Drosophila Male Germline Research Articles

Drosophila male germline stem cells and their transit amplifying daughters depend on G-protein signaling for increasing their mitotic indices in response to mating

Drosophila male germline stem cells and their transit amplifying daughters depend on G-protein signaling for increasing their mitotic indices in response to mating

Cyclin E and Cdk1 regulate the termination of germline transit-amplification process in Drosophila testis

ABSTRACT An extension of the G1 is correlated with stem cell differentiation. The role of cell cycle regulation during the subsequent transit amplification (TA) divisions is, however, unclear. Here, we report for the first time that in the Drosophila male germline lineage, the transit amplification divisions accelerate after the second TA division. The cell cycle phases, marked by Cyclin E and Cyclin B, are progressively altered during the TA. Antagonistic functions of the bag-of-marbles and the Transforming-Growth-Factor-β signaling regulate the cell division rates after the second TA division and the extent of the Cyclin E phase during the fourth TA division. Furthermore, loss of Cyclin E during the fourth TA cycle retards the cell division and induces premature meiosis in some cases. A similar reduction of Cdk1 activity during this stage arrests the penultimate division and subsequent differentiation, whereas enhancement of the Cdk1 activity prolongs the TA by one extra round. Altogether, the results suggest that modification of the cell cycle structure and the rates of cell division after the second TA division determine the extent of amplification. Also, the regulation of the Cyclin E and CDK1 functions during the penultimate TA division determines the induction of meiosis and subsequent differentiation.

Single-cyst transcriptome analysis of Drosophila male germline stem cell lineage.

The Drosophila male germline stem cell (GSC) lineage provides a great model to understand stem cell maintenance, proliferation, differentiation and dedifferentiation. Here, we use the Drosophila GSC lineage to systematically analyze the transcriptome of discrete but continuously differentiating germline cysts. We first isolated single cysts at each recognizable stage from wild-type testes, which were subsequently applied for RNA-seq analyses. Our data delineate a high-resolution transcriptome atlas in the entire male GSC lineage: the most dramatic switch occurs from early to late spermatocyte, followed by the change from the mitotic spermatogonia to early meiotic spermatocyte. By contrast, the transit-amplifying spermatogonia cysts display similar transcriptomes, suggesting common molecular features among these stages, which may underlie their similar behavior during both differentiation and dedifferentiation processes. Finally, distinct differentiating germ cell cyst samples do not exhibit obvious dosage compensation of X-chromosomal genes, even considering the paucity of X-chromosomal gene expression during meiosis, which is different from somatic cells. Together, our single cyst-resolution, genome-wide transcriptional profile analyses provide an unprecedented resource to understand many questions in both germ cell biology and stem cell biology fields.

Asymmetric Centromeres Differentially Coordinate with Mitotic Machinery to Ensure Biased Sister Chromatid Segregation in Germline Stem Cells.

Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. How non-genic information could be inherited differentially to establish distinct cell fates is not well understood. Here, we report a series of spatiotemporally regulated asymmetric components, which ensure biased sister chromatid attachment and segregation during ACD of Drosophila male germline stem cells (GSCs). First, sister centromeres are differentially enriched with proteins involved in centromere specification and kinetochore function. Second, temporally asymmetric microtubule activities and polarized nuclear envelope breakdown allow for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. Abolishment of either the asymmetric sister centromeres or the asymmetric microtubule activities results in randomized sister chromatid segregation. Together, these results provide the cellular basis for partitioning epigenetically distinct sister chromatids during stem cell ACDs, which opens new directions to study these mechanisms in other biological contexts.

DREF Genetically Counteracts Mi-2 and Caf1 to Regulate Adult Stem Cell Maintenance.

Active adult stem cells maintain a bipotential state with progeny able to either self-renew or initiate differentiation depending on extrinsic signals from the surrounding microenvironment. However, the intrinsic gene regulatory networks and chromatin states that allow adult stem cells to make these cell fate choices are not entirely understood. Here we show that the transcription factor DNA Replication-related Element Factor (DREF) regulates adult stem cell maintenance in the Drosophila male germline. A temperature-sensitive allele of DREF described in this study genetically separated a role for DREF in germline stem cell self-renewal from the general roles of DREF in cell proliferation. The DREF temperature-sensitive allele caused defects in germline stem cell self-renewal but allowed viability and division of germline stem cells as well as cell viability, growth and division of somatic cyst stem cells in the testes and cells in the Drosophila eye. Germline stem cells mutant for the temperature sensitive DREF allele exhibited lower activation of a TGF-beta reporter, and their progeny turned on expression of the differentiation factor Bam prematurely. Results of genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, genetically antagonize the role of DREF in germline stem cell maintenance. Taken together, these data suggest that DREF contributes to intrinsic components of the germline stem cell regulatory network that maintains competence to self-renew.

