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.

Premeiotic pairing of homologous chromosomes during Drosophila male meiosis.

In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper segregation. These events can vary drastically between species and between males and females of the same species. In Drosophila, in contrast to females, males do not form synaptonemal complexes (SCs), do not recombine, and have no crossing over; yet, males are able to segregate their chromosomes properly. Here, we investigated the early steps of homolog pairing in Drosophila males. We found that homolog centromeres are not paired in germline stem cells (GSCs) and become paired in the mitotic region before meiotic entry, similarly to females. Surprisingly, male germline cells express SC proteins, which localize to centromeres and promote pairing. We further found that the SUN/KASH (LINC) complex and microtubules are required for homolog pairing as in females. Chromosome movements in males, however, are much slower than in females and we demonstrate that this slow dynamic is compensated in males by having longer cell cycles. In agreement, slowing down cell cycles was sufficient to rescue pairing-defective mutants in female meiosis. Our results demonstrate that although meiosis differs significantly between males and females, sex-specific cell cycle kinetics integrate similar molecular mechanisms to achieve proper centromere pairing.

Centromeric SMC1 promotes centromere clustering and stabilizes meiotic homolog pairing.

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.

The replicative histone chaperone CAF1 is essential for the maintenance of identity and genome integrity in adult stem cells.

Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. Here, we have investigated the role of CAF1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. We depleted P180, the large subunit of CAF1, in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, our work demonstrates that chromatin dynamics mediated by CAF1 play an important role in both the regulation of stem cell identity and genome integrity.

tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription.

RNase P is a conserved endonuclease that processes the 5' trailer of tRNA precursors. We have isolated mutations in Rpp30, a subunit of RNase P, and find that these induce complete sterility in Drosophila females. Here, we show that sterility is not due to a shortage of mature tRNAs, but that atrophied ovaries result from the activation of several DNA damage checkpoint proteins, including p53, Claspin, and Chk2. Indeed, we find that tRNA processing defects lead to increased replication stress and de-repression of transposable elements in mutant ovaries. We also report that transcription of major piRNA sources collapse in mutant germ cells and that this correlates with a decrease in heterochromatic H3K9me3 marks on the corresponding piRNA-producing loci. Our data thus link tRNA processing, DNA replication, and genome defense by small RNAs. This unexpected connection reveals constraints that could shape genome organization during evolution.

Abscission is regulated by the ESCRT-III protein shrub in Drosophila germline stem cells.

Abscission is the final event of cytokinesis that leads to the physical separation of the two daughter cells. Recent technical advances have allowed a better understanding of the cellular and molecular events leading to abscission in isolated yeast or mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood. Here, we characterized the function of the ESCRT-III protein Shrub during cytokinesis in germ cells undergoing a series of complete and incomplete divisions. We found that Shrub is required for complete abscission, and that levels of Shrub are critical for proper timing of abscission. Loss or gain of Shrub delays abscission in germline stem cells (GSCs), and leads to the formation of stem-cysts, where daughter cells share the same cytoplasm as the mother stem cell and cannot differentiate. In addition, our results indicate a negative regulation of Shrub by the Aurora B kinase during GSC abscission. Finally, we found that Lethal giant discs (lgd), known to be required for Shrub function in the endosomal pathway, also regulates the duration of abscission in GSCs.

ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo.

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.

Synaptonemal complex components promote centromere pairing in pre-meiotic germ cells.

Mitosis and meiosis are two distinct cell division programs. During mitosis, sister chromatids separate, whereas during the first meiotic division, homologous chromosomes pair and then segregate from each other. In most organisms, germ cells do both programs sequentially, as they first amplify through mitosis, before switching to meiosis to produce haploid gametes. Here, we show that autosomal chromosomes are unpaired at their centromeres in Drosophila germline stem cells, and become paired during the following four mitosis of the differentiating daughter cell. Surprisingly, we further demonstrate that components of the central region of the synaptonemal complex are already expressed in the mitotic region of the ovaries, localize close to centromeres, and promote de novo association of centromeres. Our results thus show that meiotic proteins and meiotic organization of centromeres, which are key features to ensure reductional segregation, are laid out in amplifying germ cells, before meiosis has started.

lethal giant larvae is required with the par genes for the early polarization of the Drosophila oocyte.

