• Author / Creator
    Zagozewski, Jamie
  • Development of the vertebrate ocular structures requires the cooperative interactions of transcription factors, signalling proteins, external growth factors, and epigenetic regulatory factors. Consequently, misregulation of these cues can result in a number of developmental and functional defects. We were interested in the roles of the distal-less homeobox transcription factors (Dlx) Dlx1 and Dlx2, and the transmembrane receptor neuropilin-2 (Nrp2) in the development of the vertebrate retina and ocular vasculature. In the retina, Dlx genes are known to be involved in the differentiation of retinal ganglion cells (RGC) by transactivating expression of genes required for both differentiation and survival of RGC. The role of Nrp2 in the developing retina, on the other hand, is unknown. In addition to defects in RGC differentiation and survival in Dlx1/Dlx2 double knockout (DKO) retinas, an increase in expression of a critical photoreceptor gene, Crx has been reported. This led us to hypothesize that Dlx genes are required to make binary cell fate decisions between RGC and photoreceptors through transcriptional repression of genes critical for photoreceptor development, including the basic helix-loop-helix transcription factor Olig2. We identified Olig2 as a transcriptional target of DLX2 both in vivo and in vitro. We determined that Olig2 expression is limited to photoreceptor precursors and excluded from mature photoreceptors. Utilizing Dlx1/Dlx2 DKO animals we observed strain-dependent ectopic expression of OLIG2 in the ganglion cell layer (GCL). We suggest that Dlx1/Dlx2 expression is required for retinal progenitors to adopt an RGC fate and restrict the specification of photoreceptor cell fate. We also carried out studies of the role of Dlx1/Dlx2 in neuronal differentiation in the developing forebrain, which provided further insight into the mechanisms by which Dlx genes regulate differentiation in the central nervous system (CNS). Nrp2 is widely known for its role in axonal guidance in the CNS. Neuropilins have also been shown to be critical for both developmental and pathological angiogenesis. However, the function of Nrp2 in the developing retina has not been previously explored. We postulated that Nrp2 would also be critical for developmental angiogenesis and axonal guidance during ocular development. Nrp2 expression was observed in the choroid and hyaloid vessels starting at early retinal development and in the RGC during later developmental stages. We observed increased expression of amacrine cell markers in the embryonic and adult Nrp2 knockout eye, resulting in an expanded inner plexiform layer (IPL) in postnatal stages of retinal development. In addition, the hyaloid vasculature persisted into much later developmental stages when compared with wildtype (WT) littermate controls, which is reminiscent of the human ocular disease known as persistent fetal vasculature (PFV). In addition, the Nrp2 null mice had a number of secondary ocular pathologies that are concurrent with PFV including retinal folding and microphthalmia. Our results indicated that Nrp2 plays a critical role in both transient embryonic vascular development and amacrine cell genesis in the developing retina. Collectively, we found that both Dlx1/Dlx2 and Nrp2 have critical roles in the development of early-born cells of the inner retina including RGC and amacrine cells, respectively. In addition, we discovered a novel role for Nrp2 in hyaloid vasculature development. Future work clarifying the functional consequence of PFV and IPL expansion in the Nrp2 knockout eye will be of great interest, as will the role of Dlx genes in postnatal retina development.

  • Subjects / Keywords
  • Graduation date
    Fall 2017
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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