Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells.

Immune signalling pathways need to be tightly regulated as overactivation of these pathways can result in chronic inflammatory diseases and cancer. NF-κB signalling and associated innate immune pathways are crucial in the first line of defense against infection in all animals. In a genome-wide RNAi screen for modulators of Drosophila immune deficiency (IMD)/NF-κB signalling, we identified components of the Ras/MAPK pathway as essential for suppression of IMD pathway activity, even in the absence of an immune challenge. Downregulation of Ras/MAPK activity mimics the induction of innate immune responses by microbial patterns. Conversely, ectopic Ras/MAPK pathway activation results in the suppression of Drosophila IMD/NF-κB signalling. Mechanistically, we show that the Ras/MAPK pathway acts by inducing transcription of the IMD pathway inhibitor Pirk/Rudra/PIMS. Finally, in vivo experiments demonstrate a requirement for Ras/MAPK signalling in restricting innate immune responses in haemocytes, fat body and adult intestinal stem cells. Our observations provide an example of a pathway that promotes cell proliferation and has simultaneously been utilized to limit the immune response.

p24 proteins are required for secretion of Wnt ligands.

During development and disease, the exocytosis of signalling molecules, such as Wnt ligands, is essential to orchestrate cellular programs in multicellular organisms. However, it remains a largely unresolved question whether signalling molecules follow specialized transport routes through the exocytic pathway. Here we identify several Drosophila p24 proteins that are required for Wnt signalling. We demonstrate that one of these p24 proteins, namely Opossum, shuttles in the early secretory pathway, and that the Drosophila Wnt proteins are retained in the absence of p24 proteins. Our results indicate that Wnt secretion relies on a specialized anterograde secretion route with p24 proteins functioning as conserved cargo receptors.

Non-autonomous modulation of heart rhythm, contractility and morphology in adult fruit flies.

The outermost layer of the vertebrate heart originates from migratory mesothelial cells (epicardium) that give rise to coronary vascular smooth muscles and fibroblasts. The role of the epicardium in myocardial morphogenesis and establishment of normal heart function is still largely unknown. Here, we use Drosophila to investigate non-autonomous influences of epicardial-like tissue surrounding the heart tube on the structural and functional integrity of the myocardium. It has previously been shown that during Drosophila heart formation, mesodermal expression of the homeobox transcription factor even-skipped (eve) is required for specification of a subset of non-myocardial progenitors in the precardiac mesoderm. These progenitors may share some similarities with the vertebrate epicardium. To investigate a non-autonomous epicardial-like influence on myocardial physiology, we studied the consequences of reduced mesodermal Eve expression and epi/pericardial cell numbers on the maturation of the myocardial heart tube, its contractility, and acquisition of a normal heart rhythm in the Drosophila model. Targeting the eve repressor ladybird early (lbe) with the minimal eve mesodermal enhancer efficiently eliminates the mesodermal Eve lineages. These flies exhibit defects in heart structure, including a reduction in systolic and diastolic diameter (akin to 'restrictive cardiomyopathy'). They also exhibit an elevated incidence of arrhythmias and intermittent asystoles, as well as compromised performance under stress. These abnormalities are restored by eve reexpression or by lbe-RNAi co-overexpression. The data suggest that adult heart function in Drosophila is likely to be modulated non-autonomously, possibly by paracrine influences from neighboring cells, such as the epi/pericardium. Thus, Drosophila may serve as a model for finding genetic effectors of epicardial-myocardial interactions relevant to higher organisms.

REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1.

TORC1 regulates growth and metabolism, in part, by influencing transcriptional programs. Here, we identify REPTOR and REPTOR-BP as transcription factors downstream of TORC1 that are required for ∼ 90% of the transcriptional induction that occurs upon TORC1 inhibition in Drosophila. Thus, REPTOR and REPTOR-BP are major effectors of the transcriptional stress response induced upon TORC1 inhibition, analogous to the role of FOXO downstream of Akt. We find that, when TORC1 is active, it phosphorylates REPTOR on Ser527 and Ser530, leading to REPTOR cytoplasmic retention. Upon TORC1 inhibition, REPTOR becomes dephosphorylated in a PP2A-dependent manner, shuttles into the nucleus, joins its partner REPTOR-BP to bind target genes, and activates their transcription. In vivo functional analysis using knockout flies reveals that REPTOR and REPTOR-BP play critical roles in maintaining energy homeostasis and promoting animal survival upon nutrient restriction.

Wnt signaling signaling at and above the receptor level.

Wnt signaling is one of the most important developmental signaling pathways that controls cell fate decisions and tissue patterning during early embryonic and later development. It is activated by highly conserved Wnt proteins that are secreted as palmitoylated glycoproteins and act as morphogens to form a concentration gradient across a developing tissue. Wnt proteins regulate transcriptional and posttranscriptional processes depending on the distance of their origin and activate distinct intracellular cascades, commonly referred to as canonical (ß-catenin-dependent) and noncanonical (ß-catenin-independent) pathways. Therefore, the secretion and the diffusion of Wnt proteins needs to be tightly regulated to induce short- and long-range downstream signaling. Even though the Wnt signaling cascade has been studied intensively, key aspects and principle mechanisms, such as transport of Wnt growth factors or regulation of signaling specificity between different Wnt pathways, remain unresolved. Here, we introduce basic principles of Wnt/Wg signal transduction and highlight recent discoveries, such as the involvement of vacuolar ATPases and vesicular acidification in Wnt signaling. We also discuss recent findings regarding posttranslational modifications of Wnts, trafficking through the secretory pathway and developmental consequences of impaired Wnt secretion. Understanding the detailed mechanism and regulation of Wnt protein secretion will provide valuable insights into many human diseases based on overactivated Wnt signaling.

Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor.

Wnt/Wg signaling pathways are of key importance during development and disease. Canonical and noncanonical Wnt/Frizzled (Fz) pathways share a limited number of signaling components that are part of the membrane proximal signaling complex. In Drosophila, Fz and Dishevelled (Dsh) are the only two components known to be involved in both Wnt/beta-catenin and planar cell polarity (PCP) signaling. PCP signaling is required for the planar polarization of epithelial cells, which occurs, for instance, during hair orientation and gastrulation in vertebrates. Both pathways have been studied intensively in the past years. However, it still remains unresolved whether additional components are required at the receptor complex. Here we identify the Drosophila homolog of the mammalian prorenin receptor (dPRR) as a conserved modulator of canonical Wnt/beta-cat and Fz/PCP signaling. We show that dPRR depletion affects Wg target genes in cultured cells and in vivo. PRR is required for epithelial planar polarity in Drosophila and for convergent extension movements in Xenopus gastrulae. Furthermore, dPRR binds to Fz and Fz2 receptors. In summary, our data suggest that dPRR has an evolutionarily conserved role at the receptor level for activation of canonical and noncanonical Wnt/Fz signaling pathways.