Primordial Germ Cell - an overview (2023)

Abstract

Primordial germ cells (PGCs) must complete a complex and dynamic developmental program during embryogenesis to establish the germline. This process is highly conserved and involves a diverse array of tasks required of PGCs, including migration, survival, sex differentiation, and extensive epigenetic reprogramming. A common theme across many organisms is that PGC success is heterogeneous: only a portion of all PGCs complete all these steps while many other PGCs are eliminated from further germline contribution. The differences that distinguish successful PGCs as a population are not well understood. Here, we examine variation that exists in PGCs as they navigate the many stages of this developmental journey. We explore potential sources of PGC heterogeneity and their potential implications in affecting germ cell behaviors. Lastly, we discuss the potential for PGC development to function as a multistage selection process that assesses heterogeneity in PGCs to refine germline quality.

XIST EXPRESSION

In PGCs, Xist may code for a stable transcript (migrating XX PGCs) or expression may be entirely absent (XY PGCs; XX PGCs after X chromosome reactivation in the genital ridge). In either case, undifferentiated EGC lines derived from the PGCs are characterized by an unstable Xist transcript, visualized by RNA FISH as a small dot overlying the locus. A similar situation is seen in ES cells. Once the EGCs or ES cells start to differentiate, the unstable transcript disappears and is replaced by a stable transcript or absence of expression, as appropriate.

30.1.1 Primordial Germ Cells

PGCs are the sole means of genetic transmission between parent and offspring, as they generate eggs and sperm. In many species, such as C. elegans, germ cells are segregated very early in development, during the first embryonic cleavages, and are marked by deposition of ribonucleoprotein P-granules. In mammals, the process occurs later in development, and seems to be directed more by extrinsic factors than by preprogrammed intrinsic differences. For example, in mice, cells that generate PGCs are located close to extra-embryonic ectoderm during gastrulation. Rather than having a previously determined fate, cells in this location receive external signals to further differentiate into PGCs, as demonstrated by the observation that transplantation of cells from other parts of the epiblast to this region can take on a PGC fate. Several components of this signaling process have been identified. Initially, bone morphogenetic protein 4 (BMP4) and BMP8b are produced by extraembryonic ectoderm program cells from the epiblast to become extraembryonic mesoderm precursors or PGCs. Cells destined to become PGCs express higher levels of membrane protein fragilis than nuclear protein stella.

In the mouse, PGCs are visible as alkaline phosphatase (AP) positive cells at the base of the allantois at 7.5 to 8.0 days postcoitus (dpc). They begin to associate with the endoderm that is invaginating to form the hindgut at 8.5dpc. By 10.5dpc, PGCs are associated with dorsal mesenteries and are translocated to the genital ridges. The migration of PGCs is caused by both cellular migration and association with moving tissues. Throughout this migration, PGCs expand from approximately 130 cells at 8.5dpc to more than 25,000 at 13.5dpc. Once they arrive at the genital ridge, PGCs continue proliferating until they enter prophase of the first meiotic division. In males, entry into meiosis is inhibited by signals from the developing testis, blocking PGCs at G0 until after birth. In the absence of inhibitory signals, female PGCs undergo oogenesis. Although not as thoroughly studied, much is known regarding the migratory path of human PGCs, including their association with gut endoderm and migration into developing genital ridges.

PGCs do not survive well under standard tissue culture conditions, and are not pluripotent stem cells in vivo or in vitro. Early attempts to use various growth factors and feeder layers succeeded in prolonging their survival, but proliferation was limited. The combination of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and c-kit ligand (KL, also known as stem cell factor, mast cell factor, or steel factor) proved to result in an immortal cell population, especially if the KL was presented in the transmembrane form by a layer of ‘feeder’ cells (see the section ‘Feeder Layer’). Instead of simply encouraging PGC proliferation, these factors cause the normally solitary PGCs to congregate and proliferate as multicellular colonies, known as EG cells, and to gain pluripotency. Mouse EG cell lines have been derived from PGCs prior to migration around 8.0–8.5dpc, during migration at 9.5dpc, and after entry into the genital ridges between 11.5 and 12.5dpc.

