Investigating the Role of the JAK/STAT Pathway in the Drosophila Eye Disc

The JAK/STAT pathway plays numerous roles in Drosophila development, including eye development. JAK/STAT signaling was previously thought to only regulate growth of the eye disc. However, our work shows that the JAK/STAT pathway plays an important role in patterning the eye disc: it promotes formation of the eye field through repression of the wingless (wg) gene. Overexpression of the JAK/STAT activating ligand Unpaired leads to loss of wg expression and ectopic morphogenetic furrow initiation from the lateral margins. Conversely, tissue lacking stat92E is transformed from retinal tissue into head cuticle, a phenotype that is caused by ectopic wg expression. Furthermore, we show that the JAK/STAT pathway regulates a small enhancer in the wg 3' cis genomic region. As this enhancer is devoid of Stat92E-binding elements, we conclude that Stat92E represses wg through another, unidentified factor that is likely a direct target of Stat92E. Additionally, we performed a structure-function analysis of the Drosophila STAT using a variety of in vivo and in vitro assays to determine the domains of Stat92E that are required for its function. We find that expression of a full-length Stat92E protein (stat92EFL) can completely rescue the small eye phenotype and larval lethality of stat92E mutants and can activate a stat92E-lucifererase reporter while stat92E with the critical Tyr711 mutated to Phe ( stat92E Y711F) cannot. Variants lacking either the first 133 (3HA stat92EDeltaN) or the last 36 (stat92EDeltaC) residues function with the same efficiency as stat92EFL in these assays, demonstrating that these domains are not required for Stat92E function. Stat92E that lacks both domains simultaneously (stat92EDeltaNDeltaC ) also functions as well as wild type and exhibits mild constitutive activity that is tyrosine dependent. Furthermore, we found that Arg 442, which resides in the DNA-binding domain and is conserved in most mammalian STATs, is required for Stat92E function. Structural modeling suggests that Arg442 forms a critical Stat92E-DNA contact point. Therefore, a substitution at this position abolishes Stat92E's ability to bind DNA and activate transcription.