Regulation of cell fate decisions in the developing forebrain by DLX transcription factors

• Author / Creator

  Nevin, Mikaela

• Introduction
  The Dlx1/Dlx2 double knockout (DKO) mouse exhibits major defects in forebrain development including a block in differentiation and migration of GABAergic interneurons from the subpallium to the cortex. In addition, the Dlx1/Dlx2 DKO mouse generates more oligodendrocyte progenitor cells (OPC) during forebrain development, and this occurs at the expense of interneuron generation. This phenotype suggests a role for DLX genes in mediating the neuronal-glial cell fate switch in the developing forebrain. Furthermore, expression of many oligodendroglial lineage genes is increased and occurs earlier in the DKO forebrain. As such, we hypothesized that DLX2 actively represses acquisition of oligodendroglial cell fate in a subset of forebrain neural progenitor cells by direct transcriptional repression of multiple genes required for oligodendroglial development. Here, I aimed to determine whether DLX2 represses expression of the oligodendroglial lineage genes Myt1 and Plp1 during forebrain development. In order to further characterise this gene regulatory network, I also investigated regulation of Plp1 by MYT1. I also investigated whether DLX2 may play a role in regulating progenitor cell fate in the pediatric brain tumour Diffuse Intrinsic Pontine Glioma (DIPG).
  Methods
  Chromatin immunoprecipitation with a polyclonal DLX2 antibody was performed on E13.5 mouse forebrain chromatin to determine whether DLX2 occupied candidate promoter regulatory regions of the Myt1 and Plp1 promoters, and with an MYT1 antibody on E14.5 and E18.5 forebrain to determine if MYT1 occupied candidate regulatory regions of the Plp1 promoter. Electrophoretic mobility shift assays were carried out to determine whether ChIP-positive candidate regions were directly bound by DLX2 in vitro. Luciferase reporter assays were used to determine whether DLX2 binding to candidate regulatory regions affected reporter gene transcriptional output in vitro. Quantitative polymerase chain reaction was used to compare Myt1 and Plp1 transcript levels in DKO and WT E13.5 forebrain, and immunofluorescence was used to compare PLP1 protein expression in DKO and WT E13.5 forebrain. To examine the effect of DLX2 expression on DIPG differentiation status and phenotype, a patient-derived DIPG cell line (SF8628) was transfected with a DLX2 expression plasmid and expression levels of DLX2 target and oligodendroglial genes were assessed with qPCR.
  Results
  The promoter-proximal regions of Myt1 and Plp1 were found to contain multiple candidate homeodomain binding sites, and DLX2 occupied both promoters in vivo. Electrophoretic mobility shift assays showed that recombinant DLX2 directly bound multiple regulatory regions of the Plp1, but not the Myt1 promoter, in vitro. Reporter assays showed that co-expression of DLX2 and Plp1 regulatory regions may affect reporter transcription in vitro. Although Plp1 and Myt1 transcripts were not significantly increased in the DKO forebrain, PLP protein was expressed earlier and in an expanded domain in DKO forebrain. MYT1 may occupy the Plp1 promoter in vivo. SF8628 cells did not show a transcriptional response to DLX2 overexpression.
  
  Conclusions
  DLX2 may negatively regulate Plp1 during forebrain development supporting our hypothesis that DLX2 promotes the acquisition of GABAergic interneuron cell fates and actively represses oligodendroglial differentiation in neuronal progenitor subsets.

• Subjects / Keywords

  • Transcription factor
  • Cell fate
  • Homeodomain

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-3cf4-wa60

• License

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