In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge.
During embryonic development, stem cell-like primordial germ cells travel across the developing embryo to the genital ridge, which gives rise to the gonad. Around the time of their arrival, the primordial germ cells gain the capacity to undertake sexual specialization and meiosis—a process called germ cell licensing. Based on the observation that meiosis and sexual differentiation can occur when primordial germ cells stray into the area of the adrenal gland, the primordial germ cell has been thought to be responsible for its own licensing. We tested this notion by examining the licensing process in mutant mouse embryos that did not form a genital ridge. We discovered that in the absence of the genital ridge, primordial germ cells migrate across the developing embryo properly, but instead of undergoing licensing, these cells retain their primordial germ cell characteristics. We conclude that licensing of embryonic primordial germ cells for gametogenesis is dependent on signaling from the genital ridge.
Citation: Hu Y-C, Nicholls PK, Soh YQS, Daniele JR, Junker JP, van Oudenaarden A, et al. (2015) Licensing of Primordial Germ Cells for Gametogenesis Depends on Genital Ridge Signaling. PLoS Genet 11(3): e1005019. https://doi.org/10.1371/journal.pgen.1005019
Editor: R. Scott Hawley, Stowers Institute for Medical Research, UNITED STATES
Received: August 26, 2014; Accepted: January 22, 2015; Published: March 4, 2015
Copyright: © 2015 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Howard Hughes Medical Institute, where DCP is an Investigator. PKN holds an Australian National Health and Medical Research Council CJ Martin Fellowship (#1053776). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In mammals, both the testis and ovary derive from a common precursor structure, the bipotential gonad . The development of the bipotential gonad involves two simultaneously occurring processes. The coelomic epithelium on the ventromedial surface of the mesonephros transforms from a monolayer into a thickened, multilayer epithelial structure, the genital ridge. Meanwhile, primordial germ cells (PGCs) that have migrated from the base of the allantois start arriving at the genital ridge, as early as the monolayer stage, and multiply as the genital ridge thickens. The formation of the bipotential gonad in mouse embryos begins at embryonic (E) day 10.0 and continues until E11.5-E12.0, when sexual differentiation takes place [2–4].
Migratory PGCs maintain a genomic program associated with pluripotency [5,6]. They express core pluripotency genes (Oct4, Nanog, and Sox2) and are able to form teratomas following their injection into postnatal mouse testes . Around the time of their arrival at the genital ridge, PGCs undergo a global change in gene expression [8–10]. Specifically, the PGCs turn on a set of genes that enable them to undergo sexual differentiation and gametogenesis, and to switch off their pluripotency program. Following this transition, germ cells are referred to as gametogenesis-competent cells (GCCs), and are poised to initiate meiosis as well as male or female differentiation [11–13]. Upon the development of the genital ridge into either a testis or an ovary (at ~E12.5 in mouse embryos), GCCs respond to cues from the somatic environment and enter either the spermatogenic or oogenic pathway accordingly. The transition from PGC to GCC is referred to as germ cell licensing , and it represents a critical transformation of germ cells to a sexually competent state.
One of the genes upregulated in germ cells at the time of licensing is Dazl, which encodes an evolutionarily conserved and germ-cell-specific RNA-binding protein . In mouse embryos of C57BL/6 genetic background, germ cell licensing is dependent on Dazl [11,15]. In Dazl-null embryos, germ cells retain characteristics of PGCs and fail to embark upon the pathways to oogenesis or spermatogenesis in the fetal ovary or testis, respectively. However, what triggers Dazl expression and germ cell licensing remains unknown.
One hypothesis, based on observational studies, states that licensing is triggered in a cell-autonomous and gonad-independent manner. As PGCs migrate to the genital ridge, a fraction of them are left in places along the migratory path, such as in the allantois, tail, midline, spinal cord, and adrenal gland [16,17]. While most of these ectopic PGCs die, those migrating to the adrenal gland survive until ~3 weeks after birth [16,18–20]. Upadhyay and Zamboni  observed that these adrenal germ cells, regardless of the sex of the fetus, enter meiosis according to the schedule of normal ovarian germ cell development. Based on these findings, the authors hypothesized that PGCs transition into meiotic germ cells (oocytes) in a gonad-independent, and therefore cell-autonomous, manner. This hypothesis was further supported by several in vitro studies [13,21–23], showing, for instance, that PGCs isolated from E10.5 mouse embryos of both sexes continue to develop in vitro and initiate meiosis at approximately the same time as meiotic entry occurs in vivo [13,22,23].
Previous studies from our lab and others led us to question this hypothesis and suggest an alternative: PGCs undergo germ cell licensing in response to external signals, upon migration to the genital ridge. The authors who proposed the cell-autonomous hypothesis considered E10.5 PGCs to be pre-gonadal germ cells [22,23]. However, we recently showed that the marker of genital ridge formation, GATA4, is expressed as early as E10.0 . It is plausible that the E10.5 PGCs used in the in vitro studies had already been exposed to gonadal factors. In addition, the claim that the PGCs in the adrenal gland transition to meiotic germ cells without exposure to the genital ridge belies the fact that the adrenal anlagen and genital ridge derive from a common precursor, called the adrenogonadal primordium. These two organs are not segregated completely until ~E11.5 [24,25]. Adrenal PGCs would therefore be exposed to the genital ridge, or its equivalent, during a short interval in their development. These findings raise doubts about whether the transition of PGCs to meiosis-competent cells is gonad-independent, or induced by factors shared by the developing gonad and adrenal gland.
Germ cell licensing precedes meiotic entry [11,15]. Since the occurrence of licensing coincides with the arrival of PGCs at the genital ridge, we suspected that the genital ridge provides extrinsic signals required for inducing germ cell licensing. Initiation of genital ridge formation depends on the transcription factor GATA4, which is expressed in the somatic compartment, but not in germ cells . We therefore utilized Gata4 conditional knockout (cKO) embryos, which lack the genital ridge, to test the hypothesis of genital ridge-dependent licensing. If true, we would expect that in the absence of the genital ridge, PGCs would fail to undergo licensing and subsequent meiotic entry. The result of this study would provide fundamental insight into how germ cells switch off their pluripotency program and acquire competence for meiosis and sexual differentiation.
Anterior-to-posterior expression of the germ cell licensing marker Dazl
The genital ridge develops in an anterior-to-posterior (A-P) direction starting at E10.0 [1,2], as PGCs are entering the region. Dazl is expressed in germ cells during licensing for gametogenesis [8,9,11,14,15]. If the genital ridge regulates germ cell licensing, we would expect to find a similar A-P induction of licensing, along with Dazl expression. To test this prediction, we quantified Dazl transcript levels in individual germ cells along the A-P axis of the genital ridge using single-molecule fluorescence in situ hybridization (smFISH) . We first confirmed that Dazl expression was below the detectable level in migratory PGCs at E9.5, as expected (S1A Fig). When examining