Most cell types in an organism show some degree of polarization, which relies on a surprisingly limited number of proteins. The underlying molecular mechanisms depend, however, on the cellular context. Mutual inhibitions between members of the Par genes are proposed to be sufficient to polarize the C. elegans one-cell zygote and the Drosophila oocyte during mid-oogenesis. By contrast, the Par genes interact with cellular junctions and associated complexes to polarize epithelial cells. The Par genes are also required at an early step of Drosophila oogenesis for the maintenance of the oocyte fate and its early polarization. Here we show that the Par genes are not sufficient to polarize the oocyte early and that the activity of the tumor-suppressor gene lethal giant larvae (lgl) is required for the posterior translocation of oocyte-specific proteins, including germline determinants. We also found that Lgl localizes asymmetrically within the oocyte and is excluded from the posterior pole. We further demonstrate that phosphorylation of Par-1, Par-3 (Bazooka) and Lgl is crucial to regulate their activity and localization in vivo and describe, for the first time, adherens junctions located around the ring canals, which link the oocyte to the other cells of the germline cyst. However, null mutations in the DE-cadherin gene, which encodes the main component of the zonula adherens, do not affect the early polarization of the oocyte. We conclude that, despite sharing many similarities with other model systems at the genetic and cellular levels, the polarization of the early oocyte relies on a specific subset of polarity proteins.

Conserved meiotic mechanisms in the cnidarian Clytia hemisphaerica revealed by Spo11 knockout.

During meiosis, DNA recombination allows the shuffling of genetic information between the maternal and paternal chromosomes. Recombination is initiated by double-strand breaks (DSBs) catalyzed by the conserved enzyme Spo11. How this crucial event is connected to other meiotic processes is unexpectedly variable depending on the species. Here, we knocked down Spo11 by CRISPR in the jellyfish Clytia hemisphaerica. Germ cells in Clytia Spo11 mutants fail to assemble synaptonemal complexes and chiasmata, and in consequence, homologous chromosome pairs in females remain unassociated during oocyte growth and meiotic divisions, creating aneuploid but fertilizable eggs that develop into viable larvae. Clytia thus shares an ancient eukaryotic dependence of synapsis and chromosome segregation on Spo11-generated DSBs. Phylogenetically, Clytia belongs to Cnidaria, the sister clade to Bilateria where classical animal model species are found, so these results provide fresh evolutionary perspectives on meiosis regulation.

Integrin-independent repression of cadherin transcription by talin during axis formation in Drosophila.

The Drosophila melanogaster anterior-posterior axis becomes polarized early during oogenesis by the posterior localization of the oocyte within the egg chamber. The invariant position of the oocyte is thought to be driven by an upregulation of the adhesion molecule DE-cadherin in the oocyte and the posterior somatic follicle cells, providing the first in vivo example of cell sorting that is specified by quantitative differences in cell-cell adhesion. However, it has remained unclear how DE-cadherin levels are regulated. Here, we show that talin, known for its role in linking integrins to the actin cytoskeleton, has the unexpected function of specifically inhibiting DE-cadherin transcription. Follicle cells that are mutant for talin show a strikingly high level of DE-cadherin, due to elevated transcription of DE-cadherin. We demonstrate that this deregulation of DE-cadherin is sufficient to attract the oocyte to lateral and anterior positions. Surprisingly, this function of talin is independent of integrins. These results uncover a new role for talin in regulating cadherin-mediated cell adhesion.

The deubiquitinase USP8 targets ESCRT-III to promote incomplete cell division.

In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. We found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. Our results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division.

Cytoplasmic forces functionally reorganize nuclear condensates in oocytes.

Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

Collective Cell Sorting Requires Contractile Cortical Waves in Germline Cells.

Encapsulation of germline cells by layers of somatic cells forms the basic unit of female reproduction called primordial follicles in mammals and egg chambers in Drosophila. How germline and somatic tissues are coordinated for the morphogenesis of each separated unit remains poorly understood. Here, using improved live imaging of Drosophila ovaries, we uncovered periodic actomyosin waves at the cortex of germ cells. These contractile waves are associated with pressure release blebs, which project from germ cells into somatic cells. We demonstrate that these cortical activities, together with cadherin-based adhesion, are required to sort each germline cyst as one collective unit. Genetic perturbations of cortical contractility, bleb protrusion, or adhesion between germline and somatic cells induced encapsulation defects resulting from failures to encapsulate any germ cells, or the inclusion of too many germ cells per egg chamber, or even the mechanical split of germline cysts. Live-imaging experiments revealed that reducing contractility or adhesion in the germline reduced the stiffness of germline cysts and their proper anchoring to the somatic cells. Germline cysts can then be squeezed and passively pushed by constricting surrounding somatic cells, resulting in cyst splitting and cyst collisions during encapsulation. Increasing germline cysts activity or blocking somatic cell constriction movements can reveal active forward migration of germline cysts. Our results show that germ cells play an active role in physical coupling with somatic cells to produce the female gamete.

Isolation of stage-specific germ cells using FACS in Drosophila germarium.

Drosophila melanogaster oogenesis is a versatile model system to address many fundamental questions of cell and developmental biology, such as stem cell biology, mitosis, meiosis or cell polarity. Many mutagenesis and powerful genetic tools have contributed massively to identify and dissect in vivo gene functions in a stage and tissue specific manner. However, the small number of germ cells during the early steps of oogenesis have hampered a systematic description of RNA and protein contents at each stage. We describe here a protocol for isolating and comparing two small subpopulations of cells in the ovary for the purpose of RNA sequence profiling. The method is based on fluorescence-activated cell sorting (FACS) of GFP- and RFP-labeled proteins that are expressed in distinct and mostly non-overlapping regions of the germline. We used a transgene expressing a GFP-tagged Bam protein driven by its own promoter, labeling specifically the mitotic region of the germarium. We also took advantage of the short-lived Wicked protein tagged with RFP and expressed under the nanos promoter to label the meiotic region. We generated flies expressing both markers and were able to sort enough cells from each region to extract total RNAs and small RNAs. Total RNA or small RNA extracted from sorted cells were then used to generate deep-sequencing libraries that show specificity toward each compartment. This method of isolating a very small number of cells and the data generated from comparing distinct cell populations within the germline should further our understanding of these conserved steps of oogenesis.

Mixing and Matching Chromosomes during Female Meiosis.

Meiosis is a key event in the manufacturing of an oocyte. During this process, the oocyte creates a set of unique chromosomes by recombining paternal and maternal copies of homologous chromosomes, and by eliminating one set of chromosomes to become haploid. While meiosis is conserved among sexually reproducing eukaryotes, there is a bewildering diversity of strategies among species, and sometimes within sexes of the same species, to achieve proper segregation of chromosomes. Here, we review the very first steps of meiosis in females, when the maternal and paternal copies of each homologous chromosomes have to move, find each other and pair. We explore the similarities and differences observed in C. elegans, Drosophila, zebrafish and mouse females.

tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation.

tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. Here we describe a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, we used small RNAs Illumina sequencing datasets extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (dTrm7_34 and dTrm7_32). Using our pipeline, we show how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology.

The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization.

The Staufen-dependent localization of oskar mRNA to the posterior of the Drosophila oocyte induces the formation of the pole plasm, which contains the abdominal and germline determinants. In a germline clone screen for mutations that disrupt the posterior localization of GFP-Staufen, we isolated three missense alleles in the hnRNPA/B homolog, Hrp48. These mutants specifically abolish osk mRNA localization, without affecting its translational control or splicing, or the localization of bicoid and gurken mRNAs and the organization of the microtubule cytoskeleton. Hrp48 colocalizes with osk mRNA throughout oogenesis, and interacts with its 5' and 3' regulatory regions, suggesting that it binds directly to oskar mRNA to mediate its posterior transport. The hrp48 alleles cause a different oskar mRNA localization defect from other mutants, and disrupt the formation of GFP-Staufen particles. This suggests a new step in the localization pathway, which may correspond to the assembly of Staufen/oskar mRNA transport particles.