Retinal Axon Guidance Requires Integration of Eya and the Jak/Stat Pathway into Phosphotyrosine-Based Signaling Circuitries in Drosophila

Abstract

The transcriptional coactivator and phosphatase eyes absent (Eya) is dynamically compartmentalized between the nucleus and cytoplasm. Although the nuclear transcriptional circuits within which Eya operates have been extensively characterized, understanding of its cytoplasmic functions and interactions remains limited. Our previous work showed that phosphorylation of Drosophila Eya by the Abelson tyrosine kinase can recruit Eya to the cytoplasm and that eya-abelson interactions are required for photoreceptor axons to project to correct layers in the brain. Based on these observations, we postulated that photoreceptor axon targeting might provide a suitable context for identifying the cytoplasmic signaling cascades with which Eya interacts. Using a dosesensitive eya misexpression background, we performed an RNA interference-based genetic screen to identify suppressors. Included among the top 10 hits were nonreceptor tyrosine kinases and multiple members of the Jak/Stat signaling network (hop, Stat92E, Socs36E, and Socs44A), a pathway not previously implicated in axon targeting. Individual loss-of-function phenotypes combined with analysis of axonal projections in Stat92E null clones confirmed the importance of photoreceptor autonomous Jak/Stat signaling. Experiments in cultured cells detected cytoplasmic complexes between Eya and Hop, Socs36E and Socs44A; the latter interaction required both the Src homology 2 motif in Socs44A and tyrosine phosphorylated Eya, suggesting direct binding and validating the premise of the screen. Taken together, our data provide new insight into the cytoplasmic phosphotyrosine signaling networks that operate during photoreceptor axon guidance and suggest specific points of interaction with Eya.KEYWORDS SH2; genetic screen; retinal determination network; eye development; Socs44A A remarkable feature of multicellular animal development is that the complexity and diversity in tissue type and patterning is achieved using fewer than a dozen signaling pathways, including Notch, receptor tyrosine kinase, Wnt, Hedgehog, TGFb, Jak/Stat, and Hippo. These cascades are repeatedly deployed in different contexts to regulate the majority of the critical proliferation, survival, specification, and differentiation decisions (reviewed in Voas and Rebay 2004;Housden and Perrimon 2014). One strategy to achieve context-specific developmental regulation is for proteins and pathways to function together in interconnected networks. Further, individual proteins within these networks may encode multiple independent functions that can be executed in distinct parts of the cell, cell types, or stages of development. In this way, even a modest number of individual proteins and core pathways can create vast combinatorial possibilities.Eyes absent (Eya), a protein conserved from plants to humans, presents an ideal model to study integration because its multifunctionality and dynamic subcellular localization provide opportunities for interaction with many signaling pathways (reviewed in Jemc andRebay 2007 an...

The Crk adapter protein is essential forDrosophilaembryogenesis, where it regulates multiple actin-dependent morphogenic events

“…Intriguingly, abl (Rogers et al, 2016), SCAR (Zallen et al, 2002), and sponge/mbc loss (Biersmith et al, 2011) all can lead to CNS defects similar to those seen after Crk knockdown. Crk knockdown also modifies photoreceptor axon targeting defects arising from overexpression of Eyes absent (Hoi et al, 2016), an event also regulated by Abl (Xiong et al, 2009). Our data also suggest that not all Robopositive axons respect the normal midline repulsive signals after Crk knockdown, although basic Robo and Slit expression patterns are intact.…”

Maheshvara regulates JAK/STAT signaling by interacting and stabilizing hopscotch transcripts which leads to apoptosis in Drosophila melanogaster

“…JAK/STAT signaling is an important pathway required for Drosophila eye development and for proper axon targeting in a cell autonomous manner during photoreceptor formation 32 . Further, genetic studies have previously shown that overexpression of upd and hop Tumorous-lethal which encodes constitutively active form of hop results in dramatically overgrown adult eyes.…”

The Crk adapter protein is essential forDrosophilaembryogenesis, where it regulates multiple actin-dependent morphogenic events

“…Intriguingly, abl (Rogers et al , 2016), SCAR (Zallen et al , 2002), and sponge/mbc loss (Biersmith et al , 2011) all can lead to CNS defects similar to those seen after Crk knockdown. Crk knockdown also modifies photoreceptor axon targeting defects arising from overexpression of Eyes absent (Hoi et al , 2016), an event also regulated by Abl (Xiong et al , 2009). Our data also suggest that not all Robo-positive axons respect the normal midline repulsive signals after Crk knockdown, although basic Robo and Slit expression patterns are intact.…”