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In many organisms, the regenerative capacity of tissues progressively decreases as development progresses. However, the developmental mechanisms that restrict regenerative potential remain unclear. In Drosophila, wing imaginal discs become unable to regenerate upon damage during the third larval stage (L3). Here, we show that production of ecdysone after larvae reach their critical weight (CW) terminates the window of regenerative potential by acting on a bistable loop composed of two antagonistic Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (ZBTB) genes: chinmo and broad (br). Around mid L3, ecdysone signaling silences chinmo and activates br to switch wing epithelial progenitors from a default self-renewing to a differentiation-prone state. Before mid L3, Chinmo promotes a strong regenerative response upon tissue damage. After mid L3, Br installs a nonpermissive state that represses regeneration. Transient down-regulation of ecdysone signaling or Br in late L3 larvae enhances chinmo expression in damaged cells that regain the capacity to regenerate. This work unveils a mechanism that ties the self-renewing and regenerative potential of epithelial progenitors to developmental progression.

Macrophages are a major immune cell type infiltrating tumors and promoting tumor growth and metastasis. To elucidate the mechanism of macrophage recruitment, we utilize an overgrowth tumor model (“undead” model) in larval Drosophila imaginal discs that are attached by numerous macrophages. Here we report that changes to the microenvironment of the overgrown tissue are important for recruiting macrophages. First, we describe a correlation between generation of reactive oxygen species (ROS) and damage of the basement membrane (BM) in all neoplastic, but not hyperplastic, models examined. ROS and the stress kinase JNK mediate the accumulation of matrix metalloproteinase 2 (Mmp2), damaging the BM, which recruits macrophages to the tissue. We propose a model where macrophage recruitment to and activation at overgrowing tissue is a multi-step process requiring ROS- and JNK-mediated Mmp2 upregulation and BM damage. These findings have implications for understanding the role of the tumor microenvironment for macrophage activation. The molecular mechanisms regulating macrophage recruitment to tumors are unclear. Here, the authors use a Drosophila overgrowth model to show how damaged basement membranes recruit macrophages to undead tissue, via an interdependent effect of reactive oxygen species and matrix metalloproteinase 2.

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.

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation.

A new role for the endosomal sorting complex required for transport (ESCRT) is identified in fly larvae, where it is shown to be essential for the secretion and long-range signalling of the embryonic development morphogen Hedgehog. ESCRT involved in Hedgehog signalling ESCRT, the endosomal sorting complex required for transport, is best known for its roles in vesicular trafficking, cell division and mediating viral escape from cells. Pascal Thérond and co-workers now report an unexpected role for this complex. During embryonic development, Hedgehog proteins control tissue patterning and differentiation over short and long distances. The authors find that ESCRT activity is essential for Hedgehog secretion in fly larvae. Pools of Hedgehog and ESCRT are secreted together into the extracellular space and both can subsequently be detected together at the surface of receiving cells, where ESCRT activity seems to be required for long-range Hedgehog signalling. The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development 1 . Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading 2 . Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT) 3 . In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.

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.

Significance The larval imaginal discs of the fruit fly are capable of fully regenerating mechanically damaged parts. Wound healing is initiated by the JNK signaling pathway. We followed the subsequent formation of the regenerating blastema by transcriptome profiling and identified the JAK/STAT pathway as a central regulatory node controlling local cellular and global physiological responses. This signaling cascade induces, together with the Wingless pathway, proliferation of cells forming the blastema. However, JAK/STAT also up-regulates Drosophila insulin-like peptide 8 (Dilp8), a paracrine factor involved in organismal developmental delay, thereby allowing regenerative recovery. Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. Here, we profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we showed that the expression of Drosophila insulin-like peptide 8 ( dilp8 ), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing.

Even seemingly homogeneous populations of cells can express phenotypic diversity in response to environmental changes. Thus, X-ray irradiation of tissues composed of diverse cell types can have complex outcomes. We have used single-cell RNA sequencing to study the effects of X-ray radiation on the Drosophila wing imaginal disc, a relatively simple tissue composed mostly of epithelial cells. Transcriptomic clustering of cells collected from the wing disc generates clusters that are mainly grouped based on proximodistal cell location. To quantify heterogeneity of gene expression among clusters, we adapted a metric used to study market concentration, the Herfindahl-Hirschman Index. Genes involved in DNA damage repair, defense against reactive oxygen species, cell cycle progression, and apoptosis are expressed relatively uniformly. In contrast, genes encoding a subset of ligands, notably cytokines that activate the JAK/STAT pathway, some transcription factors, including Ets21C , previously implicated in regeneration, and several signaling proteins are expressed more regionally. Though the radiation-responsive transcription factor p53 is expressed relatively uniformly in the wing disc, several regionally induced genes still require p53 function, indicating that regional and radiation-induced factors combine to regulate their expression. We also examined heterogeneity within regions using a clustering approach based on cell cycle gene expression. A subpopulation of cells, characterized by high levels of tribbles expression, is amplified in irradiated discs. Remarkably, this subpopulation accounts for a considerable fraction of radiation-induced gene expression, indicating that cellular responses are non-uniform even within regions. Thus, both inter-regional and intra-regional heterogeneity are important features of tissue responses to X-ray radiation.

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.

Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFβ/Activin-like ligands in disc growth control remains ill-defined. Here, we demonstrate that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. We propose that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages.