The mammalian central nervous system (CNS) has a high degree of complexity and cell type diversity that enables sophisticated processing of sensory information, circuit formation, and behaviour. While much is known about the patterning and specification of the major neuronal classes in the CNS, through processes such as morphogen gradient signaling and transcription factor combinatorial coding, much less is known about how subtypes within each cell class are specified. Bipolar cells are one of the main classes of interneurons in the vertebrate retina and consist of fifteen different subtypes based on their physiological function, morphology, and unique gene expression. The cellular mechanisms behind the specification of these subtypes are not fully known. In this thesis, I examine these mechanisms by investigating the role of extrinsic and intrinsic factors on the specification and differentiation of bipolar cell subtypes. We hypothesize that the specification of bipolar cell subtypes occurs in a multi-step manner and is dependent on non-cell autonomous (extrinsic) signals. To test this hypothesis, I conducted a series of experiments on the early postnatal mouse retina, which is the period when bipolar cells are generated. First, I examined whether bipolar cell marker onset was temporally ordered as would be predicted in a multi-step model. Postnatal day 3 (P3) mice were injected with EdU (5-ethynyl-2’-deoxyuridine), a thymidine analog that labels proliferating cells and then dissociated and fixed the retinal cells 24-120 hours after injection. My results show that Vsx2-5.3-PRE-Cre, a marker of pan-bipolar cells specification, is first detected 36 hrs after cell cycle exit, whereas specialized bipolar subtype-specific markers are expressed 48-60 hrs post-EdU injection. This observation is consistent with the idea that bipolar cells develop in a stepwise manner, first as an unspecified, pan-bipolar cell intermediate and then into one of the 15 subtypes. To further investigate this possibility, I developed a novel dissociated retinal culture assay that enabled me to accurately track retinal progenitor cells and postmitotic precursor cells and determine the requirement of cell autonomous and non-cell autonomous mechanisms during bipolar cell subtype specification. This assay involves culturing dissociated retinal cells from P3 EdU-injected mice at high density (abundant cell contact) or low density (scarce cell contact) at various timepoints, thereby allowing me to probe the role of these mechanisms in RPCs, early postmitotic cells, and late postmitotic cells. My findings revealed the first 24-48 hrs post cell cycle exit to be a critical, cell contact-dependent period for the specification of bipolar cell subtypes. This assay also allowed us to test the effect of blocking or activating the Notch and the Sonic Hedgehog (Shh) signal transduction pathways by using pharmacological compounds and recombinant ligands. Co-activation of Notch and Shh pathways increased the specification of Vsx1+ subtypes suggesting they play a role in their specification. Altogether, our results suggest that bipolar cell subtype specification follows a multi-step model, through an undifferentiated bipolar cell intermediate, and that cell contact plays a role in the specification mechanisms of bipolar cell subtype development. This is a novel finding that provides insight into the mechanisms underlying retinal neuronal subtype development and possibly in other neuronal cell types throughout the CNS. / Graduate / 10000-01-01
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/14194 |
| Date | 07 September 2022 |
| Creators | Ruiz de Chavez Ginzo, Alberto |
| Contributors | Chow, Robert Lewis |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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