Primordial Germ Cell Determination Research Articles

Nutritional and metabolic control of germ cell fate through O-GlcNAc regulation.

Fate determination of primordial germ cells (PGCs) is regulated in a multi-layered manner, involving signaling pathways, epigenetic mechanisms, and transcriptional control. Chemical modification of macromolecules, including epigenetics, is expected to be closely related with metabolic mechanisms but the detailed molecular machinery linking these two layers remains poorly understood. Here, we show that the hexosamine biosynthetic pathway controls PGC fate determination via O-linked β-N-acetylglucosamine (O-GlcNAc) modification. Consistent with this model, reduction of carbohydrate metabolism via a maternal ketogenic diet that decreases O-GlcNAcylation levels causes repression of PGC formation in vivo. Moreover, maternal ketogenic diet intake until mid-gestation affects the number of ovarian germ cells in newborn pups. Taken together, we show that nutritional and metabolic mechanisms play a previously unappreciated role in PGC fate determination.

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Linking a Biological Mechanism to Evolvability

Evolvability has become a concept used to explain the common observation of clade asymmetry. However, the concept of evolvability means different things to different workers. Recent work has formalized the concept and we apply the formalized concept to a developmental system (primordial germ cell determination mechanism) that has been proposed to explain clade size disparity. In a simplified view, there are two general primordial germ cell (pgc) determination mechanisms: determinative and induced. The determinative mechanism is associated with species rich clades and the induced mechanism with species poor clades. The formal equations of evolvability provide a theoretical framework under which we can assess the relative influence of pgc determination mechanisms on clade evolvability. We propose that the determinative mechanism has enhanced evolvability in most clades that possess the trait.

Diversification and Germ-Line Determination Revisited: Linking Developmental Mechanism with Species Richness

Abstract.– Background: Explanations for asymmetric patterns of diversification continue to challenge paleontologists and neontologists with competing hypotheses within genetic-development and ecological frameworks. In 1988, a hypothesis was proposed that tied a primordial germ cell (PGC) determination mechanism to clade (phyla) diversification. Two general mechanisms for PGC determination are recognized: one is termed induced because induction signals are required for the production of primordial germ cells. The other mechanism is cell-autonomous, i.e. determinative, because the cells that develop in response to specific cytoplasmic determinants in the oocyte are pre-destined to become PGCs. We revisited the hypothesis and analyzed phyla diversity with germ cell determination mechanisms and examined sister clade asymmetry. Results: After 25 years of additional data accumulation, the hypothesis that high levels of species diversification are associated with the induced mode is falsified, with the determinative mode revealed as associated with higher rates of diversification. The greater species numbers are significantly associated (ANOVA p>0.003) with the determinative mode. Analysis with appropriate sister clades is unanimous in showing the clade with the determinative mode has a significantly greater number of species relative to its induced sister clade . Conclusions: The primordial germ cell determination mechanism hypothesis explains asymmetrical species diversity and morphological disparity at the phylum level. We argue that the determinative mode of primordial germ cell determination is a constraint release that has enhanced evolvability and increased rates of speciation and morphological disparity among clades. Knowledge of the mechanism for extant theropods allows speculation that its sister clade, the Sauropodomorpha would have exhibited the induced mode. Results: After 25 years of additional data accumulation, the hypothesis that high levels of species diversification are associated with the induced mode is falsified, with the determinative mode revealed as associated with higher rates of diversification. The greater species numbers are significantly associated (ANOVA p>0.003) with the determinative mode. Analysis with appropriate sister clades is unanimous in showing the clade with the determinative mode has a significantly greater number of species relative to its induced sister clade . Conclusions: The primordial germ cell determination mechanism hypothesis explains asymmetrical species diversity and morphological disparity at

Gradual recruitment and selective clearing generate germ plasm aggregates in the zebrafish embryo.

Determination of primordial germ cells (PGCs) is one of the earliest decisions in animal embryogenesis. In many species, PGCs are determined through maternally-inherited germ plasm ribonucleoparticles (RNPs). In zebrafish, these are transmitted during oogenesis as dispersed RNPs, which after fertilization multimerize and become recruited as large aggregates at furrows for the first and second cell cycles. Here, we show that the number of recruited germ plasm RNPs is halved every cell cycle. We also show that germ plasm RNPs are recruited during the third cell cycle, but only transiently. Our data support a mechanism in which systematic local gathering of germ plasm RNPs during cytokinesis and threshold-dependent clearing contribute to forming germ plasm aggregates with the highest RNP number and germ cell-inducing potential.

