A Drosophila PIAS homologue negatively regulates stat92E

Abstract

Transcriptional activation by, and therefore the physiologic impact of, activated tyrosine-phosphorylated STATs (signal transducers and activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by previous experiments with mammalian cells in culture. Here, by using the genetic modifications in Drosophila, we demonstrate the in vivo functional interaction of the Drosophila homologues stat92E and a Drosophila PIAS gene (dpias). To this end we use a LOF allele and conditionally overexpressed dpias in JAK-STAT pathway mutant backgrounds. We conclude that the correct dpias/stat92E ratio is crucial for blood cell and eye development.

Figure 1

Semiquantitative RT-PCR of dpias⁰³⁶⁹⁷/CyO;Actin-GFP,dpias⁺ (Left) and homozygous dpias⁰³⁶⁹⁷ (Right) first instar larvae. +RT, reverse transcriptase added and primers amplifying dpias mRNA. −RT, no reverse transcriptase added and primers amplifying dpias mRNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), reverse transcriptase added and primers amplifying gapdh mRNA.

Figure 2

The conserved central domain of dPIAS interacts directly with vanadate/H₂O₂-activated STAT92E (see text for details).

Figure 3

Eye phenotypes and genetic interaction demonstrated by various gene dosages of dpias, stat92E, and os. (A) ey-Gal4/TM2 flies appear WT. (B) Overexpression of dpias(537) by using one copy of UAS-dpias(537) driven by ey-Gal4 results in small and rough eyes (UAS-dpias(537)/CyO;ey-Gal4/TM2). (C) The majority of flies with two copies of UAS-dpias(537) have no eyes (UAS-dpias(537)/UAS-dpias(537);ey-Gal4/TM2). (D) Eye size and texture of eyes as in B can be rescued with simultaneous overexpression of STAT92E (UAS-dpias(537)/hs-stat92E;ey-Gal4/TM2). (E) Flies heterozygous for stat⁰⁶³⁴⁶ have phenotypically WT eyes (stat⁰⁶³⁴⁶/TM2). (F) When heterozygous stat⁰⁶³⁴⁶ flies are crossed to flies (as in B), an additional antennae can develop in place of the eye. The two resulting antennae (aristae) are marked by white arrows (UAS-dpias(537)/CyO;stat⁰⁶³⁴⁶/ey-Gal4). (G) dpias⁰³⁶⁹⁷/+ eyes have WT appearance. (H) The eyes of os¹;CyO/+;stat⁰⁶³⁴⁶/+ flies are small but can be partially rescued in size by reducing the gene dosage of dpias as shown in a sibling (I) with the genotype os¹;dpias⁰³⁶⁹⁷/+;stat⁰⁶³⁴⁶/+.

Figure 4

(A Left) SEM of a larger equatorial dpias^−/− eye clone extending along the equator (marked by an arrow), which can be seen to extend horizontally along the equator. In the blow up (A Right), normal lenses outside the clone, located dorsally and ventrally of the clone (two are marked with an asterisk), can be seen. In the center of the clone (marked by the bracket), the lens architecture is completely lost and the surface is bulged out. The Inset marks an example of a “partial lens phenotype” (37) with dotted lines, straddling the clonal border with ectopic hairs. (B Left) Diagram of a sectioned dpias^−/− eye clone delineating the clonal area in gray. In the blow up (B Right), partial (arrow) or completely failed cellular differentiation (center) can be observed in the clonal area.