The Dlg Module and Clathrin-Mediated Endocytosis Regulate EGFR Signaling and Cyst Cell-Germline Coordination in the Drosophila Testis.

SummaryTissue homeostasis and repair relies on proper communication of stem cells and their differentiating daughters with the local tissue microenvironment. In the Drosophila male germline adult stem cell lineage, germ cells proliferate and progressively differentiate enclosed in supportive somatic cyst cells, forming a small organoid, the functional unit of differentiation. Here we show that cell polarity and vesicle trafficking influence signal transduction in cyst cells, with profound effects on the germ cells they enclose. Our data suggest that the cortical components Dlg, Scrib, Lgl and the clathrin-mediated endocytic (CME) machinery downregulate epidermal growth factor receptor (EGFR) signaling. Knockdown of dlg, scrib, lgl, or CME components in cyst cells resulted in germ cell death, similar to increased signal transduction via the EGFR, while lowering EGFR or downstream signaling components rescued the defects. This work provides insights into how cell polarity and endocytosis cooperate to regulate signal transduction and sculpt developing tissues.

The hpRNA/RNAi Pathway Is Essential to Resolve Intragenomic Conflict in the Drosophila Male Germline

Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D.simulans null mutants of Dcr-2 and AGO2. Strikingly, these coreRNAi knockouts massively derepress Dox andMDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.

Centrosome Inheritance Does Not Regulate Cell Fate in Granule Neuron Progenitors of the Developing Cerebellum.

An inherent asymmetry exists between the two centrosomes of a dividing cell. One centrosome is structurally more mature (mother centrosome) than the other (daughter centrosome). Post division, one daughter cell inherits the mother centrosome while the other daughter cell inherits the daughter centrosome. Remarkably, the kind of centrosome inherited is associated with cell fate in several developmental contexts such as in radial glial progenitors in the developing mouse cortex, Drosophila neuroblast divisions and in Drosophila male germline stem cells. However, the role of centrosome inheritance in granule neuron progenitors in the developing cerebellum has not been investigated. Here, we show that mother and daughter centrosomes do exist in these progenitors, and the amount of pericentriolar material (PCM) each centrosome possesses is different. However, we failed to observe any correlation between the fate adopted by the daughter cell and the nature of centrosome it inherited.

Cytokine receptor-Eb1 interaction couples cell polarity and fate during asymmetric cell division

Asymmetric stem cell division is a critical mechanism for balancing self-renewal and differentiation. Adult stem cells often orient their mitotic spindle to place one daughter inside the niche and the other outside of it to achieve asymmetric division. It remains unknown whether and how the niche may direct division orientation. Here we discover a novel and evolutionary conserved mechanism that couples cell polarity to cell fate. We show that the cytokine receptor homolog Dome, acting downstream of the niche-derived ligand Upd, directly binds to the microtubule-binding protein Eb1 to regulate spindle orientation in Drosophila male germline stem cells (GSCs). Dome's role in spindle orientation is entirely separable from its known function in self-renewal mediated by the JAK-STAT pathway. We propose that integration of two functions (cell polarity and fate) in a single receptor is a key mechanism to ensure an asymmetric outcome following cell division.

Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells.

rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

Enhancer of polycomb maintains germline activity and genome integrity in Drosophila testis.

Tissue homeostasis depends on the ability of tissue-specific adult stem cells to maintain a balance between proliferation and differentiation, as well as ensure DNA damage repair. Here, we use the Drosophila male germline stem cell system to study how a chromatin factor, enhancer of polycomb [E(Pc)], regulates the proliferation-to-differentiation (mitosis-to-meiosis) transition and DNA damage repair. We identified two critical targets of E(Pc). First, E(Pc) represses CycB transcription, likely through modulating H4 acetylation. Second, E(Pc) is required for accumulation of an important germline differentiation factor, Bag-of-marbles (Bam), through post-transcriptional regulation. When E(Pc) is downregulated, increased CycB and decreased Bam are both responsible for defective mitosis-to-meiosis transition in the germline. Moreover, DNA double-strand breaks (DSBs) accumulate upon germline inactivation of E(Pc) under both physiological condition and recovery from heat shock-induced endonuclease expression. Failure of robust DSB repair likely leads to germ cell loss. Finally, compromising the activity of Tip60, a histone acetyltransferase, leads to germline defects similar to E(Pc) loss-of-function, suggesting that E(Pc) acts cooperatively with Tip60. Together, our data demonstrate that E(Pc) has pleiotropic roles in maintaining male germline activity and genome integrity. Our findings will help elucidate the in vivo molecular mechanisms of E(Pc).

All are equal, but some are more equal than others: Epigenetic regulation of germline stem cell fate in <i>Drosophila</i> <i>melanogaster</i>

Adult stem cells are capable of both symmetric and asymmetric cell divisions. Asymmetric cell division allows self-renewal and gives rise to intermediate cells that ultimately differentiate into specific cell types. Consequently, adult stem cells play a key role in development and tissue homeostasis during the life span of an organism. Typically, adult stem cell divisions are regulated through coordination between non-autonomous signaling from the niche and cell-autonomous influences from stem cell-intrinsic factors. Although localized distribution of proteins, RNA and organelles during cell division contributes significantly to the differences between fates of daughter cells, recent studies have also implicated epigenetic factors in this process. A number of epigenetic modifications remain associated with the chromosomes during mitosis and serve as a template to reestablish fates after mitosis. Whether the distribution of epigenetic modifications is random on each chromatid or there is a bias in their distribution is therefore under extensive investigation. The nonrandom distribution of epigenetic modifications on mitotic chromosomes provides an attractive possible explanation of how bias is generated during chromatid segregation. In Drosophila male germline stem cells, the histone modifications present in the stem cells are distinct from those in the differentiating daughter cells. These modifications help to retain pre-existing histones in the mother cell while imparting newly synthesized histones to the daughter cell. Importantly, the retention of pre-existing histones in the stem cells is a prerequisite to maintain their ability to self-renew. Here we summarize recent studies that focus on the role of different epigenetic modifications in the regulation of asymmetric adult germline stem cell divisions in Drosophila. We further describe how epigenetic modifications potentially lead to variations in the otherwise equivalent chromatids, and discuss the role of biased chromatid segregation in asymmetric cell divisions.

Symmetry from Asymmetry or Asymmetry from Symmetry?

The processes of DNA replication and mitosis allow the genetic information of a cell to be copied and transferred reliably to its daughter cells. However, if DNA replication and cell division were always performed in a symmetric manner, the result would be a cluster of tumor cells instead of a multicellular organism. Therefore, gaining a complete understanding of any complex living organism depends on learning how cells become different while faithfully maintaining the same genetic material. It is well recognized that the distinct epigenetic information contained in each cell type defines its unique gene expression program. Nevertheless, how epigenetic information contained in the parental cell is either maintained or changed in the daughter cells remains largely unknown. During the asymmetric cell division (ACD) of Drosophila male germline stem cells, our previous work revealed that preexisting histones are selectively retained in the renewed stem cell daughter, whereas newly synthesized histones are enriched in the differentiating daughter cell. We also found that randomized inheritance of preexisting histones versus newly synthesized histones results in both stem cell loss and progenitor germ cell tumor phenotypes, suggesting that programmed histone inheritance is a key epigenetic player for cells to either remember or reset cell fates. Here, we will discuss these findings in the context of current knowledge on DNA replication, polarized mitotic machinery, and ACD for both animal development and tissue homeostasis. We will also speculate on some potential mechanisms underlying asymmetric histone inheritance, which may be used in other biological events to achieve the asymmetric cell fates.

Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division.

Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. Here we identify Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. We propose that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions.

Evaluation of the Asymmetric Division of Drosophila Male Germline Stem Cells.

Asymmetric cell division (ACD) is utilized in many stem cell systems to produce two daughter cells with different cell fates. Despite the fundamental importance of ACD during development and tissue homeostasis, the nature of ACD is far from being fully understood. Step-by-step observation of events during ACD allows us to understand processes that lead to ACD. Here we describe examples of how we evaluate ACD in vivo using the Drosophila male germline stem cell system.

Sex Chromosome-wide Transcriptional Suppression and Compensatory Cis-Regulatory Evolution Mediate Gene Expression in the Drosophila Male Germline.

The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower—approximately 3-fold or more—for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis.

Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl.

Regulation of Stem Cells in Their Niche

Stem cells are characterized by their ability to asymmetrically divide, generating another self-renewing stem cell and a differentiating daughter cell. They reside within the local niche microenvironment, which, together with systemic signals, regulates intrinsic stem cell function. Stem cells are finely regulated, with even slight deregulation leading to gain of stem cell function and tumorigenesis or loss of stem cell function and tissue degeneration in aging. In this review, we highlight how stem cells are maintained within their niche and what is known about their deregulation in aging. To highlight these points, we look at the Drosophila male germline stem cell niche as a model system, specifically focusing on cell cycle progression, signaling pathways, epigenetics, and the control of spindle orientation by centrosomes. Finally, we sum up pertinent questions to be addressed in understanding stem cell function and their malfunction in aging.

Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin

The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis.

Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells.

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub-GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.