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Culture of Drosophila expressing the steroid-dependent GeneSwitch transcriptional activator under the control of the ubiquitous α-tubulin promoter was found to produce extensive pupal lethality, as well as a range of dysmorphic adult phenotypes, in the presence of high concentrations of the inducing drug RU486. Prominent among these was cleft thorax, seen previously in flies bearing mutant alleles of the nuclear receptor Ultraspiracle and many other mutants, as well as notched wings, leg malformations, and bristle abnormalities. Neither the α-tubulin-GeneSwitch driver nor the inducing drug on their own produced any of these effects. A second GeneSwitch driver, under the control of the daughterless promoter, which gave much lower and more tissue-restricted transgene expression, exhibited only mild bristle abnormalities in the presence of high levels of RU486. Coexpression of the alternative oxidase (AOX) from Ciona intestinalis produced a substantial shift in the developmental outcome toward a wild-type phenotype, which was dependent on the AOX expression level. Neither an enzymatically inactivated variant of AOX, nor GFP, or the alternative NADH dehydrogenase Ndi1 from yeast gave any such rescue. Users of the GeneSwitch system should be aware of the potential confounding effects of its application in developmental studies.

Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In Drosophila pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax. Here we report that concomitant expression of the Ciona intestinalis alternative oxidase (AOX) was able to compensate for JNK pathway downregulation, substantially correcting the cleft thorax phenotype. AOX expression also promoted wound-healing behavior and single-cell migration in immortalized mouse embryonic fibroblasts (iMEFs), counteracting the effect of JNK pathway inhibition. However, AOX was not able to rescue developmental phenotypes resulting from knockdown of the AP-1 transcription factor, the canonical target of JNK, nor its targets and had no effect on AP-1-dependent transcription. The migration of AOX-expressing iMEFs in the wound-healing assay was differentially stimulated by antimycin A, which redirects respiratory electron flow through AOX, altering the balance between mitochondrial ATP and heat production. Since other treatments affecting mitochondrial ATP did not stimulate wound healing, we propose increased mitochondrial heat production as the most likely primary mechanism of action of AOX in promoting cell migration in these various contexts.

Highlights • Scarface is up-regulated in peripodial epithelium and peripodial stalk cells in wing disc. • Overexpression of JNK pathway causes up-regulation of scarface. • Down regulation of JNK pathway results in down regulation of scarface. • Scarface knockdown in wing disc phenocopies JNK pathway defect., The importance of the Jun N-terminal Kinase (JNK) pathway during normal development and tumor invasion has been well documented in Drosophila. Here, this pathway plays important roles in epithelial morphogenesis, wound healing, apoptosis, immunity and regulation of lifespan. However, which downstream molecules facilitate these effects is not very well elucidated. In this study, data are presented on a serine protease homolog (SPH), scarface. These data show that scarface is under regulatory control of the JNK pathway and that this pathway is both necessary and sufficient for its expression within the context of thoracic development. Consequently, down-regulation of scarface results in a thoracic-cleft phenotype that phenocopies the JNK pathway defect. A possible role of scarface during thoracic development in Drosophila is discussed.