Unanticipated domain requirements for Drosophila Wnk kinase in vivo.

WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. Here, we show that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. We show that the only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. Our data thus provide novel insights into unexpected in vivo roles of specific WNK domains.

Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila.

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences1-3. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases4. Here we report that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size.

Damage sensing by a Nox-Ask1-MKK3-p38 signaling pathway mediates regeneration in the adult Drosophila midgut.

Epithelia are exposed to diverse types of stress and damage from pathogens and the environment, and respond by regenerating. Yet, the proximal mechanisms that sense epithelial damage remain poorly understood. Here we report that p38 signaling is activated in adult Drosophila midgut enterocytes in response to diverse stresses including pathogenic bacterial infection and chemical and mechanical insult. Two upstream kinases, Ask1 and Licorne (MKK3), are required for p38 activation following infection, oxidative stress, detergent exposure and wounding. Ask1-p38 signaling in enterocytes is required upon infection to promote full intestinal stem cell (ISC) activation and regeneration, partly through Upd3/Jak-Stat signaling. Furthermore, reactive oxygen species (ROS) produced by the NADPH oxidase Nox in enterocytes, are required for p38 activation in enterocytes following infection or wounding, and for ISC activation upon infection or detergent exposure. We propose that Nox-ROS-Ask1-MKK3-p38 signaling in enterocytes integrates multiple different stresses to induce regeneration.

Tissue-specific function of Patj in regulating the Crumbs complex and epithelial polarity.

Patj is described as a core component of the Crumbs complex. Along with the other components, Crumbs and Stardust, Patj has been proposed as essential for epithelial polarity. However, no proper in vivo genetic analysis of Patj function has been performed in any organism. We have generated the first null mutants for Drosophila Patj. These mutants are lethal. However, Patj is not required in all epithelia where the Crumbs complex is essential. Patj is dispensable for ectoderm polarity and embryonic development, whereas more severe defects are observed in the adult follicular epithelium, including mislocalisation of the Crumbs complex from the apical domain, as well as morphogenetic defects. These defects are similar to those observed with crumbs and stardust mutants, although weaker and less frequent. Also, gain-of-function of Crumbs and Patj mutation genetically suppress each other in follicular cells. We also show that the first PDZ domain of Patj associated with the Stardust-binding domain are sufficient to fully rescue both Drosophila viability and Crumbs localisation. We propose that the only crucial function of Patj hinges on the ability of its first two domains to positively regulate the Crumbs complex, defining a new developmental level of regulation of its dynamics.