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.

Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

Current Approaches and Applications in Avian Genome Editing.

Advances in genome-editing technologies and sequencing of animal genomes enable researchers to generate genome-edited (GE) livestock as valuable animal models that benefit biological researches and biomedical and agricultural industries. As birds are an important species in biology and agriculture, their genome editing has gained significant interest and is mainly performed by using a primordial germ cell (PGC)-mediated method because pronuclear injection is not practical in the avian species. In this method, PGCs can be isolated, cultured, genetically edited in vitro, and injected into a recipient embryo to produce GE offspring. Recently, a couple of GE quail have been generated by using the newly developed adenovirus-mediated method. Without technically required in vitro procedures of the PGC-mediated method, direct injection of adenovirus into the avian blastoderm in the freshly laid eggs resulted in the production of germ-line chimera and GE offspring. As more approaches are available in avian genome editing, avian research in various fields will progress rapidly. In this review, we describe the development of avian genome editing and scientific and industrial applications of GE avian species.

DND protein functions as a translation repressor during zebrafish embryogenesis.

Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish.

Strategies for the Generation of Gene Modified Avian Models: Advancement in Avian Germline Transmission, Genome Editing, and Applications.

Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. However, in birds, the introduction of the CRISPR system into primordial germ cells (PGCs), a germline-competent stem cell, is considered a much more reliable approach for the development of genome-edited models. After genome editing, PGCs are transplanted into the embryo to establish germline chimera, which are crossed to produce genome-edited birds. In addition, various methods, including delivery by liposomal and viral vectors, have been employed for gene editing in vivo. Genome-edited birds have wide applications in bio-pharmaceutical production and as models for disease resistance and biological research. In conclusion, the application of the CRISPR system to avian PGCs is an efficient approach for the production of genome-edited birds and transgenic avian models.

Visualization of X chromosome reactivation in mouse primordial germ cells in vivo.

X chromosome inactivation (XCI), determined during development, remains stable after embryonic cell divisions. However, primordial germ cells (PGCs) are exceptions in that XCI is reprogrammed and inactivated X chromosomes are reactivated. Although interactions between PGCs and somatic cells are thought to be important for PGC development, little is known about them. Here, we performed imaging of X chromosome reactivation (XCR) using the 'Momiji' mouse system, which can monitor the X chromosome's inactive and active states using two color fluorescence reporter genes, and investigated whether interactions would affect XCR in PGCs. Based on their expression levels, we found that XCR of the Pgk1 locus began at embryonic day (E)10.5 and was almost complete by E13.5. During this period, PGCs became distributed uniformly in the genital ridge, proliferated, and formed clusters; XCR progressed accordingly. In addition, XCR of the Pgk1 locus preceded that of the Hprt locus, indicating that the timing of epigenetic memory erasure varied according to the locus of each of these X-linked genes. Our results indicate that XCR proceeds along with the proliferation of PGCs clustered within the genital ridge. This article has an associated First Person interview with the first author of the paper.

Ligand-Receptor Interactions Elucidate Sex-Specific Pathways in the Trajectory From Primordial Germ Cells to Gonia During Human Development.

The human germ cell lineage originates from primordial germ cells (PGCs), which are specified at approximately the third week of development. Our understanding of the signaling pathways that control this event has significantly increased in recent years and that has enabled the generation of PGC-like cells (PGCLCs) from pluripotent stem cells in vitro. However, the signaling pathways that drive the transition of PGCs into gonia (prospermatogonia in males or premeiotic oogonia in females) remain unclear, and we are presently unable to mimic this step in vitro in the absence of gonadal tissue. Therefore, we have analyzed single-cell transcriptomics data of human fetal gonads to map the molecular interactions during the sex-specific transition from PGCs to gonia. The CellPhoneDB algorithm was used to identify significant ligand-receptor interactions between germ cells and their sex-specific neighboring gonadal somatic cells, focusing on four major signaling pathways WNT, NOTCH, TGFß/BMP, and receptor tyrosine kinases (RTK). Subsequently, the expression and intracellular localization of key effectors for these pathways were validated in human fetal gonads by immunostaining. This approach provided a systematic analysis of the signaling environment in developing human gonads and revealed sex-specific signaling pathways during human premeiotic germ cell development. This work serves as a foundation to understand the transition from PGCs to premeiotic oogonia or prospermatogonia and identifies sex-specific signaling pathways that are of interest in the step-by-step reconstitution of human gametogenesis in vitro.

In vitro culture and characterization of duck primordial germ cells.

This study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

On the Origin of Testicular Germ Cell Tumors: From Gonocytes to Testicular Cancer.

Human primordial germ cells (PGCs) have been described in the yolk sac wall around the beginning of the third week. From week 4 to 5, they migrate under control of SCF/c-KIT signaling pathway to the genital ridge, where they become gonocytes. PGCs and gonocytes express classic pluripotency markers, such as KIT, NANOG, and OCT3/4 that, during spermatogonia differentiation, are gradually suppressed, and substituted by the expression of some germ cell specific genes, such as VASA, SOX17, and TSPY. These genes, during normal development of germ cells, are tightly regulated by epigenetic modification, in terms of microRNA expression and DNA methylation. In adolescents and young adults, testicular germ cell tumors (TGCT) have a common precursor, the germ cell neoplasia in situ (GCNIS); the hypothesis of their origin from PGCs or gonocytes, whose maturation is altered, is widely accepted. The origin of TGCT, probably starting at early stages of embryogenesis, seems to be a part of the Testicular Dysgenesis Syndrome (TDS) where some early PGC/gonocytes, for still unclear reasons, are blocked in their differentiation, retaining their early marker profile. In this paper, current knowledge on the combination of epidemiological and genomic factors, involved in the development of testicular germ cell tumors, is reviewed.

Primordial Germ Cell Isolation from Xenopus laevis Embryos.

Primordial germ cells (PGCs) are the precursors to the gametes and have the unique ability to retain full developmental potential. However, the mechanism(s) and gene-network(s) necessary for their proper specification and development are poorly understood. This is due, in part, to the challenges that must be overcome in order to identify and isolate PGCs during critical stages of development. Two distinct mechanisms have been characterized to specify the germ cell lineage in vertebrates: induction and inheritance. Regardless of mechanism, there are common developmental features shared among all vertebrates in forming the germ cell lineage. Xenopus offers several advantages for understanding the molecular mechanisms necessary to establish the germ line. Here, we provide detailed methods for isolating live PGCs at different time points: 1) just after they have segregated from the endodermal lineage, and 2) while they are migrating towards the presumptive gonad. Isolation of PGCs at these critical developmental stages will allow for the investigation of the mechanism(s) and gene-network(s) necessary for their proper specification and development.