Ascl1 genetics reveals insights into cerebellum local circuit assembly.

Two recently generated targeted mouse alleles of the neurogenic gene Ascl1 were used to characterize cerebellum circuit formation. First, genetic inducible fate mapping (GIFM) with an Ascl1(CreER) allele was found to specifically mark all glial and neuron cell types that arise from the ventricular zone (vz). Moreover, each cell type has a unique temporal profile of marking with Ascl1(CreER) GIFM. Of great utility, Purkinje cells (Pcs), an early cohort of Bergmann glia, and four classes of GABAergic interneurons can be genetically birth dated during embryogenesis using Ascl1(CreER) GIFM. Astrocytes and oligodendrocytes, in contrast, express Ascl1(CreER) throughout their proliferative phase in the white matter. Interestingly, the final position each neuron type acquires differs depending on when it expresses Ascl1. Interneurons (including candelabrum) attain a more outside position the later they express Ascl1, whereas Pcs have distinct settling patterns each day they express Ascl1. Second, using a conditional Ascl1 allele, we discovered that Ascl1 is differentially required for generation of most vz-derived cells. Mice lacking Ascl1 in the cerebellum have a major decrease in three types of interneurons with a tendency toward a loss of later-born interneurons, as well as an imbalance of oligodendrocytes and astrocytes. Double-mutant analysis indicates that a related helix-loop-helix protein, Ptf1a, functions with Ascl1 in generating interneurons and Pcs. By fate mapping vz-derived cells in Ascl1 mutants, we further discovered that Ascl1 plays a specific role during the time period when Pcs are generated in restricting vz progenitors from becoming rhombic lip progenitors.

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets.

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells.

BACKGROUND: While the diversity and spatio-temporal origin of olfactory bulb (OB) GABAergic interneurons has been studied in detail, much less is known about the subtypes of glutamatergic OB interneurons. RESULTS: We studied the temporal generation and diversity of Neurog2-positive precursor progeny using an inducible genetic fate mapping approach. We show that all subtypes of glutamatergic neurons derive from Neurog2 positive progenitors during development of the OB. Projection neurons, that is, mitral and tufted cells, are produced at early embryonic stages, while a heterogeneous population of glutamatergic juxtaglomerular neurons are generated at later embryonic as well as at perinatal stages. While most juxtaglomerular neurons express the T-Box protein Tbr2, those generated later also express Tbr1. Based on morphological features, these juxtaglomerular cells can be identified as tufted interneurons and short axon cells, respectively. Finally, targeted electroporation experiments provide evidence that while the majority of OB glutamatergic neurons are generated from intrabulbar progenitors, a small portion of them originate from extrabulbar regions at perinatal ages. CONCLUSIONS: We provide the first comprehensive analysis of the temporal and spatial generation of OB glutamatergic neurons and identify distinct populations of juxtaglomerular interneurons that differ in their antigenic properties and time of origin.

Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signaling.

Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.