GENETIC REGULATION OF CELL FATE DECISIONS IN THE DEVELOPING RETINA

• Author / Creator

  Li, Zixuan

• Rationale: The Dlx1/Dlx2 double knockout (DKO) mouse demonstrates major defects during retina development including significantly reduced number of retinal ganglion cells (RGCs) due to enhanced apoptosis of RGCs and consequently thinning of the optic nerve. In addition, the Dlx1/Dlx2 DKO mouse has increased expression of Otx2 and ectopic expression of Crx in the ganglion cell layer (GCL). Crx, the cone and rod photoreceptor homeobox gene, is a gene required for photoreceptor development. Otx2, the orthodenticle homeobox2 gene, belongs to the same transcription factor family as Crx and is a major determinant of photoreceptor specification. Otx2 has been shown to be bound by DLX2 in vivo. The Dlx1/Dlx2 DKO retina phenotype indicates that the cell fate of uncommitted retina progenitor cells (RPCs) may be altered. This suggests that Dlx genes may play a role in mediating cell fate decisions between retinal ganglion cells and photoreceptor cells in the developing retina.
  
  Hypothesis: We hypothesize that OTX2 represses the acquisition of retinal ganglion cell fate in retinal progenitor cells by directly repressing Dlx1/Dlx2 transcription, which is essential for retinal ganglion cell differentiation. The goal is to determine whether OTX2 represses retinal ganglion cell fate by repressing Dlx2 expression.
  Methods: Electrophoretic mobility shift assays were conducted to determine if OTX2 directly binds candidate regulatory subregions in vitro. Chromatin immunoprecipitation with a polyclonal OTX2 antibody was performed on chromatin isolated from E18.5 mouse retinal tissues to determine whether OTX2 occupied candidate EMSA-positive candidate regulatory subregions in vivo. Luciferase reporter assays were performed to determine whether OTX2 binding to candidate Dlx1/Dlx2 regulatory subregions affects reporter gene transcriptional expression in vitro. Histology and immunofluorescence using polyclonal DLX2 antibody was performed to compare retina morphology and DLX2 protein expression in Otx2 heterozygous mutant (Otx2+/GFP) and wildtype P100 retina.
  Results: We discovered that 10kb upstream of the Dlx2 and Dlx1 promoter region and the Dlx1/Dlx2 intergenic region contain multiple candidate homeodomain binding sites for OTX2. Electrophoretic mobility shift assays demonstrated that recombinant OTX2 specifically and directly bound multiple binding sites upstream the Dlx2 promoter, in the Dlx1/Dlx2 intergenic region, and upstream the Dlx1 promoter in vitro. OTX2 also occupied several of these regulatory subregions upstream the Dlx2 promoter, as well as the Dlx1/Dlx2 intergenic enhancer, I12a in vivo. Luciferase reporter assays demonstrated that co-expression of OTX2 and Dlx2 regulatory subregions regulates reporter transcription both positively and negatively in vitro. Luciferase reporter assays demonstrated that OTX2 activates reporter gene transcription via the I12a intergenic enhancer and a proximal regulatory subregion upstream the Dlx2 promoter. OTX2 may also repress reporter gene transcription via a distal regulatory subregion upstream the Dlx2 promoter. The heterozygous Otx2 mutant retina may have an expansion of the retinal ganglion cell layer and a thinning of the inner nuclear layer. We also observed that DLX2 expression-domain may expand in the heterozygous Otx2 mutant retina.
  Conclusions: These results suggest that OTX2 may regulate Dlx2 both positively and negatively during retina development which is partially consistent with our hypothesis that OTX2 represses retinal ganglion cell differentiation genes such as Dlx2 to promote the acquisition of photoreceptor cell fate. Furthermore, OTX2 may also activate Dlx2 to facilitate developmental programing in other cell type precursors.

• Subjects / Keywords

  • gene regulatory networks

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-hpn3-bq06

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.