A cis-element in the Notch1 locus is involved in the regulation of gene expression in interneuron progenitors

Abstract

Interneurons comprise approximately one third of the total cortical neurons in the mammalian cerebral cortex. Studies have revealed many details in the generation of this cell type. However, the mechanism that defines interneuron-lineage specific gene expression is not well understood. Gene regulatory elements, e.g., promoters, enhancers, and trans-acting factors, are essential for the proper control of gene expression. Here, we report that a novel evolutionarily conserved cis-element in the second intron of the Notch1 locus play an important role in regulating gene expression in interneuron progenitors. The spatiotemporal activity of the cis-element in the developing central nervous system (CNS) was determined by both transient reporter expression in the developing chick and a transgenic mouse model. Its activity is well correlated with neurogenesis in both the chick and mouse and restricted to neural progenitor cells in the ganglionic eminence that are fated to differentiate into GABAergic interneurons of the neocortex. We further demonstrate that the cis-element activity requires the binding motif for trans-acting factors Gsh1/Barx2/Brn3. Deletion of this binding motif abolishes reporter gene expression. Together, these data provide new insights into the regulatory mechanisms of interneuron development in the vertebrate CNS.

Transcriptional Regulation of Notch1 Expression by Nkx6.1 in Neural Stem/Progenitor Cells during Ventral Spinal Cord Development

“…CR2 is a fragment of evolutionarily conserved non-coding DNA located in the second intron of Notch1 gene locus and its gene regulatory activity has been shown in the interneuron progenitors of the developing neocortex13. We first determined the minimum sequence and protein factors required for CR2 activity and analyzed protein binding and gene regulatory activities within sub-regions of CR2.…”

“…GFP protein has a relatively long half-life of ~26 hrs22. The GFP signal is visible for about seven days after its production13 and the remnant GFP protein in cells can be detected by immunohistochemistry for additional seven days (expression level reduce to <1%; calculation was based on half-life of 26 hrs). This allows us to trace the fate of GFP+ cells even when CR2 ceased its activity in the differentiated cells.…”

“…1). Although a 18 bp piece of CR2 (86–104 bp in CR2) functions as an enhancer in the developing brain13, it lacks the ability to drive GFP expression in the spinal cord (Fig. 1b,d, CR2.d).…”

“…All animal work was conducted in accordance with relevant guidelines and regulations of the IACUC. The CR2-GFP transgenic mouse ( Mus musculus ) strain was generated as previously described13 and maintained in our lab. Embryonic and neonatal spinal cords were obtained from the transgenic animals via microsurgical dissection.…”

“…Often these cis -elements are evolutionarily conserved1112. We have previously identified an evolutionarily conserved cis -element in the second intron of Notch1 gene (Notch1CR2, or CR2, a 399 bp non-coding DNA fragment)13. CR2 regulates gene expression in neural stem/progenitor cells (NSPCs) and was predominantly active in the GABAergic interneuron progenitors in the ganglionic eminence during neocortical development13.…”