Browsing by Author "McFarlane, Sarah"
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Item Open Access Channeling Vision: Voltage-Gated Calcium Channels of Rods and Cones(2018-11-27) Waldner, Derek; Stell, William K.; Bech-Hansen, N. Torben; McFarlane, Sarah; Altier, ChristopheCongenital stationary night blindness (CSNB) is a set of inherited diseases characterized by defects in neurotransmission from photoreceptors to second-order neurons in the retina. CSNB2A, specifically, is caused by mutations in Cacna1f – the pore-forming sub-unit of the voltage-gated calcium channel (VGCC), CaV1.4, which is responsible for calcium-mediated glutamate release at photoreceptor ribbon synapses. In this work, we initially expanded the characterization of the Cacna1f-KO mouse retina with a particular focus on cones using our own Cacna1fG305X mouse line. We have comprehensively characterized cone morphology and viability throughout the murine lifespan, and also verified several findings reported in an alternative Cacna1f-KO model which suggest possibility of late-stage rescue. We also identified ectopic, synapse-like cone-rod bipolar cell contacts, which have been described in no other model of retinal disease. We then sought to investigate whether late-stage rescue of retinal morphology and function is feasible in the Cacna1f-KO retina. To this end, we have designed a strategy employing transgenic mice that, with appropriate gene combinations, will allow for inducible expression of Cacna1f. We have characterized several transgenic mice relevant to these experimental aims, and provided some proof-of-concept for future experiments which may provide insight into the plausibility of therapeutic interventions. We also sought to establish an alternative model of CSNB2A in the post-embryonic chick. The relative lack of tools for genetic manipulation of this model led to us developing a novel means – avian adeno-associated viral (A3V) vectors. Using A3V vectors we were able to achieve highly efficient local transduction of photoreceptors following sub-retinal injection, thus providing us with a new tool for investigation of chicken retinal circuitry. Finally, in an attempt to replicate CSNB2A in the chicken retina, we sought to characterize VGCC expression to definitively establish the target for gene knockdown. We were able to definitively prove expression of a Cacna1f orthologue in the chicken retina, and establish its sequence and mRNA expression patterns. Unfortunately, shRNA-encoding A3Vs were unable to achieve significant knockdown, but we have established a powerful framework for future investigations in this model.Item Open Access Characterization of embryonic hypothalamic neurospheres and the role of sex steroids in the developing hypothalamus(2017) Thornton, Hayley; Kurrasch, Deborah; Biernaskie, Jeff; McFarlane, SarahThere is emerging evidence indicating sex steroids may function as neurotrophic factors during brain development. Our objective was to determine whether sex steroids influence murine hypothalamic development during peak neurogenesis. We hypothesized during development hypothalamic neural stem and progenitor cells (NSPCs) respond to hormones to influence proliferation and/or self-renewal. Initially, we characterized the neurosphere assay as an in vitro model system to study hypothalamic NSPCs. We isolated hypothalamic NSPCs and assessed various culturing parameters such as media, density, growth factors, and extrinsic signals. We then utilized this assay to test whether exposure to androgens and estrogens alters their growth. Pharmacological treatment of DHT enhanced the sphere-forming capacity of primary neurospheres. Self-renewal capacity was also increased with physiologically-relevant doses of DHT and E2 exposed only as secondary cultures. Overall, we provide a working protocol for studying embryonic hypothalamic neurospheres and evidence that hypothalamic NSPC behaviour can be modulated by sex steroids.Item Open Access Class 3 semaphorins and retinal ganglion cell development(2011) Kita, Elizabeth Maria; McFarlane, SarahItem Open Access Class 3 semaphorins and zebrafish retinal ganglion cell development(2007) Callander, Davon Christina; McFarlane, SarahItem Open Access Elucidating the Role of Pten in Retinal Development and Pattern Formation(2017) Tachibana, Nobuhiko; Schuurmans, Carol; Mains, Paul; McFarlane, Sarah; Chan, JenniferThe retina is a neural tissue that