Maternally inherited intron coordinates primordial germ cell homeostasis during Drosophila embryogenesis

Primordial germ cells (PGCs) give rise to the germline stem cells (GSCs) in the adult Drosophila gonads. Both PGCs and GSCs need to be tightly regulated to safeguard the survival of the entire species. During larval development, a non-cell autonomous homeostatic mechanism is in place to maintain PGC number in the gonads. Whether such germline homeostasis occurs during early embryogenesis before PGCs reach the gonads remains unclear. We have previously shown that the maternally deposited sisRNA sisR-2 can influence GSC number in the female progeny. Here we uncover the presence of a homeostatic mechanism regulating PGCs during embryogenesis. sisR-2 represses PGC number by promoting PGC death. Surprisingly, increasing maternal sisR-2 leads to an increase in PGC death, but no drop in PGC number was observed. This is due to ectopic division of PGCs via the de-repression of Cyclin B, which is governed by a genetic pathway involving sisR-2, bantam and brat. We propose a cell autonomous model whereby germline homeostasis is achieved by preserving PGC number during embryogenesis.Subject terms: Development, Gene regulation     

CHCHD2 inhibits apoptosis by interacting with Bcl-x L to regulate Bax activation

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.Apoptosis is a tightly regulated form of programmed cell death that is critical for proper embryonic development, tissue homeostasis and immune response. Aberrant regulation of apoptosis contributes to a wide range of ailments including autoimmune disorders, neurodegenerative diseases and cancer. Unlike necrotic cell death, apoptosis is a genetic program that is characterized by distinct morphological features such as membrane blebbing, chromatin condensation, DNA fragmentation and cell shrinkage.¹ In vertebrates, apoptosis can occur through two pathways: extrinsic, or receptor-mediated apoptosis, and intrinsic, or mitochondria-mediated apoptosis. Intrinsic apoptosis is induced by cellular stressors such as DNA damage, which lead to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release from the mitochondrial intermembrane space, activation of cysteine proteases (caspases) and induction of apoptosis. Once MOMP occurs, cell death is thought to be inevitable. Therefore, much research has been devoted to elucidating the mechanisms and signaling pathways that govern this critical regulatory point in apoptosis.MOMP is controlled largely by the B-cell lymphoma 2 (Bcl-2) family of proteins,² all of which contain at least one of four BH (Bcl-2 homology) domains designated BH1–4. During apoptosis, the pro-apoptotic Bcl-2 proteins Bax and/or Bak become activated and oligomerize on the mitochondrial outer membrane³ increasing mitochondrial membrane permeabilization through a mechanism that is not entirely clear. Bax and Bak are activated by BH3-only Bcl-2 family proteins such as Bim, t-Bid and Puma.^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13} Conversely, Bax and Bak are inhibited by pro-survival Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL.^{2, 14, 15, 16} Of the pro-survival Bcl-2 family proteins, Bcl-2 is found at the outer mitochondrial membrane, whereas Bcl-xL and Mcl-1 localize to the outer mitochondrial membrane and the mitochondrial matrix.^{17, 18} Matrix-localized Bcl-xL and Mcl-1 have been shown to promote mitochondrial respiration,¹⁹ suggesting that crosstalk exists between apoptotic pathways and other mitochondria-based biological events. Based on this recent discovery, one might reason that other mitochondrial proteins previously characterized as structural proteins or metabolism-associated enzymes could play an additional intermediate role in the regulation of apoptosis by interacting with Bcl-2 family proteins.We identified CHCHD2 in a mass spectrometry-based screen for binding partners of p32, a mitochondrial protein previously shown by our lab to bind and mediate the apoptotic effects of the tumor suppressor p14ARF.²⁰ CHCHD2 was subsequently detected in independent screens for proteins that regulate cellular metabolism and migration;^{21, 22} however, the functions of CHCHD2 remain unknown. CHCHD2 is encoded by the chchd2 gene (coiled-coil helix coiled-coil helix domain-containing 2), which spans 4921 base pairs, contains 4 exons, and is located on human chromosome 7p11.2, a chromosomal region that is often amplified in glioblastomas.²³ The protein encoded by the chchd2 gene is ubiquitously expressed²⁴ and is relatively small, as it codes for only 151 amino acids. CHCHD2 is well-conserved among different species from humans to yeast, and mouse and human CHCHD2 share 87% amino acid sequence identity (Supplementary Figures S1A and S1B). CHCHD2 contains a C-terminal CHCH (coiled-coil helix coiled-coil helix) domain, which is characterized primarily by four cysteine residues spaced 10 amino acids apart from one another (CX(9)C motif).²⁵ The function of the CHCH domain is not well understood, and the few characterized proteins that harbor this domain have diverse functions. Many CHCH domain-containing proteins localize to the mitochondrial inner membrane or the intermembrane space, including Cox12, Cox17, Cox19, Cox23, Mia40 (yeast homolog of human CHCHD4), CHCHD3 and CHCHD6. Cox17 and Cox19 aid in the assembly of the COX complex,^{26, 27} whereas Mia40/Tim40 has been shown to transport proteins into the mitochondrial intermembrane space.^{28, 29} Furthermore, CHCHD3 and CHCHD6 are essential for maintaining the integrity of mitochondrial cristae and thus mitochondrial function.^{30, 31, 32} Interestingly, a recent report has shown that CHCHD6 is regulated by DNA damage stress, and alterations in CHCHD6 expression affect the viability of breast cancer cells in response to genotoxic anticancer drugs.³²Despite advances in our understanding of how MOMP and apoptosis are regulated by the Bcl-2 family of proteins, much remains unknown with respect to the mechanisms that lead to Bax activation and oligomerization particularly concerning the roles that mitochondria-associated proteins play in the process. In this study, we characterize the small, mitochondria-localized protein CHCHD2 as a novel regulator of Bax oligomerization and apoptosis. Furthermore, we show evidence that CHCHD2 binds to Bcl-xL at the mitochondria under unstressed conditions. In response to apoptotic stimuli, CHCHD2 decreases and loses its mitochondria localization, which is accompanied by decreased Bcl-xL–Bax interaction and increased Bax homo-oligomerization and Bax–Bak hetero-oligomerization. Collectively, our results suggest that CHCHD2 negatively regulates the apoptotic cascade upstream of Bax oligomerization.     

