A fundamental question in developmental neuroscience addresses how an organism acquires a nervous system with the correct composition and spatial distribution of neurons and glia. The basic helix-loop- helix (bHLH) transcription factor Ascl1 is one factor critical for neuronal differentiation and subtype specification of multiple neuronal cell-types throughout the development of the central and peripheral nervous systems. More recently, genetic fate mapping experiments have shown that early embryonic Ascl1 expressing progenitor cells are fated to become only neurons;however, at a later embryonic stage, Ascl1 expressing progenitor cells are fated to become oligodendrocytes. The transcriptional programs regulated by Ascl1 in the generation of these two cell types remain largely unknown. Therefore, the overall goal of this project is to identify the gene networks controlled by Ascl1 during neurogenic and oligodendrogenic stages of the developing spinal cord. To uncover what roll Ascl1 has generating the appropriate number and type of neural cells in the CNS, the following specific aims will be pursued. 1) Determine the genome-wide interactions of Ascl1 with chromatin during neurogenic and oligodendrogenic stages of the developing spinal cord. The cis-regulatory elements under the control of Ascl1 in each temporally distinct progenitors will be identified by biochemically isolating the chromatin bound to Ascl1 using Chromatin Immunoprecipitation followed by the massive parallel sequencing of all bound chromatin (ChIP-seq). Some unique regulatory elements found in each progenitor population will be tested for their activity in driving the appropriate expression of GFP reporter constructs in mice. 2) Identify and compare Ascl1 target genes during neurogenic and oligodendrogenic stages of the developing spinal cord. Ascl1 expressing progenitors from E11.5 and E15.5 neural tubes will be isolated by FACS and their mRNA profile will be characterizes by RNA-seq. A comparison of the expressed genes from each stage will provide information on the cellular content in a lineage restricted neuronal progenitor versus a glial restricted progenitor cell. The RNA-seq data will be coupled with the ChIP-seq data to identify Ascl1 target genes in the two temporally distinct progenitor populations.
As Ascl1 is an essential transcription regulator during neural development, elucidation of targets regulated by Ascl1 will further our understanding of how neurological disorders and cancers misuse endogenous developmental signaling pathways, and will increase our chances of identifying novel therapeutic targets. The results of this study will impact several fields of concern to public health such as neural stem research, neural regeneration, neural cell-derived cancers (glioblastoma, neuroblastoma, and neuroendocrine cancers), and understanding the fundamental biology underlying developmental disorders such as autism and schizophrenia.
|Meredith, David M; Borromeo, Mark D; Deering, Tye G et al. (2013) Program specificity for Ptf1a in pancreas versus neural tube development correlates with distinct collaborating cofactors and chromatin accessibility. Mol Cell Biol 33:3166-79|