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ROLE OF DLX GENES IN FOREBRAIN DEVELOPMENT

  • Author / Creator
    Sara Japoni
  • AbstractNervous system development is a highly regulated and complex process in which neurons and glial cells are generated and through migration arrive at their final destinations. Dlx genes are required for central neural development. They encode transcription factors that bind to TAAT/ATTA motifs of regulatory regions and regulate target gene expression. Dlx1/2 genes are important factors involved in neuronal versus glial cell fate switch favoring GABAergic interneuron differentiation. DLX2 represses oligodendrocyte differentiation by negatively regulating the oligodendrocyte marker Olig2. Expression of another oligodendrocyte differentiation gene, Nkx2.2 is increased in mice lacking Dlx1/2. Based on these observations, I hypothesized that DLX2 directly mediates transcriptional repression of oligodendroglial differentiation in determination of neuronal versus glial cell fates during forebrain development, in part through inhibition of Nkx2.2.Differentiation and tangential migration of inhibitory interneurons from the ganglionic eminences (GE) to the neocortex is disrupted in the Dlx1/Dlx2 double knockout (DKO) mouse. CXCR4/CXCL12 signalling also has an important role in the guidance of interneurons from the ganglionic eminences to the neocortex and mice lacking Cxcr4 or Cxcl12 have defects in positioning of interneurons in cortical layers. Moreover, in the absence of Dlx1/2 function, expression of Cxcr4 is significantly decreased in GABAergic interneurons in the medial and caudal GEs. However, the mechanism by which CXCR4/CXCL12 is activated is not well known. I therefore sought to understand the upstream activation of Cxcr4 expression. I hypothesized that DLX2 directly mediates transcriptional activation of GABAergic interneuron migration, in part, through activation of Cxcr4 during forebrain development.Little is known about posttranslational modifications of the DLX transcription factors and whether these modifications affect DLX’s subcellular localization or transcriptional activity. A regulatory subunit of DNA-dependent protein kinase interacts with DLX2, which has the potential to phosphorylate DLX2. Moreover, previous experiments in Eisenstat lab also showed the presence of posttranslational modifications in DLX2 extracted from E13.5 GE. Furthermore, DLX2 is both expressed in the nucleus and cytoplasm of the ventral thalamus revealing the capability of DLX2 to translocate between the nucleus and cytoplasm. Therefore, I proposed to identify the key amino acid residues that are phosphorylated in DLX2. I hypothesized that DLX2 functions as a phosphoprotein and is localized to the nucleus in determining progenitor fate.ChIP-based PCR of embryonic mouse forebrain demonstrated that DLX2 occupies regions containing putative DLX2 binding sites upstream of the Nkx2.2 and Cxcr4 genes. DLX2 significantly affected luciferase reporter gene expression in vitro when co-expressed with the regulatory regions of Cxcr4 and Nkx2.2 occupied by DLX2 in vivo. Quantitative RT-PCR showed an increase in transcript levels of Nkx2.2 and a decrease in transcript levels of Cxcr4 in the Dlx1/2 DKO tissues compared to the wild-type supporting a repressing and activating role of DLX2 on Nkx2.2 and Cxcr4 expression, respectively. Conducting Boyden Transwell assays on SK-N-BE (2) neuroblastoma cells revealed that there was a significant increase in cell migration in the presence of CXCL12; however, this migration was significantly decreased in Dlx2-siRNA treated cells compared to untreated cells, supporting the contribution of DLX2 to cell migration in vitro. These results were corroborated by pharmacological inhibition of CXCR4 signalling. Western Blotting on tissues extracted from E13.5 GE using a specific DLX2 antibody supported the presence of posttranslational modifications in the detected DLX2. Treating the samples with lambda protein phosphatase eliminated the upper band suggesting that phosphorylation is a major posttranslational modification present in DLX2.My results support the hypothesis that DLX2 regulates expression of Cxcr4 and Nkx2.2 in order to maintain proper differentiation and migration of interneurons and concurrently repress oligodendrocyte cell fate in the developing forebrain. This research will contribute to the emerging evidence supporting a role for the DLX transcription factors and their downstream target genes in maintaining the balance of excitation to inhibition during brain development by actively repressing oligodendrocyte differentiation while promoting interneuron differentiation and migration.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-d621-ag73
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.