The long term goal of this research is to elucidate molecular mechanisms by which cell-extrinsic cues influence retinal progenitor cell fate choices and differentiation during normal development, thereby providing a basis for the prevention and treatment of retinal diseases. The proposed study focuses on the cytokine-signaling pathway in the mouse retina. Ciliary neurotrophic factor (CNTF), a member of the cytokine superfamily, has a variety of effects on neuronal development and survival. In the developing retina, CNTF profoundly affects photoreceptor, bipolar, and amacrine cell differentiation as well as promoting ganglion cell axonal growth and survival. In addition, CNTF prevents retinal photoreceptor cell degeneration caused by mutations or light-induced damages. Despite these effects, the mechanism of CNTF action in the developing or mature retina is not understood. Cytokine signals are generally mediated through membrane receptors that activate cellular Jak tyrosine kinases and STAT transcription factors. However, the specific signaling components in the retina that mediate the effects of CNTF, and the retinal cell types that directly respond to the CNTF signal, have not been characterized. Therefore, experiments are proposed to biochemically identify the Jak kinases and STAT transcription factors activated by CNTF in the developing mouse retina using immunoprecipitation and Western blot analyses of retinal cell extracts. The distribution and cellular location of the CNTF receptor, Jak protein kinases and STAT factors in the retina will be determined by immunocytochemistry to define retinal cell types that can respond to CNTF or CNTF-like cytokines. In addition, cell type-specific activation of STAT transcription factors will be assayed by immunofluorescence microscopy of dissociated cell and explant cultures treated with CNTF using combinations of antibodies recognizing activated (phosphorylated) STAT factors and distinct retinal cell types. Lastly, the biological function of the cytokine signaling pathway during retinal neurogenesis will be examined by perturbing the activity of Jak and STAT signaling molecules. Embryonic and postnatal retinas will be infected in vivo and in vitro with murine retroviral vectors expressing mutant Jak kinases and STAT factors, and naturally-occurring Jak and STAT inhibitor proteins. Effects of these perturbations on retinal development will be examined with histological and immunocytochemical methods using molecular and cellular markers. These proposed studies will elucidate the biological function and cellular mechanisms of cytokine signal transduction during mammalian retinal neurogenesis and thus establish a foundation for therapeutic interventions of human retinal diseases using CNTF or CNTF-like cytokines.
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