Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

Transcriptome dynamics during metamorphosis of imaginal discs into wings and thoracic dorsum in Apis mellifera castes

Background Much of the complex anatomy of a holometabolous insect is built from disc-shaped epithelial structures found inside the larva, i.e., the imaginal discs, which undergo a rapid differentiation during metamorphosis. Imaginal discs-derived structures, like wings, are built through the action of genes under precise regulation. Results We analyzed 30 honeybee transcriptomes in the search for the gene expression needed for wings and thoracic dorsum construction from the larval wing discs primordia. Analyses were carried out before, during, and after the metamorphic molt and using worker and queen castes. Our RNA-seq libraries revealed 13,202 genes, representing 86.2% of the honeybee annotated genes. Gene Ontology analysis revealed functional terms that were caste-specific or shared by workers and queens. Genes expressed in wing discs and descendant structures showed differential expression profiles dynamics in premetamorphic, metamorphic and postmetamorphic developmental phases, and also between castes. At the metamorphic molt, when ecdysteroids peak, the wing buds of workers showed maximal gene upregulation comparatively to queens, thus underscoring differences in gene expression between castes at the height of the larval-pupal transition. Analysis of small RNA libraries of wing buds allowed us to build miRNA-mRNA interaction networks to predict the regulation of genes expressed during wing discs development. Conclusion Together, these data reveal gene expression dynamics leading to wings and thoracic dorsum formation from the wing discs, besides highlighting caste-specific differences during wing discs metamorphosis.

Critical genetic program for Drosophila imaginal disc regeneration revealed by single-cell analysis

Whether regeneration is primarily accomplished by re-activating gene regulatory networks used previously during development or by activating novel regeneration-specific transcriptional programs remains a longstanding question. Currently, most genes implicated in regeneration also function during development. Using single-cell transcriptomics in regenerating Drosophila wing discs, we identified two regeneration-specific cell populations within the blastema. They are each composed of cells that upregulate multiple genes encoding secreted proteins that promote regeneration. In this regenerative secretory zone, the transcription factor Ets21C controls the expression of multiple regeneration-promoting genes. While eliminating Ets21C function has no discernible effect on development, it severely compromises regeneration. This Ets21C-dependent gene regulatory network is also activated in blastema-like cells in tumorous discs, suggesting that pro-regenerative mechanisms can be co-opted by tumors to promote aberrant growth.

Illuminati, a novel form of gene expression plasticity in Drosophila neural stem cells

ABSTRACTWith the aim of developing a genetic instability (GI) sensor in vivo we used the well-established Gal80/Gal4-UAS system combined with a visual GFP marker in Drosophila. We generated a collection of 25 Drosophila lines carrying GAL80 transgenes in different locations in all major chromosomes (X, Y, II, and III). We found low rates of GFP cells in epithelial tissues such as wing discs. In contrast, in larval brains, GFP positive clusters containing neural stem cells- also called neuroblasts (NBs)- and their offspring, were highly frequent. Using genetic and imaging-based approaches, we show that GFP NBs do not result from aneuploidy or mutations in the GAL80 gene, but rather by stochastic repression of GAL80 expression. We named this novel type of gene expression instability Illuminati. Importantly, Illuminati frequency is influenced by environmental and stress conditions. Further, we found that once established, Illuminati can be propagated over many cell cycles.

Wallenda-Nmo Axis Regulates Growth via Hippo Signaling

Both Hippo signaling pathways and cell polarity regulation are critical for cell proliferation and the maintenance of tissue homeostasis, despite the well-established connections between cell polarity disruption and Hippo inactivation, the molecular mechanism by which aberrant cell polarity induces Hippo-mediated overgrowth remains underexplored. Here we use Drosophila wing discs as a model and identify the Wnd-Nmo axis as an important molecular link that bridges loss-of-cell polarity-triggered Hippo inactivation and overgrowth. We show that Wallenda (Wnd), a MAPKKK (mitogen-activated protein kinase kinase kinase) family member, is a novel regulator of Hippo pathways in Drosophila and that overexpression of Wnd promotes growth via Nemo (Nmo)- mediated Hippo pathway inactivation. We further demonstrate that both Wnd and Nmo are required for loss-of-cell polarity-induced overgrowth and Hippo inactivation. In summary, our findings provide a novel insight on how cell polarity loss contributes to overgrowth and uncover the Wnd-Nmo axis as an essential additional branch that regulates Hippo pathways in Drosophila.

Bab2 activates JNK signaling to reprogram Drosophila wing disc development

In response to cellular stress and damage, certain tissues are able to regenerate and to restore tissue homeostasis. In Drosophila imaginal wing discs, dying cells express mitogens that induce compensatory proliferation in the surrounding tissue. Here we report that high levels of the BTB/POZ transcription factor Bab2 in the posterior compartment of wing discs activates c-Jun N-terminal kinase (JNK) signaling and local, cell-autonomous apoptotic cell death. This in turn triggered the upregulation of the Dpp mitogen and cellular proliferation in the anterior compartment in a JNK-dependent manner. In the posterior compartment, however, dpp expression was suppressed, most likely by direct transcriptional repression by Bab2. This dual-mode of JNK-signaling, autocrine pro-apoptotic signaling and paracrine pro-proliferative signaling, led to opposite effects in the two compartments and reprogramming of the adult wing structure. We establish Bab2 as a regulator of wing disc development, with the capacity to reprogram development via JNK activation in a cell-autonomous and non-cell-autonomous manner.Summary statementZhao et al. shows that the BTB/POZ transcription factor Bab2 is a potent activator of JNK signaling, apoptosis and compensatory proliferation, thereby driving both pro-tumorigenic and anti-tumorigenic processes.

Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism

Dis3L2 is a highly conserved 3’-5’ exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms’ tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.

Autonomous termination of proliferation in the Drosophila wing disc is TORC1 dependent

Cells in a developing organ stop proliferating when the organ reaches a correct, final size. The underlying mechanisms are not understood. Although many signaling pathways and cell cycle components are required to sustain cell proliferation, which one of these turns off to terminate proliferation is not known. Here we study proliferation termination using Drosophila wing discs. We extend larval development to provide wing discs a constant growth-sustaining environment, allowing them to terminate proliferation autonomously. We find that the wing pouch, which forms the adult wing blade, terminates proliferation in the absence of brinker or warts, indicating that neither Dpp signaling nor Hippo/Yorkie signaling control final wing size. Instead, termination of proliferation coincides with reduced TORC1 activity and is bypassed by reactivating TORC1. Hence proliferation ceases due to reduced cell growth. Experimental manipulation of Dpp or Yki signaling can bypass proliferation termination in hinge and notum regions, suggesting that the mechanisms regulating proliferation termination may be distinct in different regions of the disc.One Sentence SummaryUsing Drosophila, Strassburger et al. investigate the termination of proliferation of an organ when it reaches its final size, and show this occurs due to a drop in TORC1 signaling.

Widely Used Mutants of eiger, Encoding the Drosophila Tumor Necrosis Factor, Carry Additional Mutations in the NimrodC1 Phagocytosis Receptor

The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells appeared to transiently accumulate in egr3 mutant wing discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr3 mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr3 mutant, its sister allele, egr1, and its parental strain, Regg1GS9830. As the egr3 and egr1 alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.