Spermatogonial Dedifferentiation into Germline Stem Cells in Drosophila Testes.

Stem cell pools are dynamic and capable of reacting to insults like injury and starvation. Recent work has highlighted the key role of dedifferentiation as a conserved mechanism for replenishing stem cell pools after their loss, thereby maintaining tissue homeostasis. The testis of the fruit fly Drosophila melanogaster offers a simple but powerful system to study dedifferentiation, the process by which differentiating spermatogonia can revert their fate to become fully functional germline stem cells (GSCs). Dedifferentiated GSCs show interesting characteristics, such as being more proliferative than their wild-type sibling GSCs. To facilitate the study of the cellular and molecular mechanisms underlying the process of germline dedifferentiation in the Drosophila testis, here we describe techniques for inducing high rates of dedifferentiation and for unambiguously labeling dedifferentiated GSCs.

chinmo-mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche.

Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, we show that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo-/- GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. Our results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution.

Proliferative stem cells maintain quiescence of their niche by secreting the Activin inhibitor Follistatin.

Aging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. We use the Drosophila testis to investigate what factors maintain niche cells. The testis niche comprises quiescent "hub" cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). We identify the cell-cycle-responsive Dp/E2f1 transcription factor as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.

The Emerging Roles of JNK Signaling in Drosophila Stem Cell Homeostasis.

The Jun N-terminal kinase (JNK) pathway is an evolutionary conserved kinase cascade best known for its roles during stress-induced apoptosis and tumor progression. Recent findings, however, have identified new roles for this pleiotropic pathway in stem cells during regenerative responses and in cellular plasticity. Here, we provide an overview of recent findings about the new roles of JNK signaling in stem cell biology using two well-established Drosophila models: the testis and the intestine. We highlight the pathway's roles in processes such as proliferation, death, self-renewal and reprogramming, and discuss the known parallels between flies and mammals.

Transcriptomic analysis of feminizing somatic stem cells in the Drosophila testis reveals putative downstream effectors of the transcription factor Chinmo.

One of the best examples of sexual dimorphism is the development and function of the gonads, ovaries and testes, which produce sex-specific gametes, oocytes, and spermatids, respectively. The development of these specialized germ cells requires sex-matched somatic support cells. The sexual identity of somatic gonadal cells is specified during development and must be actively maintained during adulthood. We previously showed that the transcription factor Chinmo is required to ensure the male sexual identity of somatic support cells in the Drosophila melanogaster testis. Loss of chinmo from male somatic gonadal cells results in feminization: they transform from squamous to epithelial-like cells that resemble somatic cells in the female gonad but fail to properly ensheath the male germline, causing infertility. To identify potential target genes of Chinmo, we purified somatic cells deficient for chinmo from the adult Drosophila testis and performed next-generation sequencing to compare their transcriptome to that of control somatic cells. Bioinformatics revealed 304 and 1549 differentially upregulated and downregulated genes, respectively, upon loss of chinmo in early somatic cells. Using a combination of methods, we validated several differentially expressed genes. These data sets will be useful resources to the community.

Next-Generation Sequencing Reveals Increased Anti-oxidant Response and Ecdysone Signaling in STAT Supercompetitors in Drosophila.

Cell competition is the elimination of one viable population of cells (the losers) by a neighboring fitter population (the winners) and was discovered by studies in the Drosophila melanogaster wing imaginal disc. Supercompetition is a process in which cells with elevated JAK/STAT signaling or increased Myc become winners and outcompete wild-type neighbors. To identify the genes that are differentially regulated in STAT supercompetitors, we purified these cells from Drosophila wing imaginal discs and performed next-generation sequencing. Their transcriptome was compared to those of control wing disc cells and Myc supercompetitors. Bioinformatics revealed that STAT and Myc supercompetitors have distinct transcriptomes with only 41 common differentially regulated genes. Furthermore, STAT supercompetitors have elevated reactive oxygen species, an anti-oxidant response and increased ecdysone signaling. Using a combination of methods, we validated 13 differentially expressed genes. These data sets will be useful resources to the community.