The roles played by KL and the tyrosine kinase receptor for KL, c-Kit, in the in vitro derivation of EG cells from PGCs have parallels in vivo. c-Kit is expressed in PGCs, and KL is expressed along the PGC migratory pathway and in the genital ridges. The roles of KL and c-Kit in PGC survival were originally characterized through several mutations at their respective loci, Sl and W, which resulted in subfertile or sterile mice. PGCs are formed in homozygous mutant embryos of W and Sl, but mitosis is severely impaired, and the few PGCs that reach the gonad do not survive. KL is produced as a membrane-bound growth factor that can undergo proteolytic cleavage to generate a soluble form. Mice lacking the membrane-bound KL, but not the soluble form, maintain low PGC numbers and are sterile, suggesting that the membrane-bound form but not the soluble form is essential for PGC survival. The mechanism involved in KL-induced PGC survival has been shown to involve suppression of apoptosis. The c-kit receptor has also been shown to be involved in the adhesion of mouse PGCs to somatic cells in vitro. Other recent studies attempting to identify signaling pathways activated by KL binding to its receptor in mouse PGCs have shown activation of AKT kinase and telomerase.

In contrast to the embryologically early and relatively undifferentiated epiblast, PGCs arise late and have a specialized role during normal development. In this regard, it is somewhat surprising that exposure to three cytokines can convert PGCs into pluripotent stem cells in vitro. It is possible that the flexibility provided by extrinsic signaling during PGC specification, rather than intrinsic preprogramming, allows for this conversion.

Primordial germ cells

Primordial germ cells (PGCs), the ancestors of the gametes, originate in the mouse at least as early as on day 7 of embryonic development (for a review see [183]). They arise from a population of pluripotent somatic cells in the proximal epiblast near the extraembryonic ectoderm [184]. Bmp signals from this tissue have been shown to select PGCs from their somatic neighbours dose-dependently via the Smad pathway [185, 186]. PGCs actively suppress differentiation programmes of somatic cells and acquire pluripotency, functions that are mediated downstream of Bmp signalling by the two zinc finger transcriptional repressors Blimp1 and Prdm14 [187, 188]. PGCs pass through the posterior primitive streak and are found first in the posterior part of the embryo at the base of the allantois [189, 190]. They are large, round cells which contain a high level of alkaline phosphatase activity [191]. More recently, a pluripotency marker, the POU transcription factor Oct4, was found as a PGC marker [192]. A truncated Oct4 promotor was used to drive expression of green fluorescent protein in transgenic animals. Thus, migration of PGCs was visualized in a living embryo [193]. From day 8.5 onwards PGCs migrate through the hindgut and mesentery wall and colonize the genital ridges. The genital ridges, which give rise to the gonads, are a paired mesodermal tissue that lies beneath the dorsal mesentery of the body. By day 12.5 of development PGCs are largely confined to the developing gonads. Invitro studies suggest that colonization of the genital ridges is brought about by active movement of the PGCs, and that PGCs lose their invasive motility after entering the gonad anlagen [194]. Directed migration of PGC is regulated by cues from their somatic environment, including chemotactic signals presumably from the gonad, as well as gradients formed by proteins in the extracellular matrix. Again depending on environmental cues, PGCs proliferate during their migration, and the population of about 10–100 PGCs present around day 7–8p.c. in the extraembryonic mesoderm increases to more than 20000 in the colonized genital ridges around day 14p.c. [195]. Once within the genital ridges massive epigenetic changes occur in PGCs including random X-chromosome inactivation in female PGCs.

At 12dp.c. differences between male and female genital ridges become apparent and male and female germ cells embark on their specific developmental programmes. This is not cell-autonomous but relies on the somatic environment of the PGCs. Male PGCs enter mitotic arrest around day 13p.c. and continue development only after birth. In contrast, female mouse PGCs enter meiosis from day 13 of development onwards and by about 3–5days after birth all germ cells have undergone oogonial development and are in the diplotene stage of meiosis (for recent reviews see [183, 196]).

Abstract

Primordial germ cells (PGCs) are the embryonic precursors of the gametes and represent the founder cells of the germline. Specification of PGCs is a critical divergent point during embryogenesis. Whereas the somatic lineages will ultimately perish, cells of the germline have the potential to form a new individual and hence progress to the next generation. It is therefore critical that the genome emerges intact and carrying the appropriate epigenetic information during its passage through the germline. To ensure this fidelity of transmission, PGC development encompasses extensive epigenetic reprogramming. The low cell numbers and relative inaccessibility of PGCs present a challenge to those seeking mechanistic understanding of the crucial developmental and epigenetic processes in this most fascinating of lineages. Here, we present an overview of PGC development in the mouse and compare this with the limited information available for other mammalian species. We believe that a comparative approach will be increasingly important to uncover the extent to which mechanisms are conserved and reveal the critical steps during PGC development in humans.

