Abstract
The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to ‘cis’-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader applicability than has been previously recognized.
Highlights
The external sensory organs of Drosophila include the compound eye and the bristles, both of which have served as paradigms to understand mechanisms underlying neurogenesis
We focused on the putative MAPK site, as this effector of EGFR signaling has been implicated in birth of patterned R8s, and because murine HES6 is modified by CK2 and MAPK [73, 75]
The studies described here reveal that the highly conserved MAPK site in E(spl)-M8 is important for repressor activity in the developing eye, a regulation that appears to be dispensable during bristle development
Summary
The external sensory organs of Drosophila include the compound eye and the bristles, both of which have served as paradigms to understand mechanisms underlying neurogenesis (reviewed in [1,2,3,4,5]). The precise hexagonal (pseudocrystalline) geometry of the Drosophila compound eye [6] has afforded an ideal system to understand the roles of signaling pathways and molecular determinants driving cell fate specification and pattern formation [3, 7,8,9,10]. The compound eye of Drosophila is composed of ~750 unit eyes (ommatidia), each of which includes eight photoreceptors (R1-R8), twelve accessory cells and an inter-ommatidial bristle (IOB). The temporal nature of the MF represents a 48-hour window, encompassing retinal neurogenesis
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