Ex utero monkey embryogenesis from blastocyst to early organogenesis.

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.

Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

Upstream open reading frames control PLK4 translation and centriole duplication in primordial germ cells.

Centrosomes are microtubule-organizing centers comprised of a pair of centrioles and the surrounding pericentriolar material. Abnormalities in centriole number are associated with cell division errors and can contribute to diseases such as cancer. Centriole duplication is limited to once per cell cycle and is controlled by the dosage-sensitive Polo-like kinase 4 (PLK4). Here, we show that PLK4 abundance is translationally controlled through conserved upstream open reading frames (uORFs) in the 5' UTR of the mRNA. Plk4 uORFs suppress Plk4 translation and prevent excess protein synthesis. Mice with homozygous knockout of Plk4 uORFs (Plk4 Δu/Δu ) are viable but display dramatically reduced fertility because of a significant depletion of primordial germ cells (PGCs). The remaining PGCs in Plk4 Δu/Δu mice contain extra centrioles and display evidence of increased mitotic errors. PGCs undergo hypertranscription and have substantially more Plk4 mRNA than somatic cells. Reducing Plk4 mRNA levels in mice lacking Plk4 uORFs restored PGC numbers and fully rescued fertility. Together, our data uncover a specific requirement for uORF-dependent control of PLK4 translation in counterbalancing the increased Plk4 transcription in PGCs. Thus, uORF-mediated translational suppression of PLK4 has a critical role in preventing centriole amplification and preserving the genomic integrity of future gametes.

Balancing the scales: fine-tuning Polo-like kinase 4 to ensure proper centriole duplication.

Polo-like kinase 4 (Plk4) is the master regulator of centriole assembly. Several evolutionarily conserved mechanisms strictly regulate Plk4 abundance and activity to ensure cells maintain a proper number of centrioles. In this issue of Genes & Development, Phan et al. (pp. 718-736) add to this growing list by describing a new mechanism of control that restricts Plk4 translation through competitive ribosome binding at upstream open reading frames (uORFs) in the mature Plk4 mRNA. Fascinatingly, this mechanism is especially critical in the development of primordial germ cells in mice that are transcriptionally hyperactive and thus exquisitely sensitive to Plk4 mRNA regulation.

Gene expression programs in mammalian spermatogenesis.

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell type-specific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies.

Niche cells regulate primordial germ cell quiescence in response to basement membrane signaling.

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates Caenorhabditis elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating that laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germline growth, suggesting that BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors.

Germ granules in development.

A hallmark of all germ cells is the presence of germ granules: assemblies of proteins and RNA that lack a delineating membrane and are proposed to form via condensation. Germ granules across organisms share several conserved components, including factors required for germ cell fate determination and maintenance, and are thought to be linked to germ cell development. The molecular functions of germ granules, however, remain incompletely understood. In this Development at a Glance article, we survey germ granules across organisms and developmental stages, and highlight emerging themes regarding granule regulation, dynamics and proposed functions.

Gastruloid-derived primordial germ cell-like cells develop dynamically within integrated tissues.

Primordial germ cells (PGCs) are the early embryonic precursors of gametes - sperm and egg cells. PGC-like cells (PGCLCs) can currently be derived in vitro from pluripotent cells exposed to signalling cocktails and aggregated into large embryonic bodies, but these do not recapitulate the native embryonic environment during PGC formation. Here, we show that mouse gastruloids, a three-dimensional in vitro model of gastrulation, contain a population of gastruloid-derived PGCLCs (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of specific exogenous PGC-specific signalling or extra-embryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, owing to their inherent co-development, serve as a novel model to study the dynamics of PGC development within integrated tissue environments.

An inducible germ cell ablation chicken model for high-grade germline chimeras.

Chicken embryos are a powerful and widely used animal model in developmental biology studies. Since the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) into recipients after genetic modifications. However, low inheritance caused by competition between host germ cells and the transferred cells is a common complication and greatly reduces production efficiency. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which nitroreductase produces cytotoxic alkylating agents from administered metronidazole, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus was used to drive the expression of the nitroreductase gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system 'germ cell-specific autonomous removal induction' (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

Single-cell RNA-sequencing analysis of early sea star development.

Echinoderms represent a broad phylum with many tractable features to test evolutionary changes and constraints. Here, we present a single-cell RNA-sequencing analysis of early development in the sea star Patiria miniata, to complement the recent analysis of two sea urchin species. We identified 20 cell states across six developmental stages from 8 hpf to mid-gastrula stage, using the analysis of 25,703 cells. The clusters were assigned cell states based on known marker gene expression and by in situ RNA hybridization. We found that early (morula, 8-14 hpf) and late (blastula-to-mid-gastrula) cell states are transcriptionally distinct. Cells surrounding the blastopore undergo rapid cell state changes that include endomesoderm diversification. Of particular import to understanding germ cell specification is that we never see Nodal pathway members within Nanos/Vasa-positive cells in the region known to give rise to the primordial germ cells (PGCs). The results from this work contrast the results of PGC specification in the sea urchin, and the dataset presented here enables deeper comparative studies in tractable developmental models for testing a variety of developmental mechanisms.

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification.

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.

The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris.