is a part of the central nervous system that resides at the back of the eye. It serves as the first sensory processing center of the visual environment. All of the cell types in the retina have been well-characterized, and their physiological functions are generally well described. As a member of a developmental biology laboratory, I have been interested in understanding the factors that dictate the events that regulate the precisely orchestrated development of the retina, such as the proliferation of neural progenitors, differentiation of neuronal cell types, migration of neuronal cells to their designated destinations, and the formation of synaptic connections. When this project was undertaken, very little was understood about the role of the Pten (phosphatase and tensin homologue) phosphatase and PI3K (phosphatidylinositol 3-kinase) signaling pathway in the retina. While PI3K signaling was known to be responsible for proliferation, differentiation, cell death, migration, neurite outgrowth, and synapse formation in different regions of the developing CNS, its role in the retina was understudied. In this thesis, I describe experiments in which I deleted Pten gene in the mouse retina with a conditional knockout (cKO) approach to elucidate its role in retinal development. During early retinal development, I found that Pten is required to regulate the differentiation of retinal amacrine cells and rod photoreceptors. I focused on amacrine cells, and found that Pten regulates amacrine cell number by modulating three different signaling pathways, Akt, TgfβII, and Erk (Chapter 3). Furthermore, I found that the deletion of Pten in the peripheral and not central retina created an animal model of Pten hamartoma tumor syndrome (PHTS), with the central ‘wild-type’-like retinal tissue forming a hamartoma-like lesion (Chapter 4). Finally, I described a role for Pten at later stages of the retinal development in regulating cellular patterning (Chapter 5). Specifically, I observed several examples of mispatterning in Pten cKO retinas, including an expansion of the inner plexiform layer, and the disrupted organization of amacrine cells in both the radial and tangential planes. I also investigated genetic relationships between Pten and the cell adhesion molecule Dscam in guiding cellular positioning, as the deletion of Dscam largely phenocopies the Pten cKO in the retina, and I found for the most part that they act in separate pathways. In summary, I have significantly expanded our knowledge of Pten function in the developing retina during my PhD studies.Item Open Access Extrinsic Factors and RPE Regeneration(2024-01-24) Selje, Sara J.; McFarlane, Sarah; Hocking, Jennifer; Ungrin, MarkThe retinal pigment epithelium (RPE) is a monolayer of pigmented cells that closely interacts with photoreceptor outer segments of the outer vertebrate retina to maintain visual function. Damage to the RPE, for instance in a disease such as Age-Related Macular Degeneration, results in photoreceptor degeneration and subsequently, vision loss. In contrast to mammals, zebrafish can intrinsically regenerate a functional RPE layer after injury. Specific molecular pathways are known to regulate RPE proliferation in culture, but the pathways that function in vivo to promote RPE regeneration remain largely unknown. My aim is to determine potential pathways that influence RPE regeneration in zebrafish. First, I examine the importance of the secreted ligand Semaphorin 3F (SEMA3F), expressed in the RPE of both mammals and zebrafish, in RPE regeneration. I use a sema3fa homozygous mutant zebrafish on a transgenic RPE injury background (Tg(rpe65a:NTR-EGFP)) where timed application of the drug metronidazole (MTZ) to the bath results in nitroreductase-mediated RPE-specific cell death. My data suggest Sema3fa has no effect on the extent of RPE injury in this model, though RPE apoptosis may be delayed and increased in the absence of Sema3fa. Further, loss of Sema3fa may induce an initial increase in proliferation in the RPE as well as increased proliferation in the photoreceptor outer nuclear layer. Second, I provide an initial assessment of the involvement of additional pathways in zebrafish RPE regeneration. These pathways impact proliferation and/or migration of cells in culture and are expressed within the RPE. I use in situ hybridization to visualize larval RPE expression of 10 candidate genes before and after RPE injury. Genes