Drosophila Germline Stem Cells Research Articles

Lethal Giant Disc is a target of Cdk1 and regulates ESCRT-III localization during germline stem cell abscission.

Abscission is the final step of cytokinesis that allows the physical separation of sister cells through the scission of the cellular membrane. This deformation is driven by ESCRT-III proteins, which can bind membranes and form dynamic helices. A crucial step in abscission is the recruitment of ESCRT-III proteins at the right time and place. Alix is one of the best characterized proteins that recruits ESCRT-III proteins from yeast to mammals. However, recent studies in vivo have revealed that pathways acting independently or redundantly with Alix are also required at abscission sites in different cellular contexts. Here, we show that Lgd acts redundantly with Alix to properly localize ESCRT-III to the abscission site in germline stem cells (GSCs) during Drosophila oogenesis. We further demonstrate that Lgd is phosphorylated at multiple sites by the CycB/Cdk1 kinase. We found that these phosphorylation events potentiate the activity of Shrub, a Drosophila ESCRT-III, during abscission of GSCs. Our study reveals that redundancy between Lgd and Alix, and coordination with the cell cycle kinase Cdk1, confers robust and timely abscission of Drosophila germline stem cells.

Niche Tet maintains germline stem cells independently of dioxygenase activity

Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their involvement in adult stem cell regulation remains unclear. Here, we identify a novel enzymatic activity-independent function of Tet in the Drosophila germline stem cell (GSC) niche. Tet activates the expression of Dpp, the fly homologue of BMP, in the ovary stem cell niche, thereby controlling GSC self-renewal. Depletion of Tet disrupts Dpp production, leading to premature GSC loss. Strikingly, both wild-type and enzyme-dead mutant Tet proteins rescue defective BMP signaling and GSC loss when expressed in the niche. Mechanistically, Tet interacts directly with Bap55 and Stat92E, facilitating recruitment of the Polybromo Brahma associated protein (PBAP) complex to the dpp enhancer and activating Dpp expression. Furthermore, human TET3 can effectively substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. Our findings highlight a conserved novel catalytic activity-independent role of Tet as a scaffold protein in supporting niche signaling for adult stem cell self-renewal.

Chromatin and gene expression changes during female Drosophila germline stem cell development illuminate the biology of highly potent stem cells

Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. Drosophila female germline stem cells (GSCs) perpetuate without change over evolutionary time and generate cystoblast daughters that develop into nurse cells and oocytes. Cystoblasts initiate differentiation by generating a transient syncytial state, the germline cyst, and by increasing pericentromeric H3K9me3 modification, actions likely to suppress transposable element activity. Relatively open GSC chromatin is further restricted by Polycomb repression of testis or somatic cell-expressed genes briefly active in early female germ cells. Subsequently, Neijre/CBP and Myc help upregulate growth and reprogram GSC metabolism by altering mitochondrial transmembrane transport, gluconeogenesis, and other processes. In all these respects GSC differentiation resembles development of the totipotent zygote. We propose that the totipotent stem cell state was shaped by the need to resist transposon activity over evolutionary timescales.

Chromatin and gene expression changes during female Drosophila germline stem cell development illuminate the biology of highly potent stem cells.

Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. Drosophila female germline stem cells (GSCs) perpetuate without change over evolutionary time and generate cystoblast daughters that develop into nurse cells and oocytes. Cystoblasts initiate differentiation by generating a transient syncytial state, the germline cyst, and by increasing pericentromeric H3K9me3 modification, actions likely to suppress transposable element activity. Relatively open GSC chromatin is further restricted by Polycomb repression of testis or somatic cell-expressed genes briefly active in early female germ cells. Subsequently, Neijre/CBP and Myc help upregulate growth and reprogram GSC metabolism by altering mitochondrial transmembrane transport, gluconeogenesis, and other processes. In all these respects GSC differentiation resembles development of the totipotent zygote. We propose that the totipotent stem cell state was shaped by the need to resist transposon activity over evolutionary timescales.

H/ACA snRNP-dependent ribosome biogenesis regulates translation of polyglutamine proteins.

Stem cells in many systems, including Drosophila germline stem cells (GSCs), increase ribosome biogenesis and translation during terminal differentiation. Here, we show that the H/ACA small nuclear ribonucleoprotein (snRNP) complex that promotes pseudouridylation of ribosomal RNA (rRNA) and ribosome biogenesis is required for oocyte specification. Reducing ribosome levels during differentiation decreased the translation of a subset of messenger RNAs that are enriched for CAG trinucleotide repeats and encode polyglutamine-containing proteins, including differentiation factors such as RNA-binding Fox protein 1. Moreover, ribosomes were enriched at CAG repeats within transcripts during oogenesis. Increasing target of rapamycin (TOR) activity to elevate ribosome levels in H/ACA snRNP complex-depleted germlines suppressed the GSC differentiation defects, whereas germlines treated with the TOR inhibitor rapamycin had reduced levels of polyglutamine-containing proteins. Thus, ribosome biogenesis and ribosome levels can control stem cell differentiation via selective translation of CAG repeat-containing transcripts.

