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Two papers in this issue demonstrate novel proteomic approaches that enhance genetic analysis. Matsuoka et al. investigated how diet-regulated... Elevated levels of human chitinase-like proteins (CLPs) are associated with numerous chronic inflammatory diseases and several cancers, often correlating with poor prognosis. Nevertheless, there is scant knowledge of their function. The CLPs normally mediate immune responses and wound healing and, when upregulated, they can promote disease progression by remodeling tissue, activating signaling cascades, stimulating proliferation and migration, and by regulating adhesion. We identified Imaginal disc growth factors (Idgfs), orthologs of human CLPs CHI3L1, CHI3L2, and OVGP1, in a proteomics analysis designed to discover factors that regulate tube morphogenesis in a Drosophila melanogaster model of tube formation. We implemented a novel approach that uses magnetic beads to isolate a small population of specialized ovarian cells, cells that nonautonomously regulate morphogenesis of epithelial tubes that form and secrete eggshell structures called dorsal appendages (DAs). Differential mass spectrometry analysis of these cells detected elevated levels of four of the six Idgf family members (Idgf1, Idgf2, Idgf4, and Idgf6) in flies mutant for bullwinkle (bwk), which encodes a transcription factor and is a known regulator of DA-tube morphogenesis. We show that, during oogenesis, dysregulation of Idgfs (either gain or loss of function) disrupts the formation of the DA tubes. Previous studies demonstrate roles for Drosophila Idgfs in innate immunity, wound healing, and cell proliferation and motility in cell culture. Here, we identify a novel role for Idgfs in both normal and aberrant tubulogenesis processes.

Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes. Epithelial folding has mainly been linked to forces acting in the apical actomyosin network of cells. Here, the authors show using live imaging that two distinct mechanisms, changes in basal surface tension and changes in lateral surface tension, drive the formation of two folds in the Drosophila wing disc.

Developing animals frequently adjust their growth programs and/or their maturation or metamorphosis to compensate for growth disturbances (such as injury or tumor) and ensure normal adult size. Such plasticity entails tissue and organ communication to preserve their proportions and symmetry. Here, we show that imaginai discs autonomously activate DILP8, a Drosophilo insulin-like peptide, to communicate abnormal growth and postpone maturation. DILP8 delays metamorphosis by inhibiting ecdysone biosynthesis, slowing growth in the imaginai discs, and generating normal-sized animals. Loss of dilp8 yields asymmetric individuals with an unusually large variation in size and a more varied time of maturation. Thus, DILP8 is a fundamental element of the hitherto ill-defined machinery governing the plasticity that ensures developmental stability and robustness.

Histone acetylation regulates gene expression, cell function and cell fate 1 . Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid β-oxidation, which is also increased in the rim. Inhibition of fatty acid β-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression. Analyses of histone acetylation in Drosophila wing imaginal discs reveal distinct patterns of acetylation and cellular metabolism that affect gene expression and cell specification.

How cells communicate to initiate a regenerative response after damage has captivated scientists during the last few decades. It is known that one of the main signals emanating from injured cells is the Reactive Oxygen Species (ROS), which propagate to the surrounding tissue to trigger the replacement of the missing cells. However, the link between ROS production and the activation of regenerative signaling pathways is not yet fully understood. We describe here the non-autonomous ROS sensing mechanism by which living cells launch their regenerative program. To this aim, we used Drosophila imaginal discs as a model system due to its well-characterized regenerative ability after injury or cell death. We genetically-induced cell death and found that the Apoptosis signal-regulating kinase 1 (Ask1) is essential for regenerative growth. Ask1 senses ROS both in dying and living cells, but its activation is selectively attenuated in living cells by Akt1, the core kinase component of the insulin/insulin-like growth factor pathway. Akt1 phosphorylates Ask1 in a secondary site outside the kinase domain, which attenuates its activity. This modulation of Ask1 activity results in moderate levels of JNK signaling in the living tissue, as well as in activation of p38 signaling, both pathways required to turn on the regenerative response. Our findings demonstrate a non-autonomous activation of a ROS sensing mechanism by Ask1 and Akt1 to replace the missing tissue after damage. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.

