High-throughput single-cell transcriptomics reveals the female germline differentiation trajectory in Arabidopsis thaliana

Female germline cells in flowering plants differentiate from somatic cells to produce specialized reproductive organs, called ovules, embedded deep inside the flowers. We investigated the molecular basis of this distinctive developmental program by performing single-cell RNA sequencing (scRNA-seq) of 16,872 single cells of Arabidopsis thaliana ovule primordia at three developmental time points during female germline differentiation. This allowed us to identify the characteristic expression patterns of the main cell types, including the female germline and its surrounding nucellus. We then reconstructed the continuous trajectory of female germline differentiation and observed dynamic waves of gene expression along the developmental trajectory. A focused analysis revealed transcriptional cascades and identified key transcriptional factors that showed distinct expression patterns along the germline differentiation trajectory. Our study provides a valuable reference dataset of the transcriptional process during female germline differentiation at single-cell resolution, shedding light on the mechanisms underlying germline cell fate determination.

Neuronal regulated ire-1-dependent mRNA decay controls germline differentiation in Caenorhabditis elegans

Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in Caenorhabditis elegans demonstrate that knockout of the germline-specific translation repressor gld-1 causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that endoplasmic reticulum (ER) stress enhances this phenotype to suppress germline tumor progression(Levi-Ferber et al., 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated inositol requiring enzyme-1 (IRE-1)-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuit’s integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation and demonstrate that regulated Ire1-dependent decay can affect animal physiology by controlling a specific neuronal circuit.

O-101 Neospermatogenesis benefits from a three-dimensional culture system

Study question Does a three-dimensional (3D) culture system increase the efficiency of male germline differentiation of mouse embryonic stem cells (mESC) over a bidimensional method? Summary answer Our 3D culture system based on direct spherification proves superior to the standard bidimensional plating in promoting neogametogenesis of mESC into post-meiotic male germ cells. What is known already Two-dimensional monolayer cell cultures are common in stem cell research. However, this method does not replicate a physiological 3D spatial relationship and may provide an inaccurate replication of in vivo environments. A 3D spherical structure allows us to mimic the seminiferous tubule, the site of in vivo spermatogenesis. By using spheroids as a scaffold to replicate cell culture systems, we can study spermatogenesis in a controlled setting. Direct spherification, a technique commonly used in molecular gastronomy, provides an opportunity to create spheroids that mimic in vivo events that materialize in the lab Study design, size, duration mESCs were initially cultured on a 6-well plate coated with fibroblasts and inserted into sodium alginate spheres. To coax differentiation, spheres (3 to 6 mm in diameter) were plunged directly into differentiation medium (DM) while the control mESC in 6-well dishes were layered with it. Cells obtained from both culture systems were tested by biomarkers for different germ cell stages Participants/materials, setting, methods Bidimensional mESC at 80% confluence were differentiated either on a plate or spherified for a 3D culture. Both systems underwent the same timeline of exposure to EpiLC medium with Activin A, bFGF and KSR for 3 days and PGCLC medium with BMP4, LIF, SCF and EGF for 7 days. Differentiated cells were retrieved from each method at day 3 and day 10 to assess for germ line differentiation markers, DAZL, VASA and BOULE Main results and the role of chance Under optic visualization through the sphere wall, cellular aggregation was seen on day 2 of culturing in EpiLC medium while this phenomenon was not observed on bidimensional plating. In the conventional method, cells expressed 7% DAZL (spermatogonium cell stage) and 1% VASA (pre-spermatid cell stage) whereas in direct spherification, cells expressed 20% DAZL (P < 0.001) and 15% VASA positivity (P < 0.0001). To further compare the different methods in later stages of germ-line differentiation, the remaining spheres were cultured in PGCLC medium for 7 days. At day 10, isolated cells were assessed for VASA and DAZL again. In the conventional method, 23% of cells expressed positivity for VASA and 29% DAZL whereas direct spherification achieved a positivity rate of 43% for VASA (P < 0.005) and 45% for DAZL (P < 0.005). This increased expression in both VASA and DAZL signify the increased number of cells undergoing germline differentiation. Additionally, BOULE was assessed for the presence of meiotic cells such as the spermatocyte. The conventional method yielded < 1% BOULE positivity whereas in direct spherification, there was 10% positivity (P < 0.005). Direct spherifcation result shows that differentiation almost doubled in comparison to the conventional method, yielding more post-meiotic cells in the same amount of time Limitations, reasons for caution Despite a higher differentiation rate in direct spherification, these cells would still need to be tested for their fertilization potential. The ability to achieve fertilization, blastocysts and live pups would provide final proof and reliability of this method of neogametogenesis Wider implications of the findings Differentiating ESCs through direct spherification provides an alternative to studying intercellular relationships. This provides an opportunity to study spermatogenesis in more detail by replicating the microenvironment of the seminiferous tubule. Once embryo developmental competence of the de novo gamete is confirmed, this may open a new chapter in human reproduction Trial registration number N/A

The emerging role of small RNAs in ovule development, a kind of magic

In plants, small RNAs have been recognized as key genetic and epigenetic regulators of development. Small RNAs are usually 20 to 30 nucleotides in length and they control, in a sequence specific manner, the transcriptional or post-transcriptional expression of genes. In this review, we present a comprehensive overview of the most recent findings about the function of small RNAs in ovule development, including megasporogenesis and megagametogenesis, both in sexual and apomictic plants. We discuss recent studies on the role of miRNAs, siRNAs and trans-acting RNAs (ta-siRNAs) in early female germline differentiation. The mechanistic complexity and unique regulatory features are reviewed, and possible directions for future research are provided.

RNA Kinase CLP1/Cbc Regulates Meiosis Initiation in Spermatogenesis

CLP1, TSEN complex, and VCP are evolutionarily conserved proteins whose mutations are associated with neurodegenerative diseases. In this study, we have found that they are also involved in germline differentiation. To optimize both quantity and quality in gametes production, germ cells expand themselves through limited mitotic cycles prior to meiosis. Stemming from our previous findings on the correlation between mRNA 3′-processing and meiosis entry, here we identify that the RNA kinase Cbc, the Drosophila member of the highly conserved CLP1 family, is a component of the program regulating the transition from mitosis to meiosis. Using genetic manipulations in Drosophila testis, we demonstrate that nuclear Cbc is required to promote meiosis entry. Combining biochemical and genetic methods, we reveal that Cbc physically and/or genetically intersects with Tsen54 and TER94 (VCP ortholog) in this process. The C-terminal half of Tsen54 is both necessary and sufficient for its binding with Cbc. Further, we illustrate the functional conservation between Cbc and mammalian CLP1 in the assays of subcellular localization and Drosophila fertility. As CLP1, TSEN complex, and VCP have also been identified in neurodegenerations of animal models, a mechanism involving these factors seems to be shared in gametogenesis and neurogenesis.

Early germline differentiation in bivalves: TDRD7 as a candidate investigational unit for Ruditapes philippinarum germ granule assembly

The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves.

The Osa-Containing SWI/SNF Chromatin-Remodeling Complex Is Required in the Germline Differentiation Niche for Germline Stem Cell Progeny Differentiation

The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. Here, we report that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, we show that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation.

Neuronal regulated Ire1-dependent mRNA decay controls germline differentiation in C. elegans

Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in C. elegans demonstrate that knockout of the germline-specific translation repressor gld-1, causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that ER stress enhances this phenotype to suppress germline tumor progression (Levi-Ferber M, 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation, and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated IRE-1-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuit's integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation, and demonstrate that RIDD can affect animal physiology by controlling a specific neuronal circuit.

Non-autonomous regulation of germline stem cell proliferation by somatic MPK-1/MAPK activity in C. elegans

Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is a major positive regulator of cell proliferation that is often upregulated in cancer. Yet few studies have addressed ERK/MAPK regulation of proliferation within a complete organism. The C. elegans ERK/MAPK ortholog MPK-1 is best known for its control of somatic organogenesis and germline differentiation, but it also stimulates germline stem cell proliferation. Here we identify tissue-specific MPK-1 isoforms and characterize their distinct roles in germline function. The germline-specific MPK-1B isoform promotes germline differentiation, but has no apparent role in germline stem cell proliferation. By contrast, the soma-specific MPK-1A isoform promotes germline proliferation non-autonomously. Indeed, MPK-1A functions in the intestine or somatic gonad to promote germline proliferation, independently of its other known roles. We propose that a non-autonomous role of ERK/MAPK in stem cell proliferation may be conserved across species and other tissue types, with major clinical implications for cancer and other diseases.

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.