Nanog Regulates Primordial Germ Cell Migration Through Cxcr4b

Gonadal development in vertebrates depends on the early determination of primordial germ cells (PGCs) and their correct migration to the sites where the gonads develop. Several genes have been implicated in PGC specification and migration in vertebrates. Additionally, some of the genes associated with pluripotency, such as Oct4 and Nanog, are expressed in PGCs and gonads, suggesting a role for these genes in maintaining pluripotency of the germ lineage, which may be considered the only cell type that perpetually maintains stemness properties. Here, we report that medaka Nanog (Ol-Nanog) is expressed in the developing PGCs. Depletion of Ol-Nanog protein causes aberrant migration of PGCs and inhibits expression of Cxcr4b in PGCs, where it normally serves as the receptor of Sdf1a to guide PGC migration. Moreover, chromatin immunoprecipitation analysis demonstrates that Ol-Nanog protein binds to the promoter region of Cxcr4b, suggesting a direct regulation of Cxcr4b by Ol-Nanog. Simultaneous overexpression of Cxcr4b mRNA and depletion of Ol-Nanog protein in PGCs rescues the migration defective phenotype induced by a loss of Ol-Nanog, whereas overexpression of Sdf1a, the ligand for Cxcr4b, does not restore proper PGC migration. These results indicate that Ol-Nanog mediates PGC migration by regulating Cxcr4b expression.

Downregulation of Reproductive Homeobox Gene 6 (Rhox6) Interferes with Male Germ Cell Differentiation.

Homeodomain proteins contain a 60 amino acid DNA binding motif called homeobox that regulates a variety of developmental events such as determination of body segment identity, limb development and organogenesis. Recently, a novel cluster of homeobox genes has been discovered on the X chromosome of the mouse. Because of their selective expression in reproductive organs, they are named Reproductive homeobox genes on the X chromosome (Rhox). Rhox6, a member of the Rhox6 family, is expressed in primordial germ cells (PGCs) in the developing gonad and the placenta, although the biological function of Rhox6 in determination of PGCs and placental cells remains unknown. To investigate the Rhox6 function, we generated male chimeric mice by injection of the gene-trapped embryonic stem cells (ESCs) harboring a hypomorphic mutation in Rhox6 into wild-type blastocysts. Almost all of the male chimeric mice exhibited infertility associated with smaller testes, reduced sperm number and motility and abnormal morphology of the epididymal sperm. Next, to obtain a better understanding of the Rhox6 function in germ cell differentiation, we took advantage of in vitro ESC culture. ESCs can differentiate into PGCs when ESCs are cultured as aggregates called embryoid bodies (EBs) in the medium containing Ascorbic acid, 1-Thioglycerol and Transferrin without the leukemia inhibitory factor. By using a mouse ESC line that expresses the enhanced green fluorescent protein (EGFP) under the promoter of Oct3/4, a central regulator of cellular pluripotency, the presence of PGCs within EBs was monitored in real time by EGFP expression. While this ESC line was induced to differentiate into PGCs, expression of Rhox6 was downregulated by stable expression of the short-hairpin (sh) RNA against the Rhox6 transcripts. The level of Rhox6 knockdown was validated by RT-PCR. As a negative control, a scrambled nucleotide sequence that does not show any match with the mouse genome was used to express shRNA molecules. EBs were generated from multiple independent clones that express either one of the shRNAs, which were isolated by electroporation of the ESC line with each vector, followed by drug selection. We found that 0% of the EBs having the shRNA against the Rhox6 transcripts expressed (n=29; the number of clones used to generate EBs is 17) had EGFP-positive cells, whereas 43% of the EBs having the control shRNA expressed (n=35; the number of clones used to generate EBs is 25) had EGFP-positive cells. To validate differentiation of PGCs, these EBs were pooled according to EGFP expression and used to examine expression of PGC markers by semi-quantitative RT-PCR. Because expression of Rex1, a marker for undifferentiated ESCs, was significantly downregulated in these EBs than in undifferentiated ESCs, EGFP expression detected in the EBs indicates the presence of PGCs, but not the presence of undifferentiated ESCs. Rhox6 as well as other PGC markers including Oct3/4, Rnh2, Piwil2, and Fgls were downregulated in the EBs expressing the shRNA against the Rhox6 transcripts, whereas the EBs expressing the control shRNA with EGFP-positive cells expressed Rhox6 and these PGC markers. Collectively, these in vivo and in vitro studies strongly suggest that downregulation of Rhox6 impairs differentiation of PGCs. This study is supported by the Departmental Startup fund including the USDA Cooperative State Research, Education and Extension Service, Hatch project (ILLU-538-323). (poster)