Oct4 is required for primordial germ cell survival

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.     

Mouse germ cell development during embryogenesis

The discrimination and differentiation of germ cells from somatic cells is a fundamental issue during development. The early specification of mouse primordial germ cells (PGCs) is achieved by the induction of Blimp1, a key regulator of germ cells. Nanos3 is one of the genes activated in early PGCs and prevents apoptosis during their migration stage. Once PGCs enter the embryonic gonads, they differentiate according to the somatic sex of the organism. During this process, Nanos2 plays an important role as it promotes male germ cell pathway by suppressing the female fate. In this review, the process of germ cell development in the mouse is discussed with a particular focus on the functions of the key proteins, Blimp1, Nanos, and Dead end1.     

Regulation of primordial germ cell development in the mouse

Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.     

Growth factors in mouse primordial germ cell migration and proliferation

Information obtained mainly from in vitro culture studies and genetic analysis of mouse mutants White spotting and Steel indicate a pivotal role of growth factors in the development of mouse primordial germ cells (PGCs). While stem cell factor (SCF) and TGFβ1 seem to have a role in PGC migration (as an adhesion factor and a chemoattractant, respectively), the former is certainly required for PGC survival in vitro and probably in vivo as well. Recent findings suggest that the mechanism by which SCF supports PGC survival is by preventing PGC apoptosis. A similar action appears to be exerted by leukemia inhibitory factor (LIF), a further growth factor influencing PGC growth in culture.PGC proliferation seems to be mainly induced by cAMP dependent mechanisms, but futther investigations are needed to clarify the interrelationships among the different molecular pathways activated by SCF, LIF, cAMP and other putative PGC growth factors (i.e. bFGF). Stimulation of long-term proliferation of PGCs, leading to derivation of ES-like cells (embryonal germ cells) obtained by using a combination of growth factors (bFGF, SCF and LIF), opens new intriguing perspectives for such studies and transgenic technology.     

The mouse gcd2 mutation causes primordial germ cell depletion

Germ cell depletion 2 (gcd2) is a chemically induced recessive mutation that causes infertility in male and female mice. The infertility is caused by germ cell depletion as early as 11.5 days post-coitum, when primordial germ cells have completed their migration to the embryonic gonads. Thus, the gcd2 mutation affects the proliferation and/or survival of germ cells after they arrive in the embryonic gonad, a developmental time when little is known about the requirements for germ cell proliferation and survival. The sterility phenotype is incompletely penetrant, has variable expressivity, and is modulated by strain background. The penetrance ranges from 37% in strain C57BL/6J to nearly 100% in CAST/EiJ. Genetic mapping localized gcd2 to a approximately 1Mb region on Chr 2. This interval contains a small number of annotated genes, of which none are known to have a role in germ cell development. Sequencing the coding regions of these genes failed to reveal a mutation, and BACs containing two of the candidate genes failed to rescue the phenotype. This raises the possibilities that the gcd2 mutation resides in non-coding sequences, and regulates genes outside the genetically defined critical region.     

A role for E-cadherin in mouse primordial germ cell development

In this study we show that mouse primordial germ cells and fetal germ cells at certain stages of differentiation express E-cadherin and alpha and beta catenins. Moreover, we demonstrate that the formation of germ cell aggregates that rapidly occurs when monodispersed germ cell populations are released from embryonic gonads in culture is E-cadherin mediated, developmentally regulated, and dependent on the sex of the germ cells. Immunoblotting analyses indicate that the lower ability to form aggregates of primordial germ cells in comparison to fetal germ cells is not due to gross changes in E-cadherin expression, altered association with beta catenin, or changes in beta catenin phosphorylation. Investigating possible functions of E-cadherin-mediated adhesion in primordial germ cell development, we found that E-cadherin-mediated adhesion may stimulate the motility of primordial germ cells. Moreover, treatment of primordial germ cells cultured on STO cell monolayers with an anti-E-cadherin antibody caused a significant decrease in their number and markedly reduced their ability to form colonies in vitro. The same in vitro treatment of explanted undifferentiated gonadal ridges cultured for 4 days results in decreased numbers and altered localization of the germ cell inside the gonads. Taken together these results suggest that E-cadherin plays an important role in primordial germ cell migration and homing and may act as a modulator of primordial germ cell development.     

From primordial germ cell to primordial follicle: mammalian female germ cell development

In mammals, the final number of oocytes available for reproduction of the next generation is defined at birth. Establishment of this oocyte pool is essential for fertility. Mammalian primordial germ cells form and migrate to the gonad during embryonic development. After arriving at the gonad, the germ cells are called oogonia and develop in clusters of cells called germ line cysts or oocyte nests. Subsequently, the oogonia enter meiosis and become oocytes. The oocyte nests break apart into individual cells and become packaged into primordial follicles. During this time, only a subset of oocytes ultimately survive and the remaining immature eggs die by programmed cell death. This phase of oocyte differentiation is poorly understood but molecules and mechanisms that regulate oocyte development are beginning to be identified. This review focuses on these early stages of female germ cell development.     

Smad5 is required for mouse primordial germ cell development

Smad5, together with Smad1 and Smad8, have been implicated as downstream signal mediators for several bone morphogenetic proteins (BMPs). Recent studies have shown that primordial germ cells (PGCs) are absent or greatly reduced in Bmp4 or Bmp8b mutant mice. To define the role of Smad5 in PGC development, we examined PGC number in Smad5 mutant mice by Oct4 whole-mount in situ hybridization and alkaline phosphatase staining. We found ectopic PGC-like cells in the amnion of some Smad5 mutant mice, however, the total number of PGCs was greatly reduced or completely absent in Smad5 mutant embryos, similar to Bmp4 or Bmp8b mutant embryos. Therefore, Smad5 is an important factor involved in PGC generation and localization.     

Isolation of mouse primordial germ cells

Primordial germ cells (PGCs) were obtained from fetal mouse gonads of both sexes days post coitum (dpc), either by collagenase treatment, or by mechanical procedures with or without prior EDTA treatment. With mechanical procedures alone, yield was relatively low and many of the cells released were dead. After EDTA treatment, both yield and viability were significantly improved. Collagenase treatment gave the best yield of cells, since the entire gonad was disaggregated, but contamination with somatic cells was substantial, and the adhesive properties of the germ cells were altered by the treatment. When cells released following EDTA treatment were fractionated on a simple Percoll gradient, several thousand viable PGCs per gonad could be obtained in 2–3 h, with not more than 10–20% somatic cell contamination.     

The role of cadherins during primordial germ cell migration and early gonad formation in the mouse

Primordial germ cells (PGCs) are the founder cells of the gametes. In mammals, PGCs migrate from the hindgut to the genital ridges, where they coalesce with each other and with somatic cells to form the primary sex cords. We show here that, in both sexes, PGCs express P- and E-cadherins during and after migration, and N-cadherin at post-migratory stages. E-Cadherin is not expressed by PGCs whilst in the hindgut, but is upregulated as they leave. Blocking antibodies against E-, but not P-cadherin cause defective PGC-PGC coalescence, and in some cases, ectopic PGCs.     

The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo

In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. We show that the pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, we show that Bax is downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages.     

Myogenic potential of mouse primordial germ cells

Primordial germ cells are the only stem cells that retain true developmental totipotency after gastrulation, express markers typical of totipotent/pluripotent status and are able both in vivo and in vitro to give rise to pluripotent stem cells as EC and EG cells. We have therefore explored the possibility of the trans-differentiation of mouse PGCs to a myogenic lineage by transplanting them directly or after in vitro culture into a regenerating muscle and by culturing them on monolayers of differentianting muscle cells. The results obtained suggest that mouse PGCs may trans-differentiate into myogenic cells, provided that their somatic environment is preserved. This occurs at an estimated frequency of 0.01%, which is no higher than that reported for stem cells of adult tissues.     

Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.     

Developmental potential of mouse primordial germ cells

There are distinctive and characteristic genomic modifications in primordial germ cells that distinguish the germ cell lineage from somatic cells. These modifications include, genome-wide demethylation, erasure of allele-specific methylation associated with imprinted genes, and the re-activation of the X chromosome. The allele-specific differential methylation is involved in regulating the monoallelic expression, and thus the gene dosage, of imprinted genes, which underlies functional differences between parental genomes. However, when the imprints are erased in the germ line, the parental genomes acquire an equivalent epigenetic and functional state. Therefore, one of the reasons why primordial germ cells are unique is because this is the only time in mammals when the distinction between parental genomes ceases to exist. To test how the potentially imprint-free primordial germ cell nuclei affect embryonic development, we transplanted them into enucleated oocytes. Here we show that the reconstituted oocyte developed to day 9.5 of gestation, consistently as a small embryo and a characteristic abnormal placenta. The embryo proper also did not progress much further even when the inner cell mass was 'rescued' from the abnormal placenta by transfer into a tetraploid host blastocyst. We found that development of the experimental conceptus was affected, at least in part, by a lack of gametic imprints, as judged by DNA methylation and expression analysis of several imprinted genes. The evidence suggests that gametic imprints are essential for normal development, and that they can neither be initiated nor erased in mature oocytes; these properties are unique to the developing germ line.