that may show changes in expression post-injury include bmp7b, caska, foxm1, her4.1, msnb, rpe65a, trpm7, and vrk1. Future work could include using loss-of-function approaches in the RPE injury model to determine potential roles of these genes in RPE regeneration. In the long-term, this work may impact gene therapies for patients suffering from retinal degenerative diseases.Item Open Access Fibroblast growth factor signalling regulates guidance gene expression(2018-01-15) Yang, Jung-Lynn Jonathan; McFarlane, Sarah; Nguyen, Minh Dang; Schuurmans, Carol; Bech-Hansen, Nils Torben; Chow, RobertNeurons need to be properly connected to form a functioning neural network. Guidance cues expressed in the neuron’s surroundings help locate postsynaptic targets and also mediate cell migration and morphology. The expression of guidance cues must be tightly controlled to precisely orchestrate the process of wiring the nervous system. These guidance cues and their receptors have been identified, but the mechanisms that control the expression of guidance genes, especially the genes for guidance cues, are relatively unknown. In particular, Slit1 and Sema3a are expressed in the forebrain of the African clawed frog, Xenopus laevis, to direct the growth of the optic tract toward the visual target in the brain, the optic tectum. Fibroblast growth factor (Fgf) signalling maintains the expression of slit1 and sema3a during optic tract development. I demonstrate how Fgf receptors (Fgfrs) regulate the slit1 and sema3a promoters and characterize the −2285+326slit1 and −2930+63sema3a sequences to locate silencers and core promoters. In the forebrain, Fgfr1 modulates slit1 expression, and Fgfr2-4 regulate sema3a expression. Then, I explore signalling pathways downstream of Fgfr activation that modify slit1 and sema3a expression. PI3K-Akt signalling promotes slit1 and sema3a expression, but active MAPK signalling is sufficient to downregulate these guidance genes. Further, because Fgf signalling controls the expression of Lhx2/9 and Etv1, I choose these as candidate transcription factors for slit1 and sema3a expression. In vitro and in vivo, Lhx2 and Etv1 repress and transactivate, respectively, slit1 and sema3a. In vitro, Lhx9 gain of function downregulates slit1. The focus of this thesis is guidance cue regulation, specifically, the mechanisms of how Fgf signalling regulates slit1 and sema3a expression through Fgfrs, intracellular signalling pathways, and transcription factors, thereby connecting extrinsinc factors to intrinsic control of guidance cue expression.Item Open Access Fibroblast growth factors and axon guidance in the developing visual system(2004) Webber, Christine A.; McFarlane, SarahItem Open Access Fibroblast growth factors and retinal cell genesis(1999) Patel, Ambreen; McFarlane, SarahItem Open Access GABA and retino-tectal development(2000) Ferguson, Shane C. D. J.; McFarlane, SarahItem Open Access The Glyoxalase 1 – Methylglyoxal Pathway Regulates Neurite Development of Cerebral Cortical Neurons in the Mammalian Brain(2020-05) Mohammad, Lamees; Yang, Guang; Guo, Jiami; McFarlane, Sarah; Huang, Carol T.L.Newborn neurons of the mammalian cerebral cortex undergo substantial morphological changes during development. Neurite elongation and branching are crucial morphological changes in early neuronal development that are necessary for the formation of dendrites and axons – structural elements that allow neurons to communicate with each other and form circuits. One factor that has received little attention as a possible regulator of neurite development is metabolism. In this thesis, I show that methylglyoxal, an intermediate metabolite of glycolysis, and its metabolizing enzyme, glyoxalase 1 (Glo1) regulate the elongation and branching of neurites. Knockdown of Glo1 expression using short-hairpin RNA or inhibiting the enzymatic activity of Glo1 in cultured mouse cortical neurons reduces neurite length and impairs branching organization. Furthermore, I found that knockdown or inhibition of Glo1 activity perturbs development of both excitatory projection neurons and inhibitory interneurons. When neurons are treated with excessive methylglyoxal, morphological perturbation is recapitulated. These results suggest a link between methylglyoxal metabolism and neuronal development and provide the foundation for future studies of the molecular mechanisms that mediate this metabolic regulation.Item Open Access Illuminating the Role of Opsin Proteins in Circadian Rhythm Regulation(2022-08) Debnath, Nilakshi; McFarlane, Sarah; Antle, Michael; Grewal, SavrajCircadian rhythms allow organisms from bacteria to humans to entrain with the environmental light: dark cycle and regulate their physiological, metabolic, and behavioral activities. The rhythms are regulated by the oscillations of four core circadian genes: Period, Cryptochrome, BMAL1 and CLOCK. These rhythms synchronize to the light: dark cycle of the environment. Detecting light is necessary not only for vision but also for the proper synchronization of circadian rhythms. Opsins are proteins that detect light and are primarily expressed in the retina for visual light detection. Studies have demonstrated that opsins synchronize the circadian rhythms of an organism; however if opsins show rhythmic expression is still being studied. Furthermore, opsins are expressed in the skin where their function is poorly understood. Some of the current treatments for circadian rhythm disorders are light exposure based. Better understanding of the regulation of light-detecting proteins will aid in the development of more effective treatment. I focus on how mRNAs and proteins for opsins in the skin are regulated over 24 hours. Specifically, I studied opsin regulation in pigmented cells of the skin known as melanocytes (mammals) and melanophores (non-mammalian vertebrates). These cells exhibit dynamic pigmentation responses to environmental light and dark. Using Xenopus laevis as a model organism, I analyzed the expression of 22 opsin genes previously identified in this species in the skin by RT-PCR. Highly expressed in melanophores were: opn5 (a neuropsin), and the two melanopsin homologues opn4 and opn4b, as well as the four core clock genes. In-situ hybridization studies showed opn5 mRNA to be expressed in a punctate manner similar to the distribution of the melanosomes (organelles containing melanin pigment), suggesting a particular intracellular distribution of the mRNA. Protein distribution of the opsins, determined by immunohistochemistry, shows a similar pattern, suggesting specific localized expression of Opn5. Preliminary data suggest that while opn4b mRNA is regulated in a circadian manner, protein expression remains relatively constant. Whereas both the mRNA and protein of Opn5 show changes over time. My results indicate that at least two opsins are expressed in a cyclic manner in the pigmented skin cells.Item Open Access Investigating the coordination of thermoregulation by thermosensory mechanisms(2021-11) Malik, Hannan Rauf; McFarlane, Sarah; Schnetkamp, Paul; Altier, Christophe; Thompson, RogerThermoregulation is a critical homeostatic process through which organisms maintain cellular and physiological integrity. Depending on how thermoregulation occurs, organisms can be categorized as either endothermic, using heat from metabolic processes for thermoregulation, and ectothermic, not relying on metabolic heat for thermoregulation. Ectotherms also link their body temperature to that of the ambient environment. Both endotherms and ectotherms utilize a superfamily of polymodal proteins known as transient receptor potential (TRP) channels to initiate the thermoregulatory response. TRPM8, a member of the TRPM subfamily, has been characterized as a transducer of cold sensor in various vertebrates through functioning as a cutaneous thermoreceptor. There is still a paucity of information on this protein, whether it is expressed in the skin of the ectotherm, Xenopus laevis and its involvement in the afferent thermoregulatory response. This thesis explores the thermoregulatory response of Xenopus laevis to cooling, as well as the expression and putative function of TRPM8. First, I demonstrate that Xenopus laevis embryos exhibit a rapid, systemic melanosome aggregation upon introduction to non-noxious cool temperatures. Second, I examine the expression of the TRPM subfamily via RT-PCR in Xenopus, and then identify the expression of TRPM8 at the protein level in the skin of this ectotherm. Lastly, I demonstrate the role of TRPM8 in the thermoregulatory response, as a TRPM8 agonist phenocopies the melanosome aggregation seen as a response in cool temperatures, while an antagonist diminishes the melanosome aggregation. My work elucidates the thermoregulatory response of Xenopus laevis, as well as the role of TRPM8 in mediating the