Primordial germ cells (PGCs) give rise to gametes - cells necessary for the propagation and fertility of diverse organisms. Current understanding of PGC development is limited to the small number of organisms whose PGCs have been identified and studied. Expanding the field to include little-studied taxa and emerging model organisms is important to understand the full breadth of the evolution of PGC development. In the phylum Tardigrada, no early cell lineages have been identified to date using molecular markers. This includes the PGC lineage. Here, we describe PGC development in the model tardigrade Hypsibius exemplaris. The four earliest-internalizing cells (EICs) exhibit PGC-like behavior and nuclear morphology. The location of the EICs is enriched for mRNAs of conserved PGC markers wiwi1 (water bear piwi 1) and vasa. At early stages, both wiwi1 and vasa mRNAs are detectable uniformly in embryos, which suggests that these mRNAs do not serve as localized determinants for PGC specification. Only later are wiwi1 and vasa enriched in the EICs. Finally, we traced the cells that give rise to the four PGCs. Our results reveal the embryonic origin of the PGCs of H. exemplaris and provide the first molecular characterization of an early cell lineage in the tardigrade phylum. We anticipate that these observations will serve as a basis for characterizing the mechanisms of PGC development in this animal.

DNA repair protein FANCD2 has both ubiquitination-dependent and ubiquitination-independent functions during germ cell development.

When DNA interstrand crosslink lesions occur, a core complex of Fanconi anemia proteins promotes the ubiquitination of FANCD2 and FANCI, which recruit downstream factors to repair the lesion. However, FANCD2 maintains genome stability not only through its ubiquitination-dependent but also its ubiquitination-independent functions in various DNA damage response pathways. Increasing evidence suggests that FANCD2 is essential for fertility, but its ubiquitination-dependent and ubiquitination-independent roles during germ cell development are not well characterized. In this study, we analyzed germ cell development in Fancd2 KO and ubiquitination-deficient mutant (Fancd2K559R/K559R) mice. We showed that in the embryonic stage, both the ubiquitination-dependent and ubiquitination-independent functions of FANCD2 were required for the expansion of primordial germ cells and establishment of the reproductive reserve by reducing transcription-replication conflicts and thus maintaining genome stability in primordial germ cells. Furthermore, we found that during meiosis in spermatogenesis, FANCD2 promoted chromosome synapsis and regulated crossover formation independently of its ubiquitination, but that both ubiquitinated and nonubiquitinated FANCD2 functioned in programmed double strand break repair. Finally, we revealed that on meiotic XY chromosomes, H3K4me2 accumulation required ubiquitination-independent functionality of FANCD2, while the regulation of H3K9me2 and H3K9me3 depended on FANCD2 ubiquitination. Taken together, our findings suggest that FANCD2 has distinct functions that are both dependent on and independent of its ubiquitination during germ cell development.

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells.

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Tissue and cell interactions in mammalian PGC development.

Primordial germ cells (PGCs) form early in embryo development and are crucial precursors to functioning gamete cells. Considerable research has focussed on identifying the transcriptional characteristics and signalling pathway requirements that confer PGC specification and development, enabling the derivation of PGC-like cells (PGCLCs) in vitro using specific signalling cocktails. However, full maturation to germ cells still relies on co-culture with supporting cell types, implicating an additional requirement for cellular- and tissue-level regulation. Here, we discuss the experimental evidence that highlights the nature of intercellular interactions between PGCs and neighbouring cell populations during mouse PGC development. We posit that the role that tissue interactions play on PGCs is not limited solely to signalling-based induction but extends to coordination of development by robust regulation of the proportions and position of the cells and tissues within the embryo, which is crucial for functional germ cell maturation. Such tissue co-development provides a dynamic, contextual niche for PGC development. We argue that there is evidence for a clear role for inter-tissue dependence of mouse PGCs, with potential implications for generating mammalian PGCLCs in vitro.

Germ cell determination and the developmental origin of germ cell tumors.

In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis - the process of germ cell determination - occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs.

Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish.

Primordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-expressed non-coding RNA loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.

Transcriptomic landscape reveals germline potential of porcine skin-derived multipotent dermal fibroblast progenitors.

According to estimations, approximately about 15% of couples worldwide suffer from infertility, in which individuals with azoospermia or oocyte abnormalities cannot be treated with assisted reproductive technology. The skin-derived stem cells (SDSCs) differentiation into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells intervention for infertility treatment in recent years. However, the cellular origin of SDSCs and their dynamic changes in transcription profile during differentiation into PGCLCs in vitro remain largely undissected. Here, the results of single-cell RNA sequencing indicated that porcine SDSCs are mainly derived from multipotent dermal fibroblast progenitors (MDFPs), which are regulated by growth factors (EGF/bFGF). Importantly, porcine SDSCs exhibit pluripotency for differentiating into three germ layers and can effectively differentiate into PGCLCs through complex transcriptional regulation involving histone modification. Moreover, this study also highlights that porcine SDSC-derived PGCLCs specification exhibit conservation with the human primordial germ cells lineage and that its proliferation is mediated by the MAPK signaling pathway. Our findings provide substantial novel insights into the field of regenerative medicine in which stem cells differentiate into germ cells in vitro, as well as potential therapeutic effects in individuals with azoospermia and/or defective oocytes.

A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification.

Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.