Live Imaging of the Drosophila Testis Stem Cell Niche.

Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here, we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 18 h allows for tracking of cell-type-specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. By using live imaging in intact niches, we can better uncover the cellular and molecular mechanisms that regulate stem cell function in vivo.

Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes.

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

The adult Drosophila testis lacks a mechanism to replenish missing niche cells.

The adult Drosophila testis contains a well-defined niche created by a cluster of hub cells, which secrete signals that maintain adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells are normally quiescent in adult flies but can exit quiescence, delaminate from the hub and convert into CySCs after ablation of all CySCs. The opposite event, CySC conversion into hub cells, was proposed to occur under physiological conditions, but the frequency of this event is debated. Here, to probe further the question of whether or not hub cells can be regenerated, we developed methods to genetically ablate some or all hub cells. Surprisingly, when flies were allowed to recover from ablation, the missing hub cells were not replaced. Hub cells did not exit quiescence after partial ablation of hub cells, and labeled cells from outside the hub did not enter the hub during or after ablation. Despite its ability to exit quiescence in response to CySC ablation, we conclude that the hub in the adult Drosophila testis does not have a mechanism to replenish missing hub cells.

Activation of the EGFR/MAPK pathway drives transdifferentiation of quiescent niche cells to stem cells in the Drosophila testis niche.

Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. Here we find that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. We propose that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation.

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly.

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

Dynamic sex chromosome expression in Drosophila male germ cells.

Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Here we use single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis we identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5.

Probing the Function of Metazoan Histones with a Systematic Library of H3 and H4 Mutants.

Replication-dependent histone genes often reside in tandemly arrayed gene clusters, hindering systematic loss-of-function analyses. Here, we used CRISPR/Cas9 and the attP/attB double-integration system to alter numbers and sequences of histone genes in their original genomic context in Drosophila melanogaster. As few as 8 copies of the histone gene unit supported embryo development and adult viability, whereas flies with 20 copies were indistinguishable from wild-types. By hierarchical assembly, 40 alanine-substitution mutations (covering all known modified residues in histones H3 and H4) were introduced and characterized. Mutations at multiple residues compromised viability, fertility, and DNA-damage responses. In particular, H4K16 was necessary for expression of male X-linked genes, male viability, and maintenance of ovarian germline stem cells, whereas H3K27 was essential for late embryogenesis. Simplified mosaic analysis showed that H3R26 is required for H3K27 trimethylation. We have developed a powerful strategy and valuable reagents to systematically probe histone functions in D. melanogaster.

Retinoblastoma Intrinsically Regulates Niche Cell Quiescence, Identity, and Niche Number in the Adult Drosophila Testis.

Homeostasis in adult tissues depends on the precise regulation of stem cells and their surrounding microenvironments, or niches. Here, we show that the cell cycle inhibitor and tumor suppressor Retinoblastoma (RB) is a critical regulator of niche cells in the Drosophila testis. The testis contains a single niche, composed of somatic hub cells, that signals to adjacent germline and somatic stem cells. Hub cells are normally quiescent, but knockdown of the RB homolog Rbf in these cells causes them to proliferate and convert to somatic stem cells. Over time, mutant hub cell clusters enlarge and split apart, forming ectopic hubs surrounded by active stem cells. Furthermore, we show that Rbf's ability to restrict niche number depends on the transcription factors E2F and Escargot and the adhesion molecule E-cadherin. Together this work reveals how precise modulation of niche cells, not only the stem cells they support, can drive regeneration and disease.

Live Imaging of the Drosophila Testis Stem Cell Niche.

Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 12.5 h allows for tracking of cell-type specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. Utilization of live imaging in intact niches will facilitate further understanding of the cellular and molecular mechanisms that regulate stem cell function in vivo.

Chinmo is sufficient to induce male fate in somatic cells of the adult Drosophila ovary.

Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (Dsx(M)) to maintain the male identity of somatic cyst stem cells and their progeny. Here we find that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a Dsx(M)-independent mechanism. Conversely, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by expression of the canonical female sex determinant Dsx(F), indicating that chinmo acts in parallel with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss-of-function phenotype, but we find no apparent phenotypes upon removal of let-7 in the adult ovary or testis. Our finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis.

Genetics of gonadal stem cell renewal.

Stem cells are necessary for the maintenance of many adult tissues. Signals within the stem cell microenvironment, or niche, regulate the self-renewal and differentiation capability of these cells. Misregulation of these signals through mutation or damage can lead to overgrowth or depletion of different stem cell pools. In this review, we focus on the Drosophila testis and ovary, both of which contain well-defined niches, as well as the mouse testis, which has become a more approachable stem cell system with recent technical advances. We discuss the signals that regulate gonadal stem cells in their niches, how these signals mediate self-renewal and differentiation under homeostatic conditions, and how stress, whether from mutations or damage, can cause changes in cell fate and drive stem cell competition.

Niche signaling promotes stem cell survival in the Drosophila testis via the JAK-STAT target DIAP1.