Centromere proteins are asymmetrically distributed between newly divided germline stem and daughter cells and maintain a balanced niche in Drosophila males.

Stem cells can undergo asymmetric cell division (ACD) giving rise to one new stem cell and one differentiating daughter cell. In Drosophila germline stem cells (GSCs), the centromeric histone CENP-A (CID in flies) is asymmetrically distributed between sister chromatids such that chromosomes that end up in the GSC harbor more CID at centromeres. A model of "mitotic drive" has been proposed in GSCs such that stronger and earlier centromere and kinetochore interactions with microtubules bias sister chromatid segregation. Here we show that in Drosophila males, centromere proteins CID, CAL1, and CENP-C are asymmetrically distributed in newly divided GSCs and daughter cells in S phase. We find that overexpression of CID (either with or without CAL1) or CENP-C depletion disrupts CID asymmetry, with an increased pool of GSCs relative to daughter cells detectable in the niche. This result suggests a shift toward GSC self-renewal rather than differentiation, important for maintaining tissue homeostasis. Overexpression of CAL1 does not disrupt asymmetry, but instead drives germ cell proliferation in the niche. Our results in male GSCs are comparable to female GSCs, indicating that despite differences in signaling, organization, and niche composition, the effects of centromere proteins on GSC maintenance are conserved between the sexes.

Mitochondrial remodelling is essential for female germ cell differentiation and survival.

Stem cells often possess immature mitochondria with few inner membrane invaginations, which increase as stem cells differentiate. Despite this being a conserved feature across many stem cell types in numerous organisms, how and why mitochondria undergo such remodelling during stem cell differentiation has remained unclear. Here, using Drosophila germline stem cells (GSCs), we show that Complex V drives mitochondrial remodelling during the early stages of GSC differentiation, prior to terminal differentiation. This endows germline mitochondria with the capacity to generate large amounts of ATP required for later egg growth and development. Interestingly, impairing mitochondrial remodelling prior to terminal differentiation results in endoplasmic reticulum (ER) lipid bilayer stress, Protein kinase R-like ER kinase (PERK)-mediated activation of the Integrated Stress Response (ISR) and germ cell death. Taken together, our data suggest that mitochondrial remodelling is an essential and tightly integrated aspect of stem cell differentiation. This work sheds light on the potential impact of mitochondrial dysfunction on stem and germ cell function, highlighting ER lipid bilayer stress as a potential major driver of phenotypes caused by mitochondrial dysfunction.

Targeting Endogenous Loci That Function in Drosophila Germline Stem Cells.

CRISPR-Cas9 genome editing technology can be used to manipulate the genome of Drosophila melanogaster. The ability to delete genes, make specific mutations, add tags, or make other genetic manipulations is useful for studying germline stem cell biology. In this chapter, we will describe a method to use CRISPR-Cas9 genome editing technology to make knock-out and knock-in flies. We will cover everything from guideRNA (gRNA) and donor plasmid design and cloning to screening for positive edits.

Stress-induced reversible cell-cycle arrest requires PRC2/PRC1-mediated control of mitophagy in Drosophila germline stem cells and human iPSCs.

Following acute genotoxic stress, both normal and tumorous stem cells can undergo cell-cycle arrest to avoid apoptosis and later re-enter the cell cycle to regenerate daughter cells. However, the mechanism of protective, reversible proliferative arrest, "quiescence," remains unresolved. Here, we show that mitophagy is a prerequisite for reversible quiescence in both irradiated Drosophila germline stem cells (GSCs) and human induced pluripotent stem cells (hiPSCs). In GSCs, mitofission (Drp1) or mitophagy (Pink1/Parkin) genes are essential to enter quiescence, whereas mitochondrial biogenesis (PGC1α) or fusion (Mfn2) genes are crucial for exiting quiescence. Furthermore, mitophagy-dependent quiescence lies downstream of mTOR- and PRC2-mediated repression and relies on the mitochondrial pool of cyclin E. Mitophagy-dependent reduction of cyclin E in GSCs and in hiPSCs during mTOR inhibition prevents the usual G1/S transition, pushing the cells toward reversible quiescence (G0). This alternative method of G1/S control may present new opportunities for therapeutic purposes.

Atg1 modulates mitochondrial dynamics to promote germline stem cell maintenance in Drosophila

Mitochondrial dynamics (fusion and fission) are necessary for stem cell maintenance and differentiation. However, the relationship between mitophagy, mitochondrial dynamics and stem cell exhaustion needs to be clearly understood. Here we report the multifaceted role of Atg1 in mitophagy, mitochondrial dynamics and stem cell maintenance in female germline stem cells (GSCs) in Drosophila. We found that depletion of Atg1 in GSCs leads to impaired autophagy and mitophagy as measured by reduced formation of autophagosomes, increased accumulation of p62/Ref (2)P and accumulation of damaged mitochondria. Disrupting Atg1 function led to mitochondrial fusion in developing cysts. The fusion resulted from an increase in Marf levels in both GSCs and cysts, and the fusion phenotype could be rescued by overexpression of Drp1 or by depleting Marf via RNAi in Atg1-depleted cyst cells. Interestingly, double knockdown of both Atg1:Drp1 led to the significant loss of germ cells (GCs) as compared to Atg1KD and Drp1KD. Strikingly, Atg1:Marf double knockdown leads to a dramatic loss of GSCs, GCs and a total loss of vitellogenic stages, suggesting a block in oogenesis. Overall, our results demonstrate that Drp1, Marf and Atg1 function together to influence female GSC maintenance, their differentiation into cysts and oogenesis in Drosophila.

CENP-C functions in centromere assembly, the maintenance of CENP-A asymmetry and epigenetic age in Drosophila germline stem cells.

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)-the epigenetic determinant of centromere identity-is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of 'mitotic drive' that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.

RpS13 controls the homeostasis of germline stem cell niche through Rho1-mediated signals in the Drosophila testis.

ObjectivesStem cell niche regulated the renewal and differentiation of germline stem cells (GSCs) in Drosophila. Previously, we and others identified a series of genes encoding ribosomal proteins that may contribute to the self‐renewal and differentiation of GSCs. However, the mechanisms that maintain and differentiate GSCs in their niches were not well understood.Materials and MethodsFlies were used to generate tissue‐specific gene knockdown. Small interfering RNAs were used to knockdown genes in S2 cells. qRT‐PCR was used to examine the relative mRNA expression level. TUNEL staining or flow cytometry assays were used to detect cell survival. Immunofluorescence was used to determine protein localization and expression pattern.ResultsHerein, using a genetic manipulation approach, we investigated the role of ribosomal protein S13 (RpS13) in testes and S2 cells. We reported that RpS13 was required for the self‐renewal and differentiation of GSCs. We also demonstrated that RpS13 regulated cell proliferation and apoptosis. Mechanistically, we showed that RpS13 regulated the expression of ribosome subunits and could moderate the expression of the Rho1, DE‐cad and Arm proteins. Notably, Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells, and recruited cell adhesions, which was mediated by the DE‐cad and Arm proteins.ConclusionThese findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis.

DRTEL1 is Essential for the Maintenance of Germline Stem Cells

Stem cells have the potential to maintain the undifferentiated state and differentiate into specialized cell types. Despite numerous progresses have been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of Drosophila germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 directly regulates a set of candidate genes controlling GSC self-renewal and differentiation. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation for GSC maintenance. Finally, dRTEL1 is required to maintain Stat92E protein levels, the key player in GSC self-renewal. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in GSC self-renewal and provide insight into the function of dRTEL1.

Centromere assembly and non-random sister chromatid segregation in stem cells.

Asymmetric cell division (ACD) produces daughter cells with separate distinct cell fates and is critical for the development and regulation of multicellular organisms. Epigenetic mechanisms are key players in cell fate determination. Centromeres, epigenetically specified loci defined by the presence of the histone H3-variant, centromere protein A (CENP-A), are essential for chromosome segregation at cell division. ACDs in stem cells and in oocyte meiosis have been proposed to be reliant on centromere integrity for the regulation of the non-random segregation of chromosomes. It has recently been shown that CENP-A is asymmetrically distributed between the centromeres of sister chromatids in male and female Drosophila germline stem cells (GSCs), with more CENP-A on sister chromatids to be segregated to the GSC. This imbalance in centromere strength correlates with the temporal and asymmetric assembly of the mitotic spindle and potentially orientates the cell to allow for biased sister chromatid retention in stem cells. In this essay, we discuss the recent evidence for asymmetric sister centromeres in stem cells. Thereafter, we discuss mechanistic avenues to establish this sister centromere asymmetry and how it ultimately might influence cell fate.

Nuclear architecture as an intrinsic regulator of Drosophila female germline stem cell maintenance.