Germ Cell Differentiation

PGCs, once they have reached the developing genital ridges, stop migrating, but keep proliferating. They start their sexual dimorphic differentiation at around 13.5–14.5dpc in mouse and at week 11–12 in human development, when PGCs in an ovary enter meiosis, whereas PGCs in a testis arrest in mitosis. This bifurcation in the differentiation pathway between males and females is solely dependent on the environment, that is, ovary or testes, PGCs are in. It has been shown that retinoic acid (RA) produced by the mesonephros is able to induce entry into meiosis. In a testis, Sertoli cells, which surround PGCs within testis cords, produce the RA-degrading enzyme CYP26B1 (cytochrome P450, family 26, subfamily b, polypeptide 1) (Bowles et al., 2006; Koubova et al., 2006; Li et al., 2009; MacLean et al., 2007). Therefore, even though RA is also produced by the mesonephros in males, PGCs do not enter meiosis until after birth.

Primordial germ cells

Primordial germ cells (PGCs), the ancestors of the gametes, originate in the mouse at least as early as on day 7 of development (for review see Wylie and Anderson, 2002). They arise from a population of pluripotent cells in the proximal epiblast close to the extraembryonic ectoderm (Lawson and Hage, 1994). They pass through the posterior primitive streak and are found first in the posterior part of the embryo at the base of the allantois (Copp et al., 1986; Ginsburg et al., 1990). They are large, round cells, which contain a high level of alkaline phosphatase activity. This enzymatic activity can be used to trace PGCs in the early embryo (Chiquoine, 1954). More recently, the POU transcription factor OCT4 was found as PGC marker (Schöler et al., 1990). A truncated Oct4 promotor was used to drive expression of green fluorescent protein in transgenic animals. Thus, migration of PGCs was visualized in a living embryo (Anderson et al., 1999, 2000). From day 8.5 onwards PGCs migrate through the hindgut and mesentery wall and colonize the genital ridges. The genital ridges, which give rise to the gonads, are a paired mesodermal tissue which lies beneath the dorsal mesentery of the body. By day 12.5 PGCs are largely confined to the developing gonads. In vitro studies suggest that colonization of the genital ridges is brought about by active, invasive movement of the PGCs and that PGCs lose their invasive motility after entering the gonad anlagen (Donovan et al., 1986). During their migration the PGCs proliferate and the population of about 10–100 PGCs present around day 7–8 increases to more than 20,000 in the colonized genital ridges around day 14 of development (Tam and Snow, 1981; Wylie et al., 1985). Genital ridges seem to release intrinsic factors which stimulate the proliferation of PGCs and which act as chemoattractants for PGCs in vitro (Godin et al., 1990).

At day 12.5 differences between male and female genital ridges become apparent and male and female germ cells embark on their specific developmental programmes. Male PGCs enter mitotic arrest around day 13 p. c. and continue development only after birth. In contrast, female mouse PGCs enter meiosis from day 13 of development onwards and already about 3–5 days after birth all germ cells have undergone oogonial development and are in the diplotene stage of meiosis (for review see Swain and Lovell-Badge, 2002; Wylie and Anderson, 2002).

Primordial germ cells

Primordial germ cells are single cells that under certain culture conditions can form colonies of cells which morphologically resemble undifferentiated embryonic stem cells (ES cells) (Resnick et al., 1992). These cells can be maintained on feeder layers for extended periods of time and can give rise to embryoid bodies and to multiple differentiated cell phenotypes in monolayer culture and in tumours in nude mice. PGC-derived ES cells can also contribute to chimeras when injected into host blastocysts (Resnick et al., 1992). Clearly, PGCs are stem cells, still having the capacity to renew themselves and to differentiate in various directions.

Abstract

Primordial germ cells (PGCs) are embryonic precursors of sperm and egg that pass on genetic and epigenetic information from one generation to the next. In mammals, they are induced from a subset of cells in peri-implantation epiblast by BMP signaling from the surrounding tissues. PGCs then initiate a unique developmental program that involves comprehensive epigenetic resetting and repression of somatic genes. This is orchestrated by a set of signaling molecules and transcription factors that promote germ cell identity. Here we review significant findings on mammalian PGC biology, in particular, the genetic basis for PGC specification in mice and human, which has revealed an evolutionary divergence between the two species. We discuss the importance and potential basis for these differences and focus on several examples to illustrate the conserved and divergent roles of critical transcription factors in mouse and human germline.