Inferring developmental constraint and constraint release: Primordial germ cell determination mechanisms as examples

Developmental constraint and its converse constraint release are significant concepts in understanding pattern and process in macroevolution. The purpose of this paper is to propose a two-step method for identifying constraints and constraint release. The first step is a phylogenetic optimization procedure to identify which trait/process is primitive and which is derived. The primitive trait is inferred to be the constraint and the convergently derived trait the release. The second criterion uses sister-clade asymmetry. Clades diagnosed by the constraint will have fewer taxa than clades diagnosed by the release. As an example, we use the process of germ cell specification, in which there are three modes of specification. Our results corroborate previous conclusions that the induced mode is the constraint and the predetermined mode is the release and we speculate on the importance of these two processes in terms of robustness and evolvability.

Critical windows of exposure for children's health: the reproductive system in animals and humans.

Drugs and environmental chemicals can adversely affect the reproductive system. Currently, available data indicate that the consequences of exposure depend on the nature of the chemical, its target, and the timing of exposure relative to critical windows in development of the reproductive system. The reproductive system is designed to produce gametes in far greater excess than would seem to be necessary for the survival of species. Ten to hundreds of millions of spermatozoa are generated daily by most adult male mammals, yet very few of these germ cells succeed in transmitting their genetic material to the next generation. Although the number of oocytes produced in mammalian females is more limited, and their production occurs only during fetal life, most ovaries contain several orders of magnitude more oocytes than ever will be fertilized. Toxicant exposures may affect critical events in the development of the reproductive system, ranging from early primordial germ cell determination to gonadal differentiation, gametogenesis, external genitalia, or signaling events regulating sexual behavior. Although there are differences between the human reproductive system and that of the usual animal models, such models have been extremely useful in assessing risks for key human reproductive and developmental processes. The objectives for future studies should include the elucidation of the specific cellular and molecular targets of known toxicants; the design of a systematic approach to the identification of reproductive toxicants; and the development of sensitive, specific, and predictive animal models, minimally invasive surrogate markers, or in vitro tests to assess reproductive system function during embryonic, postnatal, and adult life.

Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage.

The origin of germ cells and the molecular mechanisms of primordial germ cell (PGC) determination in teleosts are unclear. Vasa is a member of the DEAD protein family and plays an indispensable role in germ cell determination in Drosophila and Xenopus species. In this study, we isolated and characterized a rainbow trout vasa cDNA as a first step towards understanding the molecular mechanisms of PGC determination and development and to develop a molecular marker to identify the PGCs in rainbow trout. Cloning of vasa cDNA was performed by degenerate- and RACE-PCR. The predicted amino acid sequence of rainbow trout Vasa contained eight consensus sequences for the DEAD protein family and five arginine-glycine-glycine repeats, a common character of known Vasa homologues. Overall amino acid similarity to the Vasa of Drosophila was 79.2%. Whole-mount in situ hybridization of eyed stage embryos (eighty somite stage) revealed that signals were localized to the putative PGCs. In adult rainbow trout tissues, both ovaries and testes contained large amounts of vasa gene transcripts. A reverse transcription-polymerase chain reaction analysis of unfertilized eggs proved that trout vasa is a maternal factor. Although we have not determined whether rainbow trout vasa functions as a germ cell determinant, its limited expression in the germ cell lineage proved that rainbow trout vasa can be used as a marker molecule for PGCs. This marker will make it possible to identify the PGCs or presumptive PGCs in early trout embryos whose germ cells can not be distinguished by morphological characteristics.

Molecular anisotropy of the early Drosophila embryo.

IT is generally accepted that cellular differentiation depends chiefly on differential gene activity7. The model of differential gene activity implies the existence of different inducers/repressors activating/repressing different genes. Indirect evidence for such molecules in Drosophila comes from experiments of Illmensee and Mahowald2 who showed that the information for primordial germ cell determination is in the cytoplasm of the posterior pole of the egg and that when this cytoplasm is transferred to the anterior pole so is the information. Their experiments suggest that genes are regulated not only in time but also in space, possibly by molecules distributed unequally in the developing system3. We have analysed Drosophila eggs for such molecules.