melanosome aggregation to add to the growing literature of this ion channel in thermoregulation and transducing cold.Item Embargo Pericyte deficiencies in a foxf2 loss of function mutant(2024-06-26) Graff, Merry Faye; Childs, Sarah; McFarlane, Sarah; Grewal, SavrajPericytes are critical players in vascular development and disease but are not widely studied in the context of cerebral small vessel disease, a vascular condition related to stroke that progressively weakens brain microvessels. Foxf2, a pericyte-expressed gene, is involved in vascular stability and reduced FOXF2 is associated with increased stroke risk and CSVD prevalence. Here, I use a zebrafish foxf2a loss of function mutant as a genetic model of CSVD to show pathological alterations in brain pericytes. Not only are there fewer brain pericytes across the lifespan, but these pericytes also exhibit morphological abnormalities like increased soma size, process length and degeneration. These abnormalities manifest differently in adult brains, and blood vessel defects are evident. I show that embryonic pericyte defects are not the result of decreased pericyte density but a combination of cell-autonomous and non-cell-autonomous effects. A downstream target of foxf2a, loxl2b, is downregulated in foxf2 mutants. While loxl2b mRNA is sufficient to boost brain pericyte numbers, mutants for this gene do not show an obvious phenotype, suggesting a more complicated role. This thesis sheds light on novel lifespan defects in mutant brain pericytes, potentially contributing to vascular destabilization, and opens new avenues for studying genetic forms of human cerebral small vessel disease.Item Open Access Plxna4 and its role in neuronal survival in the developing zebrafish hindbrain(2019-09-17) Nurcombe, Zachary W.; McFarlane, Sarah; Kurrasch-Orbaugh, Deborah M.; Ousman, Shalina S.; Mains, Paul E.Plexins are a family of transmembrane proteins that bind Semaphorin ligands, and are known to function in axon guidance. In embryonic zebrafish, I find that plxna4 is expressed widely in the nervous system early in development, and localizes to the hindbrain as neurogenesis and differentiation proceed. In the hindbrain of a plxna4-/- CRISPR mutant, expression of the Islet transcription factor by cranial motor neurons appears depleted at 48 hours post fertilization, with increased apoptosis at 24 hpf. These data support a survival role for Plexina4 in the embryonic zebrafish hindbrain. Literature indicates that the secreted heat shock protein Clusterin is a candidate ligand to mediate apoptosis through Plexina4, as Clusterin is known to promote cell survival, and bind Plxna4. I find that clu is expressed by the floorplate of the embryonic zebrafish hindbrain. Epistasis experiments reveal Clusterin and Plexina4 promote cell survival in the developing zebrafish hindbrain via independent pathways.Item Open Access The Role of Ciliopathy Genes in Axonal Development(2022-01-26) Catalano, Christy Nicole; Guo, Jiami; Mains, Paul; McFarlane, Sarah; Yang, GuangPrimary cilia are tiny, microtubule-based organelles that project from the body of all mammalian cells and function as the cellular signalling hub. Genetic mutation of ciliary genes leads to multi-organ system dysfunction causing a group of diseases called ciliopathies. Notably, ciliopathy patients present with severe neurological phenotypes, including intellectual disability and prominent axon tract defects, suggesting a role for primary cilia in axonal development. However, the mechanisms behind axonal phenotypes in ciliopathies are not well understood. The formation of axons requires the assembly of very long microtubules, which are nucleated by the centrosome. As the cell’s microtubule organizing centre and the organelle that forms the base of the primary cilium, the centrosome is a common link between the axonal cytoskeleton and the cilium. Therefore, I hypothesized that the loss of ciliary proteins could impact the microtubule cytoskeleton, which in turn could influence axonal morphology and microtubule-based trafficking. This thesis investigates the role of two proteins that localize to the centrosome and base of the cilium, Ahi1 and Bbs7, in axonal development. Using targeted shRNA gene knockdown in cortical mouse neurons, I first investigated the roles of Ahi1 and Bbs7 in axonal morphology. Then, I further analyzed the role of Ahi1 in axons, using live cell imaging to examine axonal