Gb_PGCs_merge_flat

In all sexually reproducing animals, germ cell specification during embryonic development is an essential step in ensuring species survival and evolution. We are interested in the evolutionary origins of the mechanisms that specify germ cells. Our approach is to compare the components and functions of the relevant molecular mechanisms in a variety of extant animals.

Work from traditional model organisms including frogs, zebrafish, nematodes and fruit flies has shown that a common mechanism used to specify germ cells is the inheritance of maternally provided determinants. For example, in the fruit fly Drosophila melanogaster, maternally synthesized cytoplasmic proteins and mRNAs (collectively termed “germ plasm”) are localized to the oocyte posterior. During early embryogenesis, the cells that inherit this germ plasm acquire germ cell fate.

Some other insects and crustaceans also use germ plasm to specify germ cells. For example, in the amphipod crustacean Parhyale hawaiensis, germ cell also specification occurs through inheritance of a maternally supplied cytoplasmic determinant. We have shown that a morphologically distinct cytoplasmic region in the one-cell embryo contains germ line-associated RNAs and that removal of this cytoplasmic region results in a loss of embryonic germ line cells. Based on these loss-of-function experiments, we propose that we have identified a putative germ plasm in Parhyale.

However, histological studies of arthropod germ cell formation conducted over the past two centuries have suggested that basally-branching insects, and indeed most arthropods, lack germ plasm, and instead form their germ cells later in development, likely through cell signaling. To understand the mechanisms of germ cell formation that are more likely to resemble ancestral states than the relatively derived Drosophila and Parhyale models, we therefore study the developmental and genetic mechanisms underlying germ cell specification in species that branch closer to the base of the insect tree or belong to non-insect arthropod groups. Our current models include the cricket Gryllus bimaculatus, the milkweed bug Oncopeltus fasciatus, and the spider Parasteatoda tepidariorum. Using multiple molecular markers and RNAi-based functional approaches, we found no evidence of germ plasm in basally branching insects or spiders. Instead, our experiments suggest that germ cells in these organisms form during mid-embryogenesis, in close association with mesoderm. Furthermore, we find that several genes that are essential for Drosophila germ cell specification are not required for germ cell specification in these species, but instead are required for spermatogenesis in adult males or early embryonic cell divisions. Our results suggest that the Drosophila mode of germ cell specification is derived relative to a last common insect or arthropod ancestor, and confirm that germ cell specification is a remarkably labile process across evolution.

Our current work in this area is focused on determining which specific zygotic mechanisms are required to specify germ cells in basally branching arthropods. Ongoing projects include investigation of the role of BMP signaling in arthropod germ cell formation, understanding how Hox gene-mediated body plan patterning ensures than germ cells are specified in the correct location, and an in situ hybridization screen in the spider to uncover genes involved in germ cell development. We are working on creating transgenic cricket lines that will allow us to perform inducible, heritable cell labeling (clonal analysis) so that we can track subsets of cells during early development. We are also working on adapting CRISPR genome editing technology to create tissue-specific reporter lines in crickets, so that we can track germ cells and other cell types throughout development using SPIM microscopy and other high-throughput imaging methods.

References

vasa and piwi are required for mitotic integrity in early embryogenesis in the spider Parasteatoda tepidariorum. Schwager, E.E., Meng, Y. and Extavour, C.G. Developmental Biology, 402(2): 276-290 (2015). [PubMed]
BMP signaling is required for generation of primordial germ cells in an insect. Donoughe, S., Nakamura, T., Ewen-Campen, B., Green II, D.A., Henderson, L. and Extavour, C.G Proceedings of the National Academy of Sciences of the USA, 111(11): 4133-4138 (2014). [PubMed]
Evidence against a germ plasm in the milkweed bug Oncopeltus fasciatus, a hemimetabolous insect. Ewen-Campen, B.*, Jones, T.E.*. and Extavour, C.G. Biology Open, 2(6): 556-568 (2013). [PubMed]
Germ Cell Specification Requires Zygotic Mechanisms Rather Than Germ Plasm in a Basally Branching Insect. Ewen-Campen, B., Donoughe, S., Clarke, D.N., and Extavour, C.G Current Biology, 23(10): 835-842 (2013). [PubMed]
The molecular machinery of germ line specification. Ewen-Campen, B., Schwager, E.E., Extavour, C.G Molecular Reproduction and Development, 77(1): 3-18 (2010). [PubMed]