Enhancer of Polycomb and the Tip60 complex repress hematological tumor initiation by negatively regulating JAK/STAT pathway activity.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders that cause excessive production of myeloid cells. Most MPN patients have a point mutation in JAK2 (JAK2V617F ), which encodes a dominant-active kinase that constitutively triggers JAK/STAT signaling. In Drosophila, this pathway is simplified, with a single JAK, Hopscotch (Hop), and a single STAT transcription factor, Stat92E. The hopTumorous-lethal [hopTum ] allele encodes a dominant-active kinase that induces sustained Stat92E activation. Like MPN patients, hopTum mutants have significantly more myeloid cells, which form invasive tumors. Through an unbiased genetic screen, we found that heterozygosity for Enhancer of Polycomb [E(Pc)], a component of the Tip60 lysine acetyltransferase complex (also known as KAT5 in humans), significantly increased tumor burden in hopTum animals. Hematopoietic depletion of E(Pc) or other Tip60 components in an otherwise wild-type background also induced blood cell tumors. The E(Pc) tumor phenotype was dependent on JAK/STAT activity, as concomitant depletion of hop or Stat92E inhibited tumor formation. Stat92E target genes were significantly upregulated in E(Pc)-mutant myeloid cells, indicating that loss of E(Pc) activates JAK/STAT signaling. Neither the hop nor Stat92E gene was upregulated upon hematopoietic E(Pc) depletion, suggesting that the regulation of the JAK/STAT pathway by E(Pc) is dependent on substrates other than histones. Indeed, E(Pc) depletion significantly increased expression of Hop protein in myeloid cells. This study indicates that E(Pc) works as a tumor suppressor by attenuating Hop protein expression and ultimately JAK/STAT signaling. Since loss-of-function mutations in the human homologs of E(Pc) and Tip60 are frequently observed in cancer, our work could lead to new treatments for MPN patients.This article has an associated First Person interview with the first author of the paper.

JAK/STAT signaling in stem cells and regeneration: from Drosophila to vertebrates.

The JAK/STAT pathway is a conserved metazoan signaling system that transduces cues from extracellular cytokines into transcriptional changes in the nucleus. JAK/STAT signaling is best known for its roles in immunity. However, recent work has demonstrated that it also regulates critical homeostatic processes in germline and somatic stem cells, as well as regenerative processes in several tissues, including the gonad, intestine and appendages. Here, we provide an overview of JAK/STAT signaling in stem cells and regeneration, focusing on Drosophila and highlighting JAK/STAT pathway functions in proliferation, survival and cell competition that are conserved between Drosophila and vertebrates.

Super-Competitors Game the Fitness Sensing System.

Competitive interactions between neighboring cells require fitness comparison and local killing, but the signals regulating these processes are unknown. In this issue, Alpar et al. (2018) demonstrate that fitter cells secrete serine proteases to create a local burst of active Spätzle, triggering Toll signaling and apoptosis in less fit neighbors.

JNK signaling triggers spermatogonial dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis.

Exhaustion of stem cells is a hallmark of aging. In the Drosophila testis, dedifferentiated germline stem cells (GSCs) derived from spermatogonia increase during lifespan, leading to the model that dedifferentiation counteracts the decline of GSCs in aged males. To test this, we blocked dedifferentiation by mis-expressing the differentiation factor bag of marbles (bam) in spermatogonia while lineage-labeling these cells. Strikingly, blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool. However, in mated males or challenging conditions, inhibiting bam-lineage dedifferentiation markedly reduces the number of GSCs and their ability to proliferate and differentiate. We find that bam-lineage derived GSCs have significantly higher proliferation rates than sibling GSCs in the same testis. We determined that Jun N-terminal kinase (JNK) activity is autonomously required for bam-lineage dedifferentiation. Overall, we show that dedifferentiation provides a mechanism to maintain the germline and ensure fertility under chronically stressful conditions.

Chinmo prevents transformer alternative splicing to maintain male sex identity.

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT.

Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in Pdx1-Cre;LSL-KrasG12D mice, suggesting that ICMT is required for NOTCH signaling. We used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies). We identified Rab7 and Rab8 as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster.

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F ) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal(Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling.

Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues.

Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on loss of access to these signals. Here, we demonstrate that the differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot differentiate properly. We find that an insulin peptide produced by somatic cells immediately outside of the stem cell niche acts locally to promote somatic differentiation through Insulin-like receptor (InR) activation. These results indicate that there is a local 'differentiation' niche that upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, we demonstrate that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, we show that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that the local production of positive and negative InR signals regulates the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation are actively induced by signaling.

Socs36E Controls Niche Competition by Repressing MAPK Signaling in the Drosophila Testis.