Homeostasis of Drosophila germline stem cells (GSC) depends upon the integration of intrinsic and extrinsic signals. This review highlights emerging data that support nuclear architecture as an intrinsic regulator of GSC maintenance and germ cell differentiation. Here, we focus on the nuclear lamina (NL) and the nucleolus, two compartments that undergo alterations in composition upon germ cell differentiation. Loss of NL or nucleolar components leads to GSC loss, resulting from activation of GSC quality control checkpoint pathways. We suggest that the NL and nucleolus integrate signals needed for the switch between GSC maintenance and germ cell differentiation, and propose regulation of nuclear actin pools as one mechanism that connects these compartments.

Uninvited guests: how transposable elements take advantage of Drosophila germline stem cells, and how stem cells fight back.

Transposable elements (TEs) are mobile genetic parasites that spread through host genomes by replicating in germline cells. New TE copies that arise in the genomes of germline stem cells (GSCs) are of particular value, because they are potentially transmitted to multiple offspring through the plethora of gametes arising from the same progenitor GSC. However, the fidelity of GSC genomes is also of utmost importance to the host in ensuring the production of abundant and fit offspring. Here we review tactics that TEs employ to replicate in Drosophila female GSCs, as well as mechanisms those cells use to defend against TEs. We also discuss the relationship between transposition and GSC loss, which is arbitrated through reduced signaling for self renewal, increased signaling for differentiation, and DNA damage response pathways.

Survival Motor Neuron Protein Participates in Mouse Germ Cell Development and Spermatogonium Maintenance.

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance.

Asymmetric Histone Inheritance Regulates Stem Cell Fate in Drosophila Midgut

A fundamental question in developmental biology is how distinct cell fates are established and maintained through epigenetic mechanisms in multicellular organisms. Here, we report that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically inherited by the distinct daughter cells during asymmetric division of Drosophila intestinal stem cells (ISCs). By contrast, in symmetrically dividing ISCs that produce two self-renewed stem cells, old and new H3 and H4 show symmetric inheritance patterns. These results indicate that asymmetric histone inheritance is tightly associated with the distinct daughter cell fates. To further understand the biological significance of this asymmetry, we express a mutant histone that compromises asymmetric histone inheritance pattern. We find increased symmetric ISC division and ISC tumors during aging under this condition. Together, our results demonstrate that asymmetric histone inheritance is important for establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in asymmetrically dividing male germline stem cells in Drosophila. Therefore, this work sheds light on the principles of histone inheritance in regulating stem cell fate in vivo .

Heterochromatin protein 1 (HP1) is intrinsically required for post-transcriptional regulation of Drosophila Germline Stem Cell (GSC) maintenance

A very important open question in stem cells regulation is how the fine balance between GSCs self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance. Furthermore, recent studies have revealed the importance of HP1 in DNA repair, sister chromatid cohesion and, surprisingly, in positive regulation of gene expression. Here, we show that HP1 plays a crucial role in the control of GSC homeostasis in Drosophila. Our findings demonstrate that HP1 is required intrinsically to promote GSC self-renewal and progeny differentiation by directly stabilizing the transcripts of key genes involved in GSCs maintenance.

Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche

Adult stem cells are often found in specialized niches, where the constituent cells direct self-renewal of their stem cell pool. The niche is therefore crucial for both normal homeostasis and tissue regeneration. In many mammalian tissues, niche cells have classically been difficult to identify, which has hampered any understanding of how tissues first construct niches during development. Fortunately, the Drosophila germline stem cell (GSC) niche is well defined, allowing for unambiguous identification of both niche cells and resident stem cells. The testis niche first forms in the early embryo, during a late stage of gonadogenesis. Here, using live-imaging both in vivo and ex vivo, we follow pro-niche cells as they assemble and assume their final form. We show that after ex vivo culture the niche appears fully functional, as judged by enrichment of adhesion proteins, the ability to activate STAT in adjacent GSCs, and to direct GSCs to divide orthogonally to the niche, just as they would in situ. Collectively, our imaging has generated several novel insights on niche morphogenesis that could not be inferred from fixed images alone. We identify dynamic processes that constitute an assembly phase and a compaction phase during morphogenesis. The compaction phase correlates with cell neighbor exchange among the assembled pro-niche cells, as well as a burst of divisions among newly recruited stem cells. Before compaction, an assembly phase involves the movement of pro-niche cells along the outer periphery of the gonad, using the extracellular matrix (ECM) to assemble at the anterior of the gonad. Finally, live-imaging in integrin mutants allows us to define the role of pro-niche cell-ECM interaction with regard to the new assembly and compaction dynamics revealed here.