trafficking along microtubules. This thesis provides evidence that initial axonal outgrowth and axonal branching are inhibited by ciliary gene knockdown. I also present evidence that the trafficking of early endosomes and synaptic vesicles along axonal microtubules is altered by Ahi1 deficiency, which could impact axon growth, health, and synaptic function. Further research will be necessary to understand the exact cause and consequences of these changes in axonal morphology and trafficking; however, this thesis substantiates a role for primary cilia in regulating the developing axon.Item Open Access Sema6d Signaling in the Developing Visual System(2018-06-28) Cechmanek, Paula Bernice; McFarlane, Sarah; Grewal, Savraj S.; Schuurmans, CarolThe retinal pigment epithelium (RPE) is a highly specialized monolayer of epithelial cells that arise from retinal progenitors and form a tight barrier between the neural retina (eye) and the brain. Although only a thin monolayer, RPE cells perform numerous important functions and are indispensible for mature photoreceptor renewal and survival. When RPE dysfunction occurs, in diseases like Retinitis Pigmentosa and Age-related Macular Degeneration (AMD), devastating vision loss can occur. While much work has focused on understanding RPE function in adult life, little is known about how the RPE arises during early embryonic life or how it spreads to wrap around the neural retina. In this thesis I use zebrafish (Danio rerio) as a model to a) characterize early RPE development, and b) implicate the classical axon guidance cue Semaphorin6d (Sema6d), and its receptor Plexina1 (Plxna1), in RPE and optic cup morphogenesis in the developing zebrafish embryo. I show that markers of RPE differentiation turn on in RPE progenitors prior to optic cup morphogenesis, and that RPE morphogenesis occurs in two phases: First an antero-posterior expansion of the RPE progenitor domain, followed by a second phase coupled to optic cup morphogenesis, where the RPE domain is stretched to form a thin, single-cell epithelium around the eye. Through Sema6d loss-of-function analysis I show that this second phase depends on signals between the Sema6d-expressing progenitors of the temporal neural retina and RPE progenitors expressing Plxna1. This is the first signaling pathway to be identified as a driver for optic cup morphogenesis in any model system. Developing zebrafish as a model system to study early RPE development and retinal cell migration may provide insights to understanding human RPE/retinal cell migration during development and disease.Item Open Access Semaphorin3f as a cardiomyocyte derived regulator of heart chamber development(2022-08-19) Halabi, Rami; Cechmanek, Paula B.; Hehr, Carrie L.; McFarlane, SarahAbstract Background During development a pool of precursors form a heart with atrial and ventricular chambers that exhibit distinct transcriptional and electrophysiological properties. Normal development of these chambers is essential for full term survival of the fetus, and deviations result in congenital heart defects. The large number of genes that may cause congenital heart defects when mutated, and the genetic variability and penetrance of the ensuing phenotypes, reveals a need to understand the molecular mechanisms that allow for the formation of chamber-specific cardiomyocyte differentiation. Methods We used in situ hybridization, immunohistochemistry and functional analyses to identify the consequences of the loss of the secreted semaphorin, Sema3fb, in the development of the zebrafish heart by using two sema3fb CRISPR mutant alleles. Results We find that in the developing zebrafish heart sema3fb mRNA is expressed by all cardiomyocytes, whereas mRNA for a known receptor Plexina3 (Plxna3) is expressed preferentially by ventricular cardiomyocytes. In sema3fb CRISPR zebrafish mutants, heart chamber development is impaired; the atria and ventricles of mutants are smaller in size than their wild type siblings, apparently because of differences in cell size and not cell numbers. Analysis of chamber differentiation indicates defects in chamber specific gene expression at the border between the ventricular and atrial chambers, with spillage of ventricular chamber genes into the atrium, and vice versa, and a failure to restrict specialized cardiomyocyte markers to the atrioventricular canal (AVC). The hypoplastic heart chambers are associated with decreased