The founding neural cell type in the developing retina is specified by the expression of a basic Helix-Loop Helix (bHLH) transcription factor called Atonal in the fly and Atonal homolog 5 (Ath5) in vertebrates (1-11). In the fly the founder cells later differentiate as R8 photoreceptors (12) and in vertebrates they become retinal ganglion cells (RGCs, 13). In both taxa this involves an initial rising and widespread expression of Atonal followed by its clearance from all cells except the future R8/RGC (2, 14-17). The accurate execution of this patterned selection process is crucial for normal retinal histogenesis (18). In the fly this process takes place in a moving wave called the morphogenetic furrow (12, 19-21). Here we focus on the role of the Ras/MAPK pathway in this event. MAPKs (Mitogen Activated/ Microtubule Associate Protein Kinase) are the final cytoplasmic element in the Ras signal transduction cascade (22-25). MAPKs are activated by dual phosphorylation in the cytoplasm and then phosphorylate both cytoplasmic and nuclear targets including transcription factors (26, 27). Nuclear translocation is thought to be regulated by this phosphorylation, and to rapidly follow it (in minutes, 28, 29, 30). However, in the morphogenetic furrow of the developing Drosophila eye phosphorylated MAPK antigen is held in the cytoplasm for hours (31). In the last funding period we developed a reagent to detect nuclear MAPK non-antigenically and have used this reagent to show that MAPK nuclear translocation is regulated by a second mechanism that is independent of phosphorylation. We also show that if this cytoplasmic hold is overcome, Atonal expression is disrupted and the founder cells differentiate precociously as neurons. Consequentially developmental patterning in the retina is disrupted. Preliminary data suggests that MAPK cytoplasmic hold is mediated by the sequestration of a critical nuclear transport factor: Dim7 (32, 33). We now propose four specific aims: 1) Tests for MAPK cytoplasmic hold at other times in development. 2) Define the amino-acid residues in MAPK which mediate hold. 3) Test hypotheses for the function of Dim7 in MAPK cytoplasmic hold. 4) Determine which developmental signals control Dim7 sequestration. Our long-term objective is a deep understanding of retinal histogenesis leading to possible intervention for the induction of regeneration in patients with retinal degeneration or damage.
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