Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal.

A major unsolved question in cortical development is how proliferation, neurogenesis, regional growth, regional identity, and laminar fate specification are coordinated. Here we provide evidence, using loss-of-function and gain-of-function manipulations, that the COUP-TFI orphan nuclear receptor promotes ventral cortical fate, promotes cell cycle exit and neural differentiation, regulates the balance of early- and late-born neurons, and regulates the balanced production of different types of layer V cortical projection neurons. We suggest that COUP-TFI controls these processes by repressing Mapk/Erk, Akt, and beta-catenin signaling.

Tbr-1, a neuron-specific T-box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non-palindromic T-box binding sequence, namely the non-palindromic T-element, to determine the putative downstream target genes of Tbr-1. More than 20 identified genes containing the non-palindromic T-element in the 5¢ regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr-1 and CASKenhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL-7R) and OX-2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr-1 and CASK. Deletion of the nonpalindromic T-elements from NR2b promoter impaired the induction by Tbr-1 and CASK. We also examined expression of these target genes in Tbr-1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non-palindromic T-elements in its 5¢ regulatory region, were reduced in Tbr-1 knockout mice. We suggest that Tbr-1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

Neurogenic genes in the Notch receptor-mediated signaling pathway play important roles in neuronal cell fate specification as well as neuronal differentiation. The Drosophila neuralized gene is one of the neurogenic genes. We have cloned a mouse homolog of Drosophila neuralized, m-neu1, and found that the m-neu1 transcript is expressed in differentiated neurons. Mice deficient for m-neu1 are viable and morphologically normal, but exhibit specific defects in olfactory discrimination and hypersensitivity to ethanol. These findings reveal an essential role of m-neu1 in ensuring proper processing of certain information in the adult brain.M any molecular mechanisms controlling neural development and neural function are evolutionarily conserved (1-3). One example is the Notch-mediated cell-cell interaction, a mechanism to mediate signaling between adjacent cells and thereby to specify cell fates in organisms ranging from worms and flies to mammals (4). In a given organism, the Notch signaling mechanism functions in many developmental processes as well as in adult life (5, 6). In the nervous system, the Notch signaling mechanism is involved in neurogenesis (1, 3), neuronal differentiation (2, 6), axon path finding (7,8), and neurite growth (9, 10). The importance of Notch in maintaining normal neuronal function in the adult has been revealed by a number of human neurological syndromes because of defects in Notch signaling (6).Like Notch, the Drosophila neuralized gene (neu) is a neurogenic gene whose function is to limit the number of neuronal precursor cells and to specify sensory organ as well as R8 photoreceptor cell fates (11)(12)(13)(14)(15)(16)(17). Genetic analyses suggest that neuralized interacts with other neurogenic genes, such as the Notch receptor and the Delta ligand for cell-cell interaction (18). Recent studies (15)(16)(17) revealed that neuralized functions cellautonomously to regulate a subset of Notch-dependent processes. How neuralized affects Notch signaling is unknown at present. An interesting possibility is that Neu protein with its C3HC4 RING finger domain functions as an E3 ubiquitin ligase to modulate Notch signaling (15)(16)(17)19).We have cloned a mouse homolog of Drosophila neuralized, m-neu1, and found that it is expressed in differentiated neurons. We examined its functional role in the nervous system by generating a loss-of-function allele of m-neu1. Whereas the m-neu1 null mutants exhibit normal performance in a number of behavioral tests, including Morris water maze assay for learning and memory, they display specific functional lesions in olfactory discrimination and ethanol effects on motor coordination. These studies identify specific functions of the adult brain that are critically dependent on m-neu1 activity. Materials and MethodsIsolation of a Mouse Neuralized Homolog. A mouse midgestation embryonic cDNA library (Stratagene) was screened with fragments of Drosophila neuralized cDNA as probes. In brief, the duplicated nylon filters containing mouse phage cDNAs were prehybridized...