Germ cell development depends on a complex regulatory process in the embryo, but can be initiated by a single signal in the test tube
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In the process of exploring the formation of primordial germ cells (PGCs), the precursors to both sperm and ova, RIKEN researchers have uncovered an effective means for cultivating functioning germ cells. The findings could have a profound effect on fertility research and help scientists to better understand the earliest stages of reproductive development.
At the start of the second week of embryonic development, certain cells in the proximal posterior epiblast region of the mouse embryo begin to undergo radical changes that enable their transformation into PGCs. “Our studies have identified that germ cell specification involves at least three key events: repression of the somatic [gene expression] program, re-acquisition of potential pluripotency and ensuing epigenetic reprogramming,” says Mitinori Saitou, an investigator at the RIKEN Center for Developmental Biology in Kobe.
In previous work, Saitou’s team identified the protein Blimp1 as a key factor controlling all of these processes, demonstrating that it is specifically expressed at exactly the right time and place to kick off PGC development1. However, it has remained unclear which factors are the top-level triggers for expression of PGC ‘master switch’ genes like Blimp1 and Prdm14, which is another important regulator of germ cell development. To address this question, Saitou and colleagues embarked on a new study to characterize the involvement of a variety of other signaling factors previously linked to the PGC formation process2.
Bmps in the road
The team began by looking at Bmp4 and Bmp8b, two factors secreted by a region known as the extraembryonic ectoderm (ExE), which borders the posterior epiblast. Both proteins proved essential to Blimp1 expression, although both are also expressed in a far broader swathe of the embryo than Blimp1, suggesting that Bmp4 and Bmp8b are being blocked by an inhibitory factor that restricts their activity in the anterior portion of the embryo.
In order to directly observe the effects of various factors at different stages of PGC development, the researchers cultured whole embryos and embryonic fragments under serum-free conditions in the presence of different signaling molecules. These experiments confirmed that the ExE is needed to induce Blimp1 expression and that signals from the anterior visceral endoderm serve to keep Blimp1 expression in check. Unexpectedly, however, they also indicated that the presence of Bmp4 alone is sufficient to drive the development of PGC-like cells that express both Blimp1 and Prdm14.
This finding raised questions about why embryos lacking Bmp8b fail to undergo proper germ cell development, but subsequent experiments made the answer clear: Bmp8b restricts the development of the anterior visceral endoderm, keeping in check the Blimp1 inhibitory signals produced by this extraembryonic region. Only through the combined effects of both Bmp8b and Bmp4 will proper Blimp1 expression—and therefore PGC development—take place.
Although the cells derived in these experiments bore several of the hallmarks of naturally occurring PGCs, Saitou and colleagues performed another battery of experiments to confirm that these PGC-like cells also resembled their naturally occurring counterparts at a functional level.
After nearly a week of culture, cells derived from Bmp4-treated epiblasts—which the authors termed epiPGCs—continued to express not only Blimp1 and Prdm14 (Fig. 1), but also expressed a number of other key genes specific to PGCs, such as stella and SSEA1. More importantly, however, they also proved capable of producing viable sperm following transplantation into mice; this was demonstrated both with implantation of in vitro reconstructed gonads as well as direct injection of epiPGCs into the testes of neonatal recipients. In both models, the resulting spermatozoa were healthy and suitable for fertilization of oocytes, producing healthy offspring (Fig. 2).
In addition to the raw information encoded within the genomic sequence, reproduction also involves the transmission of parent-specific epigenetic ‘imprinting’: patterns of chemical modification on the chromosomes that provide an additional level of gene regulation. Parental epigenetic marks are normally wiped clean in PGCs, and then subsequently restored during spermatogenesis. Similar behavior was observed in spermatozoa generated from transplanted epiPGCs, providing further evidence that BMP4-induction is sufficient to yield cells that are apparently interchangeable with natural PGCs.
“It’s surprising that Bmp4 is sufficient to drive epiblast cells to undergo PGC specification and subsequent further differentiation, including epigenetic reprogramming and erasure of imprints,” says Saitou. “This indicates that this specification signal is sufficient to promote PGC development ... autonomously to a certain extent.”
This study has enabled Saitou’s team to identify the essential factors involved in germ cell formation, and thereby reconstruct a spatial and temporal map of these various activation and inhibition signals that achieves an unprecedented level of detail.
Importantly, this work also represents the first time that fertilization-ready gametes have been successfully derived from cultured embryonic cells—a breakthrough with potential implications for both the laboratory and the clinic. This technology could provide the means for scientists to introduce genetic modifications in animal models for which suitable stem cells are not available. Moreover, Saitou sees this method as a powerful tool for studying—and addressing—human infertility, and he indicates that a long-term objective of his team involves using these epiPGCs to recapitulate the entire process of germ cell development in vitro. “Application of these findings to human embryonic stem cells or induced pluripotent stem cells may provide a way to find critical mutations regarding human infertility,” he concludes.
Reference
1. Kurimoto, K., Yabuta, Y., Ohinata, Y., Shigeta, M., Yamanaka, K. & Saitou, M. Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes & Development 22, 1617–1635 (2008).
2. Ohinata, Y., Ohta, H., Shigeta, M., Yamanaka, K., Wakayama, T. & Saitou, M. A signaling principle for the specification of the germ cell lineage in mice. Cell 137, 571–584 (2009).
The corresponding author for this highlight is based at the RIKEN Laboratory for Mammalian Germ Cell Biology
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About the Author
Mitinori Saitou
Mitinori Saitou received his M.D. from the Kyoto University Faculty of Medicine in 1995, and was award a Ph.D. in 1999 for his study of the structure and function of tight junctions under Shoichiro Tsukita at the Kyoto University Graduate School of Medicine. He then moved to the Wellcome Trust/Cancer Research UK Institute, where he worked as a postdoctoral research associate in Azim Surani’s laboratory, focusing on his long-term interest in the origin of the germ line in the mouse. He was appointed team leader at the CDB in 2003, and received a three-year grant from the Japan Science and Technology (JST) Corporation under the PRESTO program for the development of a single-cell microarray technology. In 2004 he became Associate Professor at the Kyoto University Graduate School of Biostudies, and was subsequently appointed Professor of the Kyoto University Graduate School of Medicine in 2009. He continues to investigate the origin, properties, and regulation of the mammalian germ cell lineage with the aim of reconstituting the lineage in vitro.