Tissue-specific stem cells are thought to resist environmental insults better than their differentiating progeny, but this resistance varies from one tissue to another, and the underlying mechanisms are not well-understood. Here, we use the Drosophila testis as a model system to study the regulation of cell death within an intact niche. This niche contains sperm-producing germline stem cells (GSCs) and accompanying somatic cyst stem cells (or CySCs). Although many signals are known to promote stem cell self-renewal in this tissue, including the highly conserved JAK-STAT pathway, the response of these stem cells to potential death-inducing signals, and factors promoting stem cell survival, have not been characterized. Here we find that both GSCs and CySCs resist cell death better than their differentiating progeny, under normal laboratory conditions and in response to potential death-inducing stimuli such as irradiation or starvation. To ask what might be promoting stem cell survival, we characterized the role of the anti-apoptotic gene Drosophila inhibitor of apoptosis 1 (diap1) in testis stem cells. DIAP1 protein is enriched in the GSCs and CySCs and is a JAK-STAT target. diap1 is necessary for survival of both GSCs and CySCs, and ectopic up-regulation of DIAP1 in somatic cyst cells is sufficient to non-autonomously rescue stress-induced cell death in adjacent differentiating germ cells (spermatogonia). Altogether, our results show that niche signals can promote stem cell survival by up-regulation of highly conserved anti-apoptotic proteins, and suggest that this strategy may underlie the ability of stem cells to resist death more generally.

The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche.

Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). Here, we find that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (Dsx(M)) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of Dsx(M). The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. Our work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals. PAPERCLIP:

Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis.

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.

Steroid signaling promotes stem cell maintenance in the Drosophila testis.

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.

Conversion of quiescent niche cells to somatic stem cells causes ectopic niche formation in the Drosophila testis.

Adult stem cells reside in specialized regulatory microenvironments, or niches, where local signals ensure stem cell maintenance. The Drosophila testis contains a well-characterized niche wherein signals from postmitotic hub cells promote maintenance of adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells were considered to be terminally differentiated; here, we show that they can give rise to CySCs. Genetic ablation of CySCs triggers hub cells to transiently exit quiescence, delaminate from the hub, and convert into functional CySCs. Ectopic Cyclin D-Cdk4 expression in hub cells is also sufficient to trigger their conversion into CySCs. In both cases, this conversion causes the formation of multiple ectopic niches over time. Therefore, our work provides a model for understanding how oncogenic mutations in quiescent niche cells could promote loss of quiescence, changes in cell fate, and aberrant niche expansion.

JAK-STAT signaling in stem cells.

Adult stem cells are essential for the regeneration and repair of tissues in an organism. Signals from many different pathways converge to regulate stem cell maintenance and differentiation while preventing overproliferation. Although each population of adult stem cells is unique, common themes arise by comparing the regulation of various stem cell types in an organism or by comparing similar stem cell types across species. The JAK-STAT signaling pathway, identified nearly two decades ago, is now known to be involved in many biological processes including the regulation of stem cells. Studies in Drosophila first implicated JAK-STAT signaling in the control of stem cell maintenance in the male germline stem cell microenvironment, or niche; subsequently it has been shown play a role in other niches in both Drosophila and mammals. In this chapter, we will address the role of JAK-STAT signaling in stem cells in the germline, intestinal, hematopoietic and neuronal niches in Drosophila as well as the hematopoietic and neuronal niches in mammals. We will comment on how the study of JAK-STAT signaling in invertebrate systems has helped to advance our understanding of signaling in vertebrates. In addition to the role of JAK- STAT signaling in stem cell niche homeostasis, we will also discuss the diseases, including cancers, that can arise when this pathway is misregulated.

Stem cell competition: finding balance in the niche.

Adult stem cells reside in local microenvironments (niches) that produce signals regulating the outcome of stem cell divisions and stem cell-niche interactions. Limited space and signals in the niche often force stem cells to compete with one another. Although previous studies have uncovered several examples of genetically distinct stem cells competing for niche access, recent studies demonstrate that genetically equivalent stem cells compete under normal conditions, resulting in dynamic stem cell behavior in the niche. New work in multiple vertebrate and invertebrate tissues shows that stem cell competition occurs continuously and mutations disrupting the balance between competing stem cells can cause diseases and defects in the niche. This review discusses recent insights into stem cell competition in mammals and Drosophila.

Recent advances in Drosophila male germline stem cell biology.

The ability of stem cells to divide asymmetrically to produce both self-renewing and differentiating daughter cells sustains many adult tissues, but germline stem cells (GSCs) are unique among stem cells as they perpetuate the genome of the species. The cellular and molecular mechanisms regulating most mammalian stem cells in their endogenous local microenvironments, or niches, are quite challenging to study. However, studies of stem cell niches such as those found in the Drosophila gonads have proven very useful. In these tissues, GSCs are housed in a readily identifiable niche, and the ability to genetically manipulate these cells and their neighbors has uncovered several fundamental mechanisms that are relevant to stem cells more generally. Here, we summarize recent work on the regulation of GSCs in the Drosophila testis niche by intercellular signals, and on the intracellular mechanisms that cooperate with these signals to ensure the survival of the germline. This review focuses on GSCs within the adult Drosophila testis; somatic stem cells in this tissue are reviewed by Zoller and Schulz in this issue.(1) For a review of the testis niche as a whole, see de Cuevas and Matunis,(2) and for more comprehensive reviews of the Drosophila testis, refer to Fuller(3) and Davies and Fuller.(4).

The Drosophila BCL6 homolog Ken and Barbie promotes somatic stem cell self-renewal in the testis niche.

Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 (Leatherman and Dinardo, 2008) and Chinmo (Flaherty et al., 2010), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo (Arbouzova et al., 2006) suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas.