Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment.

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

Epigenetic priming in the male germline.

Epigenetic priming presets chromatin states that allow the rapid induction of gene expression programs in response to differentiation cues. In the germline, it provides the blueprint for sexually dimorphic unidirectional differentiation. In this review, we focus on epigenetic priming in the mammalian male germline and discuss how cellular memories are regulated and inherited to the next generation. During spermatogenesis, epigenetic priming predetermines cellular memories that ensure the lifelong maintenance of spermatogonial stem cells and their subsequent commitment to meiosis and to the production of haploid sperm. The paternal chromatin state is also essential for the recovery of totipotency after fertilization and contributes to paternal epigenetic inheritance. Thus, epigenetic priming establishes stable but reversible chromatin states during spermatogenesis and enables epigenetic inheritance and reprogramming in the next generation.

The dynamic chromatin landscape and mechanisms of DNA methylation during mouse germ cell development.

Epigenetic marks including DNA methylation (DNAme) play a critical role in the transcriptional regulation of genes and retrotransposons. Defects in DNAme are detected in infertility, imprinting disorders and congenital diseases in humans, highlighting the broad importance of this epigenetic mark in both development and disease. While DNAme in terminally differentiated cells is stably propagated following cell division by the maintenance DNAme machinery, widespread erasure and subsequent de novo establishment of this epigenetic mark occur early in embryonic development as well as in germ cell development. Combined with deep sequencing, low-input methods that have been developed in the past several years have enabled high-resolution and genome-wide mapping of both DNAme and histone post-translational modifications (PTMs) in rare cell populations including developing germ cells. Epigenome studies using these novel methods reveal an unprecedented view of the dynamic chromatin landscape during germ cell development. Furthermore, integrative analysis of chromatin marks in normal germ cells and in those deficient in chromatin-modifying enzymes uncovers a critical interplay between histone PTMs and de novo DNAme in the germline. This review discusses work on mechanisms of the erasure and subsequent de novo DNAme in mouse germ cells as well as the outstanding questions relating to the regulation of the dynamic chromatin landscape in germ cells.

Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells.

Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow [J.-L. Maître et al., Science 338, 253-256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension-stabilizing E-cadherin-actin complexes at the contact.

Germ cells commit somatic stem cells to differentiation following priming by PI3K/Tor activity in the Drosophila testis.

How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. We find that CySCs are maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, we find that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. We propose that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline.

Building RNA-protein germ granules: insights from the multifaceted functions of DEAD-box helicase Vasa/Ddx4 in germline development.

The segregation and maintenance of a dedicated germline in multicellular organisms is essential for species propagation in the sexually reproducing metazoan kingdom. The germline is distinct from somatic cells in that it is ultimately dedicated to acquiring the "totipotency" and to regenerating the offspring after fertilization. The most striking feature of germ cells lies in the presence of characteristic membraneless germ granules that have recently proven to behave like liquid droplets resulting from liquid-liquid phase separation (LLPS). Vasa/Ddx4, a faithful DEAD-box family germline marker highly conserved across metazoan species, harbors canonical DEAD-box motifs and typical intrinsically disordered sequences at both the N-terminus and C-terminus. This feature enables it to serve as a primary driving force behind germ granule formation and helicase-mediated RNA metabolism (e.g., piRNA biogenesis). Genetic ablation of Vasa/Ddx4 or the catalytic-dead mutations abolishing its helicase activity led to sexually dimorphic germline defects resulting in either male or female sterility among diverse species. While recent efforts have discovered pivotal functions of Vasa/Ddx4 in somatic cells, especially in multipotent stem cells, we herein summarize the helicase-dependent and -independent functions of Vasa/Ddx4 in the germline, and discuss recent findings of Vasa/Ddx4-mediated phase separation, germ granule formation and piRNA-dependent retrotransposon control essential for germline development.

Maternal starvation primes progeny response to nutritional stress.

Organisms adapt to environmental changes in order to survive. Mothers exposed to nutritional stresses can induce an adaptive response in their offspring. However, the molecular mechanisms behind such inheritable links are not clear. Here we report that in Drosophila, starvation of mothers primes the progeny against subsequent nutritional stress. We found that RpL10Ab represses TOR pathway activity by genetically interacting with TOR pathway components TSC2 and Rheb. In addition, starved mothers produce offspring with lower levels of RpL10Ab in the germline, which results in higher TOR pathway activity, conferring greater resistance to starvation-induced oocyte loss. The RpL10Ab locus encodes for the RpL10Ab mRNA and a stable intronic sequence RNA (sisR-8), which collectively repress RpL10Ab pre-mRNA splicing in a negative feedback mechanism. During starvation, an increase in maternally deposited RpL10Ab and sisR-8 transcripts leads to the reduction of RpL10Ab expression in the offspring. Our study suggests that the maternally deposited RpL10Ab and sisR-8 transcripts trigger a negative feedback loop that mediates intergenerational adaptation to nutritional stress as a starvation response.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans.

The germ line produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules-well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germ line. Using Caenorhabditis elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions (IDRs). An RNA interference-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two putative LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from the nuclear envelope, germline atrophy, and reduced fertility. We show that IDRs of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote the perinuclear localization of P granules. Taken together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

Germline competent mesoderm: the substrate for vertebrate germline and somatic stem cells?

In vitro production of tissue-specific stem cells [e.g. haematopoietic stem cells (HSCs)] is a key goal of regenerative medicine. However, recent efforts to produce fully functional tissue-specific stem cells have fallen short. One possible cause of shortcomings may be that model organisms used to characterize basic vertebrate embryology (Xenopus, zebrafish, chick) may employ molecular mechanisms for stem cell specification that are not conserved in humans, a prominent example being the specification of primordial germ cells (PGCs). Germ plasm irreversibly specifies PGCs in many models; however, it is not conserved in humans, which produce PGCs from tissue termed germline-competent mesoderm (GLCM). GLCM is not conserved in organisms containing germ plasm, or even in mice, but understanding its developmental potential could unlock successful production of other stem cell types. GLCM was first discovered in embryos from the axolotl and its conservation has since been demonstrated in pigs, which develop from a flat-disc embryo like humans. Together these findings suggest that GLCM is a conserved basal trait of vertebrate embryos. Moreover, the immortal nature of germ cells suggests that immortality is retained during GLCM specification; here we suggest that the demonstrated pluripotency of GLCM accounts for retention of immortality in somatic stem cell types as well. This article has an associated Future Leaders to Watch interview with the author of the paper.

MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells.

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

Bucky Ball Is a Novel Zebrafish Vasa ATPase Activator.

Many multicellular organisms specify germ cells during early embryogenesis by the inheritance of ribonucleoprotein (RNP) granules known as germplasm. However, the role of complex interactions of RNP granules during germ cell specification remains elusive. This study characterizes the interaction of RNP granules, Buc, and zebrafish Vasa (zfVasa) during germ cell specification. We identify a novel zfVasa-binding motif (Buc-VBM) in Buc and a Buc-binding motif (zfVasa-BBM) in zfVasa. Moreover, we show that Buc and zfVasa directly bind in vitro and that this interaction is independent of the RNA. Our circular dichroism spectroscopy data reveal that the intrinsically disordered Buc-VBM peptide forms alpha-helices in the presence of the solvent trifluoroethanol. Intriguingly, we further demonstrate that Buc-VBM enhances zfVasa ATPase activity, thereby annotating the first biochemical function of Buc as a zfVasa ATPase activator. Collectively, these results propose a model in which the activity of zfVasa is a central regulator of primordial germ cell (PGC) formation and is tightly controlled by the germplasm organizer Buc.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.

Conserved germ plasm characteristics across the Danio and Devario lineages.

In many animal species, germ cell specification requires the inheritance of germ plasm, a biomolecular condensate containing maternally derived RNAs and proteins. Most studies of germ plasm composition and function have been performed in widely evolutionarily divergent model organisms, such as Caenorhabditis elegans, Drosophila, Xenopus laevis, and Danio rerio (zebrafish). In zebrafish, 12 RNAs localize to germ plasm at the furrows of the early embryo. Here, we tested for the presence of these RNAs in three additional species within the Danionin clade: Danio kyathit, Danio albolineatus, and Devario aequipinnatus. By visualizing nanos RNA, we find that germ plasm segregation patterns during early embryogenesis are conserved across these species. Ten additional germ plasm RNAs exhibit localization at the furrows of early embryos in all three non-zebrafish Danionin species, consistent with germ plasm localization. One component of zebrafish germ plasm, ca15b, lacked specific localization in embryos of the more distantly related D. aequipinnatus. Our findings show that within a subset of closely related Danionin species, the vast majority of germ plasm RNA components are conserved. At the same time, the lack of ca15b localization in D. aequipinnatus germ plasm highlights the potential for the divergence of germ plasm composition across a restricted phylogenetic space.

Temperature response of enriched pre-pubertal caprine male germline stem cells in vitro.

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-ß1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (ß1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.

CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say.

Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

To Be or Not to Be a Germ Cell: The Extragonadal Germ Cell Tumor Paradigm.

In the human embryo, the genetic program that orchestrates germ cell specification involves the activation of epigenetic and transcriptional mechanisms that make the germline a unique cell population continuously poised between germness and pluripotency. Germ cell tumors, neoplasias originating from fetal or neonatal germ cells, maintain such dichotomy and can adopt either pluripotent features (embryonal carcinomas) or germness features (seminomas) with a wide range of phenotypes in between these histotypes. Here, we review the basic concepts of cell specification, migration and gonadal colonization of human primordial germ cells (hPGCs) highlighting the analogies of transcriptional/epigenetic programs between these two cell types.

A transitory signaling center controls timing of primordial germ cell differentiation.

Organogenesis requires exquisite spatiotemporal coordination of cell morphogenesis, migration, proliferation, and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis, primordial germ cells (PGCs) either are sequestered in stem cell niches and are maintained in an undifferentiated germline stem cell state or transition directly toward differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning these cells close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso receptor tyrosine kinase (RTK) signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene, bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinates ovarian morphogenesis with stem cell self-renewal and differentiation programs, highlighting a more general role for such centers in reproductive and developmental biology.

Sgpl1 deletion elevates S1P levels, contributing to NPR2 inactivity and p21 expression that block germ cell development.

Sphingosine phosphate lyase 1 (SGPL1) is a highly conserved enzyme that irreversibly degrades sphingosine-1-phosphate (S1P). Sgpl1-knockout mice fail to develop germ cells, resulting in infertility. However, the molecular mechanism remains unclear. The results of the present study showed that SGPL1 was expressed mainly in granulosa cells, Leydig cells, spermatocytes, and round spermatids. Sgpl1 deletion led to S1P accumulation in the gonads. In the ovary, S1P decreased natriuretic peptide receptor 2 (NPR2) activity in granulosa cells and inhibited early follicle growth. In the testis, S1P increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and apoptosis in Leydig cells, thus resulting in spermatogenesis arrest. These results indicate that Sgpl1 deletion increases intracellular S1P levels, resulting in the arrest of female and male germ cell development via different signaling pathways.

Metabolo-epigenetics: the interplay of metabolism and epigenetics during early germ cells development

Metabolites control epigenetic mechanisms, and conversly, cell metabolism is regulated at the epigenetic level in response to changes in the cellular environment. In recent years, this metabolo-epigenetic control of gene expression has been implicated in the regulation of multiple stages of embryonic development. The developmental potency of stem cells and their embryonic counterparts is directly determined by metabolic rewiring. Here, we review the current knowledge on the interplay between epigenetics and metabolism in the specific context of early germ cell development. We explore the implications of metabolic rewiring in primordial germ cells in light of their epigenetic remodeling during cell fate determination. Finally, we discuss the relevance of concerted metabolic and epigenetic regulation of primordial germ cells in the context of mammalian transgenerational epigenetic inheritance.