Little is known about how organ growth is monitored and coordinated with the developmental timing in complex organisms. In insects, impairment of larval tissue growth delays growth and morphogenesis, revealing a coupling mechanism. We carried out a genetic screen in Drosophila to identify molecules expressed by growing tissues participating in this coupling and identified dilp8 as a gene whose silencing rescues the developmental delay induced by abnormally growing tissues. dilp8 is highly induced in conditions where growth impairment produces a developmental delay. dilp8 encodes a peptide for which expression and secretion are sufficient to delay metamorphosis without affecting tissue integrity. We propose that Dilp8 peptide is a secreted signal that coordinates the growth status of tissues with developmental timing.

Cell competition induces the elimination of less-fit “loser” cells by fitter “winner” cells. In Drosophila , cells heterozygous mutant in ribosome genes, Rp/+ , known as Minutes , are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

Whereas complete loss of Rp function is generally lethal, most heterozygous Rp mutants grow more slowly and are subject to competitive loss from mosaics tissues that also contain wild type cells. The rpS12 gene has a special role in the cell competition of other Ribosomal Protein (Rp) mutant cells in Drosophila. Elimination by cell competition is promoted by higher RpS12 levels and prevented by a specific rpS12 mis-sense mutation, identifying RpS12 as a key effector of cell competition due to mutations in other Rp genes. Here we show that RpS12 is also required for other aspects of Rp mutant phenotypes, including hundreds of gene expression changes that occur in 'Minute' Rp heterozygous wing imaginal discs, overall translation rate, and the overall rate of organismal development, all through the bZip protein Xrp1 that is one of the RpS12-regulated genes. Our findings outline the regulatory response to mutations affecting essential Rp genes that controls overall translation, growth, and cell competition, and which may contribute to cancer and other diseases.

Growth deceleration before growth termination is a universal feature of growth during development. Transcriptomics analysis reveals that during their two-day period of growth deceleration, wing imaginal discs of Drosophila undergo a progressive metabolic shift from oxidative phosphorylation towards glycolysis. Ultra-sensitive reporters of HIF-1α stability and activity show that imaginal discs become increasingly hypoxic during development in normoxic conditions, suggesting that limiting oxygen supply could underlie growth deceleration. We confirm the expectation that rising levels of HIF-1α dampen TOR signalling activity through transcriptional activation of REDD1. Conversely, excess TOR leads, in a tissue-size-dependent manner, to hypoxia, which boosts HIF-1α levels and activity. Thus, HIF-1α mediates a negative feedback loop whereby TOR signalling triggers hypoxia, which in turn reduces TOR signalling. Abrogation of this feedback by Sima/HIF-1α knockdown leads to cellular stress, which is alleviated by reduced TOR signalling or a modest increase in environmental oxygen. We conclude that Sima/HIF-1α prevents TOR-mediated growth from depleting local oxygen supplies during normal development. During normal development, growing fly wing precursors become hypoxic, triggering HIF-1α-mediated negative feedback on TOR signalling and growth deceleration. Without this feedback, the mismatch between oxygen supply and demand leads to tissue stress.

Fasciclin 2 ( Drosophila NCAM) is a homophilic Cell Adhesion Molecule expressed at moderate levels in the proliferating epithelial cells of imaginal discs, where it engages EGFR in a cell autonomous auto-stimulatory loop that promotes growth along larval development. In addition, Fasciclin 2 is expressed at high levels in the pre-differentiating cells of imaginal discs. Gain-of-function genetic analysis shows that Fasciclin 2 acts as a non-cell autonomous repressor of EGFR when high expression levels are induced during imaginal disc growth. Loss-of-function genetic analysis shows that this Fasciclin 2 functional facet is required at the end of larval development and it is mediated by interaction with IgCAMs CG15630 (Fipi) and CG33543 (Elff). Thus, Fasciclin 2 bears two complementary functional roles which correspond with different levels of expression. The combined results from loss- and gain-of-function analyses suggest a scenario where the Fasciclin 2/EGFR cell autonomous auto-stimulatory loop promotes cell proliferation until reaching a Fasciclin 2 expression threshold where its non-cell autonomous function stops growth. Thus, cellular integration of Fasciclin 2 autonomous and non-cell autonomous signaling from neighbor cells may be a key regulator component to orchestrate the rate of intercalary cell proliferation and the final size and shape of an organ.