The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis

et al. (2009) The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis. PLoS Genet 5(6): e1000511. doi:10.1371/journal.pgen.1000511 The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self- Renewal and Neurogenesis Stephen N. Sansom 0 Dean S. Griffiths 0 Andrea Faedo 0 Dirk-Jan Kleinjan 0 Youlin Ruan 0 James 0 Smith 0 Veronica van Heyningen 0 John L. Rubenstein 0 Frederick J. Livesey 0 Jean M. Hebert, Albert Einstein College of Medicine, United States of America 0 1 Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom, 2 Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California at San Francisco , San Francisco , California, United States of America, 3 Medical Research Council Human Genetics Unit, Western General Hospital , Edinburgh , United Kingdom 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 selfrenewal. 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. - Funding: This work was supported by grants to FJL from the Medical Research Council (UK), the Wellcome Trust, and the March of Dimes. Core support for the Gurdon Institute is provided by the Wellcome Trust and Cancer Research UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. A fundamental feature of neural development is the production of defined types of neurons in a temporal order from multipotent, regionally-specified neural stem and progenitor cells [1]. During nervous system development, maintaining the balance between stem cell self-renewal and neurogenesis is essential for the generation of the correct proportions of different classes of neurons and subsequent circuit assembly. Little is known of the molecular control of the key neural stem (NS) cell properties of multipotency and self-renewal. This is in contrast to other classes of stem cells, most notably embryonic stem (ES) cells, in which a group of three transcription factors, Sox2 and the two ES-specific factors Oct4 and Nanog, co-operate to control pluripotency and self-renewal in a non-redundant manner [2,3]. The paired-domain, homeodomain-containing transcription factor Pax6 is highly conserved among vertebrate and invertebrate species and is essential for the development of much of the central nervous system, including the eye, spinal cord and cerebral cortex, as well as pancreatic islet cells [47]. Detailed analyses of neocortical and retinal development in mice mutant for Pax6 have identified defects in neural stem and progenitor cell proliferation, multipotency, neurogenesis, the generation of specific types of neurons, and marked changes in spatial pattern [819]. In the neocortex, loss of Pax6 function results in microcephaly, abnormal development of the secondary progenitor population of the subventricular zone (SVZ, also known as basal progenitor cells, BP cells) and a disproportionate reduction in the production of later-born, upper layer neurons [12,14,15,17,2023]. Neural stem cells make all of the neurons in the brain. A key feature of these cells is the ability to regulate the balance between making more neural stem cells, the process of self-renewal, and making nerve cells, the process of neurogenesis. Too much self-renewal would result in a brain with too few neurons and abnormal circuitry; too much neurogenesis would deplete all of the neural stem cells too quickly, resulting in a small brain and neurological abnormalities. Little is currently known of the how neural stem cells control this fundamental choice. We used one transcription factor, Pax6, which is important for this decision, as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in the developing mouse brain. We found that the relative amount of Pax6 controls the balance between self-renewal and neurogenesis in neural stem cells. Increasing Pax6 levels drives the system towards neurogenesis, at the expense of self-renewal, by turning on a genetic programme for making neurons, whereas decreasing Pax6 turns off the genetic programme for neural stem cell self-renewal. In both cases, altering the levels of Pax6 ultimately leads to a small brain, but through very different mechanisms. Given the functions of Pax6 in stem cell self-renewal/ proliferation and neurogenesis, a potentially fruitful approach to uncovering cellular pathways controlling these processes is to identify the downstream targets of Pax6 in neocortical stem cells. Therefore, in this study we used Pax6 as an entry point to define the cellular networks regulating neural stem cell self-renewal and neurogenesis by combining chromatin immunoprecipitation (ChIP) to identify Pa (...truncated)