Characterization of split ends, a new component of the Drosophila epidermal growth factor receptor signaling pathway

Abstract

Split ends (spen) was isolated as a strong enhancer of the rough eye phenotype associated with constitutive activation of Yan, implicating spen as a positive regulator of the receptor tyrosine kinase (RTK) signaling pathway. Molecular characterization of spen has revealed that spen encodes a protein with 5476 amino acids. It contains three tandem repeats of an RNA Recognition Motif (RRM) at its N-terminus, suggesting that Spen might function as an RNAbinding protein. Spen also contains a highly conserved SPOC (Spen Pearalogue and Orthologue C-terminal) domain at its C-terminus. Spen-like proteins exist from worms to humans, and they likely define a novel subfamily of RNA-binding proteins based on the RRM sequence similarities. Characterization of spen mutant phenotypes in the context of RTK signaling suggests that spen function is required for normal eye development and wing vein formation, both contexts where RTK signaling has been proven to play important roles. We have focused on the development of Drosophila embryonic midline glial cells (MGCs) and have demonstrated that spen is required for the normal migration and survival of MGCs. Loss of spen leads to aberrant migration and as a consequence, reduced number of midline glial cells. As a result, spen mutant embryos exhibit severe morphology and axonguidance defects in the central nervous system, a phenotype strikingly reminiscent of those seen in spitz group mutants. The phenotypic analysis of spen mutants strongly suggests that spen is a positive regulator of the RTK pathway. Further supporting this hypothesis, we have shown that spen synergistically interacts with pointed. To further investigate the relationship between spen and the RTK pathway, we have generated a dominant negative mutant protein by truncating the C-terminus of Spen including the highly conserved SPOC domain (Spen[Delta]C). Specific overexpression of Spen[Delta]C in the midline glial cells causes lethality, and we have demonstrated that the lethality associated with Spen[Delta]C can be rescued by overexpression of activated Ras vi 2 and activated DER ligand Spitz. Since Spen[Delta]C also suppresses the lethality caused by Ras v12, spen is likely to function genetically downstream of or in parallel to Ras. The implication of a putative RNA-binding protein downstream of the RTK/Ras pathway suggests that there might be post-transcriptional gene regulation mechanisms downstream of Ras to allow the cells quickly and precisely to respond to extracellular signals. In order to elucidate the molecular mechanisms underlying Spen function in the RTK pathway, we have designed a genetic screen to isolate spen-interacting genes. By overexpression of a nuclear-localization-sequence (NLS)-tagged Spen C-terminus (CspenNLS) specifically in the eye, we have generated a rough eye phenotype. Reducing endogenous spen dosage enhances this rough eye phenotype, suggesting that CspenNLS functions as a dominant negative mutant in vivo, possibly by sequestering the spen-interacting proteins. Using this phenotype as a starting background, we screened through the deficiency kit which uncovers - 80% of the Drosophila genome and have isolated 23 enhancing and 27 suppressing regions. Among the modifiers, there are regions uncovering known RTK pathway components, including Draf, sevenless, vein, sevenup, pointed and Ras, consistent with spen functioning as a component of the RTK pathway. Most interestingly, we have isolated multiple overlapping deficiencies as modifiers of CspenNLS, suggesting that these overlapping regions might contain candidate genes directly interacting with spen. Future genetic and biochemical analysis of these candidate genes will likely shed important light on the molecular mechanisms underlying Spen function.