The Drosophila testis is a well-established system for studying stem cell self-renewal and competition. In this tissue, the niche supports two stem cell populations, germ line stem cells (GSCs), which give rise to sperm, and somatic stem cells called cyst stem cells (CySCs), which support GSCs and their descendants. It has been established that CySCs compete with each other and with GSCs for niche access, and mutations have been identified that confer increased competitiveness to CySCs, resulting in the mutant stem cell and its descendants outcompeting wild type resident stem cells. Socs36E, which encodes a negative feedback inhibitor of the JAK/STAT pathway, was the first identified regulator of niche competition. The competitive behavior of Socs36E mutant CySCs was attributed to increased JAK/STAT signaling. Here we show that competitive behavior of Socs36E mutant CySCs is due in large part to unbridled Mitogen-Activated Protein Kinase (MAPK) signaling. In Socs36E mutant clones, MAPK activity is elevated. Furthermore, we find that clonal upregulation of MAPK in CySCs leads to their outcompetition of wild type CySCs and of GSCs, recapitulating the Socs36E mutant phenotype. Indeed, when MAPK activity is removed from Socs36E mutant clones, they lose their competitiveness but maintain self-renewal, presumably due to increased JAK/STAT signaling in these cells. Consistently, loss of JAK/STAT activity in Socs36E mutant clones severely impairs their self-renewal. Thus, our results enable the genetic separation of two essential processes that occur in stem cells. While some niche signals specify the intrinsic property of self-renewal, which is absolutely required in all stem cells for niche residence, additional signals control the ability of stem cells to compete with their neighbors. Socs36E is node through which these processes are linked, demonstrating that negative feedback inhibition integrates multiple aspects of stem cell behavior.

MT-Nanotubes: Lifelines for Stem Cells.

Niche cells produce secreted factors that promote the self-renewal of stem cells in their immediate proximity, but how signaling is restricted to stem cells is not well understood. Inaba et al. (2015) report that microtubule (MT) structures called MT-nanotubes control activation of the primary self-renewal pathway in Drosophila testes.

Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo.

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. Here, we show that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. We find that the driving force behind such behavior is accelerated proliferation. Our results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process.

JAK/STAT pathway dysregulation in tumors: a Drosophila perspective.

Sustained activation of the JAK/STAT pathway is causal to human cancers. This pathway is less complex in Drosophila, and its dysregulation has been linked to several tumor models in this organism. Here, we discuss models of metastatic epithelial and hematopoietic tumors that are causally linked to dysregulation of JAK/STAT signaling in Drosophila. First, we focus on cancer models in imaginal discs where ectopic expression of the JAK/STAT pathway ligand Unpaired downstream of distinct tumor suppressors has emerged as an unexpected mediator of neoplastic transformation. We also discuss the collaboration between STAT and oncogenic Ras in epithelial transformation. Second, we examine hematopoietic tumors, where mutations that cause hyperactive JAK/STAT signaling are necessary and sufficient for "fly leukemia". We highlight the important contributions that genetic screens in Drosophila have made to understanding the JAK/STAT pathway, its developmental roles, and how its function is co-opted during tumorigenesis.

Cell competition: how to eliminate your neighbours.

A conventional view of development is that cells cooperate to build an organism. However, based on studies of Drosophila, it has been known for years that viable cells can be eliminated by their neighbours through a process termed cell competition. New studies in mammals have revealed that this process is universal and that many factors and mechanisms are conserved. During cell competition, cells with lower translation rates or those with lower levels of proteins involved in signal transduction, polarity and cellular growth can survive in a homogenous environment but are killed when surrounded by cells of higher fitness. Here, we discuss recent advances in the field as well as the mechanistic steps involved in this phenomenon, which have shed light on how and why cell competition exists in developing and adult organisms.

Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression.

RAS is the most frequently mutated oncogene in human cancers. Despite decades of effort, anti-RAS therapies have remained elusive. Isoprenylcysteine carboxylmethyltransferase (ICMT) methylates RAS and other CaaX-containing proteins, but its potential as a target for cancer therapy has not been fully evaluated. We crossed a Pdx1-Cre;LSL-KrasG12D mouse, which is a model of pancreatic ductal adenocarcinoma (PDA), with a mouse harboring a floxed allele of Icmt. Surprisingly, we found that ICMT deficiency dramatically accelerated the development and progression of neoplasia. ICMT-deficient pancreatic ductal epithelial cells had a slight growth advantage and were resistant to premature senescence by a mechanism that involved suppression of cyclin-dependent kinase inhibitor 2A (p16INK4A) expression. ICMT deficiency precisely phenocopied Notch1 deficiency in the Pdx1-Cre;LSL-KrasG12D model by exacerbating pancreatic intraepithelial neoplasias, promoting facial papillomas, and derepressing Wnt signaling. Silencing ICMT in human osteosarcoma cells decreased Notch1 signaling in response to stimulation with cell-surface ligands. Additionally, targeted silencing of Ste14, the Drosophila homolog of Icmt, resulted in defects in wing development, consistent with Notch loss of function. Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling.