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Forkhead (Fox) proteins are transcription factors that function in many processes including development, metabolism and cell cycle regulation. This gene family is divided into subfamilies that appear to originate from a common ancestor. I have identified the evolutionary selection pressures acting on individual amino acid positions in the FoxA, FoxC, FoxD, FoxI, FoxO and FoxP subfamilies. The patterns of selection observed allowed for the prediction of residue function and identification of residues that differentiate orthologs and paralogs. The subfamily structure and negative selection found within the subfamilies indicates that after gene duplication, differentiation of subfamilies through amino acid changes and subsequent negative selection on these changes has occurred. Meanwhile, the observed neutral changes and positive selection allow for further protein differentiation. Within the FoxC subfamily, positive selection was identified at one amino acid site in the inhibitory domain. Mutation of this site in FOXC1 alters transactivation activity and the effects of mutants on transactivation activity are different on different reporters. The mutant effects were consistent with those of known disease causing mutations, supporting the predicted positive selection. The inhibitory domain is known to function in reducing FOXC1 transactivation activity and influences protein stability. Here I additionally show that loss of the inhibitory domain and mutation of the positively selected site can reduce FOXC1 DNA binding. Co-transfection of FOXC1 and TLE4, a repressor protein that can potentially bind to the inhibitory domain, was shown to increase FOXC1 transactivation activity. The effects of a novel disease causing FOXC1 inhibitory domain mutation on FOXC1 function were also assessed. The mutation reduced FOXC1 transactivation activity and increased protein half-life both of which may lead to disease. Regulation of FOXC1 activity is critical for normal function and this work has furthered our knowledge of how the inhibitory domain influences FOXC1 activity. I have provided biological evidence for the theory that positive selection acts at the amino acid level to optimize protein function. I have also shown that both changes in transcription factor proteins and the cis-regulatory region of target genes have the potential to contribute to evolutionary adaptation.

Our lab has shown that conditionally disrupting the tcfap2beta gene, responsible for the activating protein-2beta (AP-2beta) transcription factor, exclusively in the craniofacial neural crest cells, leads to anterior segment dysgenesis. Subsequent loss of the corneal endothelium results in the adherence of the iris to the corneal stroma, causing closure of the iridocorneal angle. The activating protein-2beta neural crest cell knockout (AP-2beta NCC KO) model involves a complete blockage of the both the conventional (through the trabecular meshwork) and non-conventional (uveoscleral) pathways for aqueous humor drainage, and therefore it could be used as a powerful experimental model for glaucoma. As shown by our previous work, elevated intraocular pressure (IOP) and a 35% decrease in the number of cells in the retinal ganglion cell (RGC) layer was observed in AP-2beta NCC KO mice by 2 months; 6 to 11 months sooner than other reported mouse models of glaucoma. These observations suggested that the AP-2beta NCC KO mouse could be a novel and cost-effective experimental model for glaucoma if the RGC loss occurred progressively rather than due to a congenital defect. The purpose of this research project was to investigate how the retinal ganglion cell layer and macroglial activity changes with respect to age in the AP-2beta NCC KO mutant through immunofluorescence. Specifically, it was investigated whether the loss of RGCs was progressive and due to the increased IOP caused by the blockage of the uveoscleral drainage pathway. A significant decrease in the number of RGCs was observed between P4 and P10 in the retinal periphery of both WT and AP-2beta NCC KO mice (p<0.05), which is indicative of the programmed cell death that occurs due to retinal pruning during development. No statistical difference between WT and AP-2beta NCC KO mice phenotypes was observed at postnatal day 4 (P4), suggesting that no developmental defect resulted in the significant loss of RGCs at 2 months. In all other time points investigated, while no statistical difference was found between WT and the AP-2 NCC KO mutant, a clear downward trend was present in the AP-2 NCC KO mutant retinal ganglion cell layer from P10 to P40. There was also an expression of glial fibrillary acidic protein (GFAP) by Müller cells, indicating the presence of neuroinflammation at P35 and P40. This substantiates the potential P42 starting point of neurodegeneration our lab previously observed. This was further corroborated with Müller cell-associated expression of GFAP at P35 and P40 exclusively in the AP-2beta NCC KO mouse. Overall, we have shown that the retinal damage observed in our AP-2beta NCC KO mouse is not due to a developmental defect, but rather occurs over time. Thus, this mouse model, which appears to block both the conventional and unconventional uveoscleral pathways, has a profound effect on aqueous humor drainage. As a result, the model requires relatively little time to observe an increase in IOP and subsequent RGC loss. Our findings suggest that the AP-2beta NCC KO mouse can be a novel, powerful, and extremely cost-effective experimental model for glaucoma. / Thesis / Master of Science (MSc)

Glaucoma is a heterogeneous group of diseases that is currently considered to be the leading cause of irreversible blindness worldwide. Of the identified risk factors, elevated intraocular pressure remains the only modifiable risk factor that can be targeted clinically. Ocular hypertension is often a result of dysregulation of aqueous humour fluid dynamics in the anterior eye segment. Aqueous humour drainage is regulated by structures located in the anterior chamber of the eye. In some circumstances dysregulation occurs due to developmental abnormalities of these structures. The malformation of structures in the anterior segment is thought to be due to a defect in the differentiation and/or migration of the periocular mesenchyme during development. Unique to vertebrates, the neural crest cell (NCC) population contributes to the periocular mesenchyme and is instrumental to the proper development of structures in the anterior segment. For many years our laboratory has examined the role of the Activating Protein-2 (AP-2) transcription factors that are expressed in the neural crest and vital during the development of the eye. The purpose of this research project is to investigate the role of AP-2β in the NCC population during the development of the anterior segment of the eye. Conditional deletion of AP-2β expression in the NCC population demonstrated that mutants have dysgenesis of structures in the anterior segment including defects of the corneal endothelium, corneal stroma, ciliary body and a closed iridocorneal angle. Loss of retinal ganglion cells and their axons was also observed, likely due to the disruption of aqueous outflow, suggesting the development of glaucoma. The data generated from this research project will be critical in elucidating the role of AP-2β in the genetic cascade dictating the development of the anterior eye segment in addition to providing scientific research with a novel model of glaucomatous optic neuropathy. / Thesis / Master of Science (MSc)

Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...

Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...