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Great Supervisor Week Mentions
Can't let great supervisor week at UBC pass without saying thank you to Dr. Cheryl Gregory-Evans. So proud to work with her. With her patience, understanding and great attitude, I built confidence and self-esteem in research and science in general. She created just the perfect environment for any individual in her lab and despite all her other commitments, she sets aside time for each member to discuss progress and be there whenever they need her. I always get inspired by her passion in running a marathon and sharing with us all the efforts she put ( proud of her). I'm grateful to have a brilliant, wise and super competent supervisor.
Thank you for making my graduate journey at UBC wonderful and invaluable.
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - April 2022)
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Aniridia and Usher syndrome 1D are rare congenital defects that lead to vision loss in childhood. Here we tested several different approaches to treat animal models of these diseases. Aniridia is a pan-ocular condition caused by deletion or mutation of the PAX6 gene itself or by downstream intragenic abnormalities. We tested two approaches to target the aniridic-glaucoma phenotype in the Pax6Sey/+ mouse model of aniridia. First, since Tgfβ2 is a direct downstream target of Pax6, we tested whether injection of Tgfβ2-secreting mesenchymal stem cells into the Pax6Sey/+ mouse eye could improve development of anterior segment tissue abnormalities. We observed complete formation of Schlemm’s canal (SC) and a partially repopulated trabecular meshwork (TM). Secondly, we tested whether nonsense suppression strategy could rescue the TM defect in the mouse model harboring a nonsense mutation. Either an aqueous suspension of Ataluren® was injected subcutaneously, or topical eyes drops were instilled twice daily from P5 - P45. We found improved structural anatomy, and increased levels of Tgfβ2, Pitx2 and Foxc1 proteins. Furthermore, nonsense suppression via the topical route rescued the developmental defects of TM and SC better than by systemic treatment. Usher syndrome 1D (USH1D) is an autosomal-recessive condition characterized by deafness, vestibular dysfunction and vision loss caused by absence of CDH23 protein. We obtained a mouse model Y2209X line (Cdh23mtblr+/-) which carries a Cdh23 nonsense mutation. Homozygous Cdh23 mice show profound head shaking, circling behavior, deafness, and reduced ERG response. Here, we used prenatal and postnatal nonsense suppression with Ataluren®. We observed a reduction in severity of phenotypic features in Ataluren-treated mice compared to mock-treated mice as well as corrected localization of photoreceptor proteins. We also studied ex-vivo nonsense suppression therapy in Usher patient-specific cells. Induced pluripotent stem cells (iPSCs) were derived from a patient’s blood cells and three-dimensional (3D) retinal eyecups were generated in culture. The retinal eye cups were treated with Ataluren® which restored CDH23 protein levels, as well as other photoreceptors proteins including arrestin, recoverin and S-opsin. These results suggest patient-derived retinal eyecups are a useful tool for preclinical testing of small molecule drugs.
Small gains and losses of chromosomal DNA, called copy number variants (CNVs), are the cause of many human developmental abnormalities detected before or after birth. Clinically-significant CNVs are found in 2-6% of developmentally arrested embryos and fetuses (termed miscarriage) and in ~15% of children with postnatal developmental abnormalities, typically including abnormal brain function and leading to neuro-developmental delay (NDD). The overall goal of my PhD project was to characterize CNVs found in both miscarriages and in children with NDD in order to identify candidate genes that cause these two aspects of abnormal development. I used a multi-faceted approach consisting of bioinformatics, human cell-line analysis and transgenic animal model investigations. I characterized CNVs reported in miscarriages from literature as well as from our laboratory by using bioinformatics approaches to determine the CNVs size, gene content, gene density and function, known gene knockout murine phenotype, and biological pathway enrichment for all miscarriage CNV genes. My analysis identified several genes from miscarriage CNVs with important functions during prenatal development and pregnancy (e.g. CDKN1C and TIMP2) and enrichment of genes from miscarriage CNVs in biological pathways and processes relevant to embryo/fetal development and feto-maternal interaction (e.g. immune response). For discovery of candidate genes responsible for childhood NDD, I characterized CNVs mapping to a chromosome region, 2p15p16.1, which are known to be associated with multiple postnatal developmental abnormalities and NDD (termed 2p15p16.1 microdeletion syndrome). I performed detailed phenotype and CNV analysis of 33 patients with 2p15p16.1 microdeletions and identified 3 candidate genes (XPO1, REL, and BCL11A) for the developmental problems. By studying their expression in patient cell-lines as well as phenotypic consequences of the loss or gain of their expression in zebrafish, I confirmed their role in developmental abnormalities associated with this syndrome. I have also explored the role of non-coding sequences from this CNV in regulation of one of the candidate genes, BCL11A. The results of my study provide a blueprint for identification of genes with a role in abnormal development by characterizing CNVs. Understanding the cause of the developmental abnormalities opens paths for exploring possibilities for their improved diagnosis, prevention, and potential cure.
Master's Student Supervision (2010 - 2021)
Ocular coloboma is a condition caused by a malformation in optic fissure formation during early eye morphogenesis. It is characterized by visual impairment and in some instances, blindness in some children. The prevalence of ocular coloboma is estimated to range from 2 to 14 per 100,000 children. Although many pathways are known to play an important role in optic fissure closure, the mechanisms and genetic regulation driving optic fissure closure during development are still not widely understood. Different factors are associated with ocular coloboma, including genetic predisposition and environmental influences. The Wingless and Int1 (WNT) signalling pathway is conserved among vertebrates and plays an important role in regulating different developmental processes, including eye development. When the pathway is in a stimulated state, presence of WNT ligand binding to its receptor, results in deactivation of the complex consisting of APC, Axin and GSK3. This deactivation leads to accumulation of the cytoplasmic β-catenin that is translocated into the nucleus and activates transcription of target genes. Fzd5 gene plays an important role in eye morphogenesis during early stages of development. We hypothesized that abnormal levels of WNT signalling is driving the ocular coloboma phenotype. Here, we over-activated the WNT signalling pathway using the small molecule Bromoindirubin-3′-oxime (BIO) and lithium chloride (LiCl), which have been previously shown to impact this pathway. Multiple doses of LiCl treatment has a sensitivity period of disrupting WNT signalling pathway that lead to coloboma phenotype. On the other hand, BIO treatment lead to molecular disruption of WNT signalling pathway with normal eye development. Over-activation of WNT signalling was confirmed by the expression level of GSK3 and ß-catenin using western blot. Secondly, we generated a stable WNT mutant zebrafish line (fzd5) using CRISPR/Cas9 technology and observed coloboma phenotype by brightfield microscopy. This study demonstrated that overexpression of WNT signalling had a dose-dependent response and sensitivity period effect that lead to abnormal eye development both coloboma and microphthalmia phenotype in zebrafish model. Moreover, we demonstrated that zebrafish is an excellent model for CRISPR gene knockout of ocular developmental genes.