underlying residual cortical plate and contained variably truncated RG fibers, many types of cortical neurons, and radial and intermediate progenitor cells. Nevertheless, layer-specific molecular expression seemed normal in heterotopic neurons, and axons projected to appropriate targets. Dendrites, however, were excessively tortuous and lacked radial orientation. These findings indicate that DG is required on RG end feet to maintain PBM integrity and suggest that cobblestone malformations involve disturbances of RG structure, progenitor distribution, and dendrite orientation, in addition to neuronal "overmigration."

Voltammetrically controlled electron transfer reactions from alkanethiol modified gold electrode surfaces to low molecular weight molecules deposited within lipid (lecithin) bilayers.

A procedure was developed for initiating electron transfer from a gold electrode to a low molecular weight electron acceptor present inside supported lipid (lecithin) bilayers, followed by further electron transfer to an electron acceptor present in an aqueous solution. The electron acceptors present in the lecithin bilayers and aqueous phase were 7,7,8,8-tetracyanoquinodimethane (TCNQ) and [Fe(III)(CN)(6)](3-), respectively. A polished planar gold disk electrode was first coated via self-assembly procedures with an alkanethiol monolayer. A phospholipid layer consisting of multiple bilayers of lecithin containing TCNQ was subsequently deposited onto the alkanethiol monolayer. The Au/alkanethiol/lecithin-TCNQ electrode was placed in an aqueous solution containing various amounts of [Fe(III)(CN)(6)](3-) and [Fe(II)(CN)(6)](4-), with 0.5 M KCl as the supporting electrolyte. In the absence of TCNQ inside the alkanethiol/lecithin layers, only a small background current was observed. When TCNQ was included in the alkanethiol/lecithin layers, the voltammetry showed features typical of a catalytic process, due to the TCNQ being reduced to TCNQ(-*) within the lecithin bilayers and then undergoing oxidation back to TCNQ via interaction with [Fe(III)(CN)(6)](3-) at the lecithin-aqueous solution interface. The procedures for preparing the alkanethiol/lecithin-TCNQ coatings were optimized in order to obtain the most reproducible voltammetric response. Experiments were also performed using tetrathiafulvalene (TTF) as an electron donor in the lipid bilayer phase.