Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation

Background During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs). Results We found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and “bivalent” (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (μChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis. Conclusion This study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells

The three-dimensional configuration of the genome ensures cell type-specific gene expression profiles by placing genes and regulatory elements in close spatial proximity. Here, we used in situ high-throughput chromosome conformation (in situ Hi-C), RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to characterize the high-order chromatin structure signature of female germline stem cells (FGSCs) and identify its regulating key factor based on the data-driven of multiple omics data. By comparison with pluripotent stem cells (PSCs), adult stem cells (ASCs), and somatic cells at three major levels of chromatin architecture, A/B compartments, topologically associating domains, and chromatin loops, the chromatin architecture of FGSCs was most similar to that of other ASCs and largely different from that of PSCs and somatic cells. After integrative analysis of the three-dimensional chromatin structure, active compartment-associating loops (aCALs) were identified as a signature of high-order chromatin organization in FGSCs, which revealed that CCCTC-binding factor was a major factor to maintain the properties of FGSCs through regulation of aCALs. We found FGSCs belong to ASCs at chromatin structure level and characterized aCALs as the high-order chromatin structure signature of FGSCs. Furthermore, CTCF was identified to play a key role in regulating aCALS to maintain the biological functions of FGSCs. These data provide a valuable resource for future studies of the features of chromatin organization in mammalian stem cells and further understanding of the fundamental characteristics of FGSCs.

Poly(ADP-ribosyl)ating pathway regulates development from stem cell niche to longevity control

The regulation of poly(ADP-ribose) polymerase, the enzyme responsible for the synthesis of homopolymer ADP-ribose chains on nuclear proteins, has been extensively studied over the last decades for its involvement in tumorigenesis processes. However, the regulation of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for removing this posttranslational modification, has attracted little attention. Here we identified that PARG activity is partly regulated by two phosphorylation sites, ph1 and ph2, in Drosophila. We showed that the disruption of these sites affects the germline stem-cells maintenance/differentiation balance as well as embryonic and larval development, but also the synchronization of egg production with the availability of a calorically sufficient food source. Moreover, these PARG phosphorylation sites play an essential role in the control of fly survivability from larvae to adults. We also showed that PARG is phosphorylated by casein kinase 2 and that this phosphorylation seems to protect PARG protein against degradation in vivo. Taken together, these results suggest that the regulation of PARG protein activity plays a crucial role in the control of several developmental processes.

A sensitized genetic screen to identify regulators of C. elegans germline stem cells

GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells (GSCs) in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all GSCs enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic GSC defect is called the “Glp” phenotype. The lst-1 and sygl-1 genes are direct targets of Notch transcriptional activation and functionally redundant. Whereas single lst-1 and sygl-1 mutants are fertile, lst-1 sygl-1 double mutants are sterile with a Glp phenotype. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain GSCs. To this end, we conducted forward genetic screens for mutants with a Glp phenotype in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated nine glp-1 alleles, two lst-1 alleles, and one allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin (ANK) repeats not previously mutated. pole-1 single mutants have a low penetrance Glp phenotype that is enhanced by loss of sygl-1. Thus, the screen uncovered one locus that interacts genetically with sygl-1 and generated useful mutations for further studies of GSC regulation.

Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin binding protein Barrier-to-autointegration factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to deformation of NL structure and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf mis-regulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained mis-expressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global mis-expression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found up-regulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

MSL3 promotes germline stem cell differentiation in female Drosophila

Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation are incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. We discovered that the H3K36me3 reader Male-specific lethal 3 (MSL3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. MSL3, acting independent from the rest of the male specific lethal complex, promotes transcription of genes including a germline enriched ribosomal protein S19 paralog, RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, MSL3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

Pre-meiotic 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 homologues pairing in Drosophila males. We found that homologues 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 homologues pairing as in females. Chromosome movements are however 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 are integrated with similar molecular mechanisms to achieve proper homologues pairing.

Regulation of Mating-Induced Increase in Female Germline Stem Cells in the Fruit Fly Drosophila melanogaster

In many insect species, mating stimuli can lead to changes in various behavioral and physiological responses, including feeding, mating refusal, egg-laying behavior, energy demand, and organ remodeling, which are collectively known as the post-mating response. Recently, an increase in germline stem cells (GSCs) has been identified as a new post-mating response in both males and females of the fruit fly, Drosophila melanogaster. We have extensively studied mating-induced increase in female GSCs of D. melanogaster at the molecular, cellular, and systemic levels. After mating, the male seminal fluid peptide [e.g. sex peptide (SP)] is transferred to the female uterus. This is followed by binding to the sex peptide receptor (SPR), which evokes post-mating responses, including increase in number of female GSCs. Downstream of SP-SPR signaling, the following three hormones and neurotransmitters have been found to act on female GSC niche cells to regulate mating-induced increase in female GSCs: (1) neuropeptide F, a peptide hormone produced in enteroendocrine cells; (2) octopamine, a monoaminergic neurotransmitter synthesized in ovary-projecting neurons; and (3) ecdysone, a steroid hormone produced in ovarian follicular cells. These humoral factors are secreted from each organ and are received by ovarian somatic cells and regulate the strength of niche signaling in female GSCs. This review provides an overview of the latest findings on the inter-organ relationship to regulate mating-induced female GSC increase in D. melanogaster as a model. We also discuss the remaining issues that should be addressed in the future.

Interchromosomal interaction of homologous Stat92E alleles regulates transcriptional switch during stem-cell differentiation

The strength of pairing of homologous chromosomes differs in a locus-specific manner and is correlated to gene expression states. However, the functional impact of homolog pairing on local transcriptional activity is still unclear. Drosophila male germline stem cells (GSCs) constantly divide asymmetrically to produce one GSC and one differentiating gonialblast (GB). The GB then enters the differentiation program in which stem cell specific genes are quickly downregulated. Here we demonstrate that a change in local pairing state of Stat92E locus is required for the downregulation of the Stat92E gene during differentiation. Using OligoPaint fluorescent in situ hybridization (FISH), we show that the interaction between homologous loci of Stat92E is always tight in GSCs and immediately loosened in GBs. When one of the Stat92E locus was absent or relocated to another chromosome, Stat92E did not pair and failed to downregulate, suggesting that the pairing is required for switching of transcriptional activity. The defect in downregulation of Stat92E was also observed upon knockdown of global pairing or anti-pairing factors. Moreover, the Stat92E enhancer element, but not cis-transcription, is required for the change in pairing state, indicating that it is not a consequence of transcriptional changes. GSCs are known to inherit pre-existing histones H3 and H4, while newly synthesized histones are distributed in GBs. When this histone inheritance was compromised, the change in Stat92E pairing did not occur, suggesting that it is an intrinsically programmed process during asymmetric stem cell division. We propose that the change of local pairing state may be a common process to reprogram gene activity during cell-differentiation.

Krüppel-like factor 4 is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk-cell-generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells, presumptive germline stem cells, and yolk-cell progenitors. klf4 knockdown animals fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ-cell-like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.