cardiac output and heart edema. Conclusions Based on our data we propose a model whereby cardiomyocytes secrete a Sema cue that, because of spatially restricted expression of the receptor, signals in a ventricular chamber-specific manner to establish a distinct border between atrial and ventricular chambers that is important to produce a fully functional heart. Video abstractItem Open Access Semaphorin3f as a Spatial Regulator of Embryogenesis(2019-01-22) Halabi, Rami; McFarlane, Sarah; Childs, Sarah J.; Huang, PengDuring embryogenesis, cells integrate both spatial and temporal information from their surroundings to influence proliferation, migration, differentiation and physiological functions. Understanding the molecular mechanisms which confer spatial identity is essential to our understanding of tissue development and human disease. In this thesis I explore multiple roles for the secreted chemotactic ligand Semaphorin3f (Sema3f) in different biological contexts. Using zebrafish (Danio rerio) as a model I take advantage of the duplicated genome to study loss of function of both orthologs, Sema3fa and Sema3fb, in discrete contexts due to their differential expression. First, I show that in the eye Sema3fa produced by progenitors is necessary for the generation of amacrine cells within the temporal retina and the spatially-organized transcriptome of stem cells in the ciliary marginal zone (CMZ). Second, I define an endogenous role of Sema3fa to maintain the avascularity of the neural retina and refine the branch pattern of intraocular vessels. Loss of Sema3fa results in the pathologic angiogenesis of leaky blood vessels into the neural retina. Last, I unveil a role for Sema3fb produced by cardiomyocyte progenitors in the differentiation of the ventricle of the developing heart. Overall, my work provides the first evidence of a Sema3 involved in retinal progenitor cell and cardiomyocyte differentiation, and elucidates the endogenous role of Sema3fa as a negative regulator of retinal blood vessels in the embryo and adult. My data exemplifies the necessity of spatial information conferred by a single chemotactic molecule, Sema3f, to impact differentiation and cellular biology.Item Open Access Semaphorin3f in the maturation of the outer retina(2020-09-25) Mori-Kreiner, Risa; McFarlane, Sarah; Childs, Sarah J.; Guo, Jiami; Ungrin, Mark D.Cells of multicellular organisms have the remarkable ability to coordinate and control dynamic cellular activities in response to changes in their environment. From development into tissue homeostasis, cells communicate with each other through a myriad of intercellular signalling mechanisms. The large family of Semaphorins is a group of well-known extracellular signalling molecules implicated in a wide range of diverse physiological functions. In particular, the expression of secreted Class III Semaphorins (Sema3s) in the retina, not only during development but also in adult tissue, raises interesting questions about their tissue-specific spatiotemporal roles. The vertebrate retina is a highly complex, light-sensitive tissue that lines the back of our eyes. Within the retina, there are two key players that enables our ability to see: the retinal pigment epithelium (RPE) and the photoreceptors. Located in the outermost layer of the retina, the RPE and photoreceptors develop and mature together throughout the lifetime of the organism, forming an interdependent relationship that is highly critical for visual function. This thesis explores, using CRISPR/Cas9-generated loss-of-function mutants (sema3faca304), cell autonomous and non-cell autonomous roles of an RPE-secreted protein, Semaphorin3f (Sema3f), in the zebrafish retina. First, I demonstrate that both cell types, the RPE and photoreceptors, express multiple members of well-known Sema3 receptors, Nrp and PlxnA families. Second, I define a cell autonomous role of Sema3fa in maturing RPE. The loss of Sema3fa does not affect the maturation of the RPE at the transcriptional and morphological level, but does result in the perturbation of appropriate physiological responses to light conditions. Last, I demonstrate the non-cell autonomous role of Sema3fa in the development and specification of maturing photoreceptors. My work elucidates one of the many endogenous roles of Sema3fa as a regulator of the maturing retina to add to the growing literature of Sema signalling in events other than development.