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Abstract

Tissue homeostasis and regeneration in vertebrate organs often depend on the precise regulation of tissue-specific stem cell populations. In the teleost retina, the maintenance of retinal stem cells (RSCs) is essential for supporting neurogenesis and sustaining tissue growth and function throughout life. While intrinsic factors governing stem cell behavior have been well-studied, the contribution of extrinsic cues, particularly those originating from immune cells such as microglia, is not clearly understood. The teleost fish, Japanese medaka (Oryzias latipes), with its active and lifelong retinal growth supported by a well-defined stem cell niche, provides an excellent model to investigate these regulatory mechanisms. This thesis investigates the role of microglia in maintaining the RSC niche and retinal tissue homeostasis in medaka, focusing on immune-stem cell interactions mediated by chemokine signaling. Preliminary work revealed that RSCs express the chemokine Ccl25b, while microglia express its receptor Ccr9a, suggesting chemokine-mediated crosstalk. First, using transgenic lines expressing fluorophore-tagged chemokine cells and a combination of live imaging and immunostaining, I demonstrate direct interactions between microglia and RSCs within the stem cell niche of the retina. These results highlight a novel role of Ccl25b-Ccr9a signaling mechanism in immune-stem cell interaction in the retina. To dissect the significance of this chemokine signaling, I utilized a CRISPR/Cas9 generated ccl25b mutant line and analyzed both microglial behavior and stem cell dynamics. The loss of Ccl25b disrupted microglial interaction with RSCs, leading to altered phagocytic responses but did not affect the size of the stem cell pool. Further investigation using microglia depleted mutants revealed that, in the absence of microglia, RSC populations were significantly increased, their lineage potential impaired, and visual function was dysregulated, underscoring the essential role of microglia in safeguarding the stem cell niche. Finally, to determine the specificity of this regulatory axis, I created a retinal progenitor cell (RPC) specific transgenic line based on single-cell RNA sequencing markers, demonstrating that the dependency on microglial input is not restricted to RSCs and extends to other cell populations within the ciliary marginal zone. Although the precise mechanism by which microglia phagocytose RSC and RPC remains unclear, this work advances our understanding of immune-stem cell crosstalk in vertebrate tissue homeostasis and highlights microglia as critical regulators of retinal stem cell niches.