Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.
Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl − clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl − clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl − clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl − clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl − clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.
Although tissue regeneration has been studied in a variety of organisms, from Hydra to humans, many of the genes that regulate the ability of each animal to regenerate remain unknown. The larval imaginal discs of the genetically tractable model organism Drosophila melanogaster have complex patterning, wellcharacterized development and a high regenerative capacity, and are thus an excellent model system for studying mechanisms that regulate regeneration. To identify genes that are important for wound healing and tissue repair, we have carried out a genetic screen for mutations that impair regeneration in the wing imaginal disc. Through this screen we identified the chromatin-modification gene trithorax as a key regeneration gene. Here we show that animals heterozygous for trithorax are unable to maintain activation of a developmental checkpoint that allows regeneration to occur. This defect is likely to be caused by abnormally high expression of puckered, a negative regulator of Jun N-terminal kinase (JNK) signaling, at the wound site. Insufficient JNK signaling leads to insufficient expression of an insulin-like peptide, dILP8, which is required for the developmental checkpoint. Thus, trithorax regulates regeneration signaling and capacity.
Dissociation of imaginal disc cells has been carried out previously to enable flow cytometry and cell sorting to analyze cell cycle progression, cell size, gene expression, and other aspects of imaginal tissues. However, the lengthy dissociation protocols employed may alter gene expression, cell behavior and overall viability. Here we describe a new rapid and gentle method of dissociating the cells of wing imaginal discs that significantly enhances cell viability and reduces the likelihood of gene expression changes. Furthermore, this method is scalable, enabling collection of large amounts of sample for high-throughput experiments without the need for data-distorting amplifications.
Many animal species have the capability to regenerate lost body parts. How regeneration takes place and why animals have varying potentials for regeneration remain active questions for biologists. The field of regenerative biology has witnessed unprecedented advances in the last several years owing to the availability of molecular and genomics tools and the establishment of many animal models. Regeneration research in arthropods has a long history, with extensive insights achieved from using model organisms from the taxa Crustacea and Insecta. Studies in animals ranging from fiddler crabs to crickets have revealed much about the different stages of regeneration, such as wound healing, blastema formation, growth, proliferation and patterning, as well as how hormonal control and systemic signalling impact regenerative capacity. The molecular and genetic insights achieved from studying these simpler model organisms have the potential to impact the field of regenerative biology by identifying conserved mechanisms of regeneration. Key Concepts Regeneration studies use the fiddler crab, crayfish, sand hopper, red flour beetle, fruit fly, cockroach, cricket and silverfish. For amputated limbs, wounds heal by a combination of rapid closure of the wound with a scab or autotomy membrane, and migration of cells into the wound. Imaginal disc wound closure involves cytoskeletal‐driven cell shape changes and zippering together of the epithelium, without cell migration. A regeneration blastema, or zone of proliferating cells, forms after both external limb amputation and imaginal disc damage. Growth of the blastema requires similar signals in multiple model organisms, including growth factor signalling in response to FGFs and EGFR activity, Wg/WNT signalling and Hippo signalling. Many developmental patterning genes are also required for patterning during regeneration. However, knockdown of these patterning genes revealed additional roles in regeneration beyond those observed during normal development. Some plasticity in cell fate enables replacement of lost cell types. Signals at the wound can alter pattern and cell fate, generating ectopic eye spots in butterflies and requiring the activity of a protective factor that stabilises cell fate gene expression during regeneration in fruit flies. Hormonal signalling, which controls moulting and metamorphosis, limits regenerative capacity. In some model organisms, tissue damage can influence hormone production and the timing of moults and metamorphosis.
structure and composition of the nucleolus. The nucleolus is a multifunctional regulatory nuclear compartment that, besides hosting ribosome biogenesis, regulates cellular growth, cell death, and the cell cycle. The Myc transcription factor stimulates tissue growth, in part by activating the expression of genes required for ribosome biogenesis. By qRT-PCR we show that Myc is necessary and sufficient for transcriptional activation of vito expression. Our results raise the hypothesis that the integration of signalling and nutritional cues that control cell growth in Drosophila requires Vito to regulate nucleolar function and architecture.
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