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Germ Line Research

Evolution of Germ Line Specification Mechanisms

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.

Gryllus PGCs

Gryllus primordial germ cells (labeled here in green with an anti-Gryllus-Piwi antibody) arise in embryonic abdominal segments A2-A4.

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
Ewen-Campen, B., Schwager, E.E., Extavour, C.G. The molecular machinery of germ line specification. Molecular Reproduction and Development 77(1): 3-18 (2010) PDF PubMed

Ewen-Campen, B., Donoughe, S., Clarke, D.N., and Extavour, C.G. Germ Cell Specification Requires Zygotic Mechanisms Rather Than Germ Plasm in a Basally Branching Insect. Current Biology 23(10): 835-842 (2013) PDF PubMed

Ewen-Campen, B.*, Jones, T.E.*., and Extavour, C.G. Evidence against a germ plasm in the milkweed bug Oncopeltus fasciatus, a hemimetabolous insect. Biology Open (Company of Biologists) 2(6):556-568 (2013) (* equal author contribution) PDF PubMed

Donoughe, S., Nakamura, T., Ewen-Campen, B., Green II, D.A., Henderson, L. and Extavour, C.G. BMP signalling is required for generation of primordial germ cells in an insect. Proceedings of the National Academy of Sciences of the USA (in press) [PDF of accepted MS]

Schwager, E.E., Meng, Y. and Extavour, C.G. vasa is required for mitotic integrity in early embryogenesis in the spider Parasteatoda tepidariorum. (in revision)



Evolution of a Novel Germ Line Determinant Gene

oskar is the only gene known to be both necessary and sufficient to form germ plasm in Drosophila. Previously, oskar was believed to be a recently-evolved, novel gene, present only in fruit flies and their close relatives. However, we identified an oskar ortholog in the cricket Gryllus, a basally-branching insect, which does not use germ plasm to specify germ cells (see above). We showed that cricket oskar is expressed in neural stem cells, and involved in embryonic neural patterning rather than germ line development. These results demonstrate that oskar arose far earlier in insect evolution than previously thought, and that its well-studied function in the Drosophila germ line is a recent evolutionary innovation.  

We are currently continuing our research on oskar in multiple ways. First, we are researching the evolutionary history of this novel gene by examining the phylogenetic distribution of oskar in insects, and identifying sequence changes that correlate with the functional changes of Oskar protein in different insect lineages. To this end, we have performed molecular evolutionary analyses of oskar and identified regions and residues that appear to be under positive selection in specific insect lineages.

GbNS

Gryllus embryonic nervous system in the head, gnathal and anterior thoracic segments.

Second, we are trying to determine the genetic origin of oskar, given that oskar genes are found only in insects. We are working on identifying the ancestral sequences that likely gave rise to oskar, and determining when in evolutionary time oskar first arose. Third, we are studying the role of cricket oskar in adult neural function, in collaboration with the Mizunami lab. Finally, in collaboration with the Leschziner lab, we are combining biochemical and structural biology approaches to shed light on the mechanism by which Drosophila Oskar assembles germ plasm. This work will provide a foundation for us to understand why fly Oskar assembles germ plasm but cricket Oskar does not. Ultimately we hope to understand not only the evolutionary origin of this novel gene, but the molecular genetic basis for its ancestral neural function and subsequent acquisition of the novel germ plasm assembly function, which permitted the evolution of a new mechanism of germ cell formation in insects.  

References

Extavour, C G. Long-Lost Relative Claims Orphan Gene: oskar in a Wasp. PLoS Genetics 7(4): e1002045 (2011) PDF PubMed

Ewen-Campen, B., Srouji, J.R., Schwager, E.E. and Extavour, C.G. oskar Predates the Evolution of Germ Plasm in Insects. Current Biology 22(23): 2278-2283 (2012) PDF PubMed Read more in the Harvard Gazette and a Dispatch in Current Biology

Ahuja, A. and Extavour, C.G. Patterns of molecular evolution of the germ line specification gene oskar suggest that a
novel domain may contribute to functional divergence in Drosophila. Development, Genes and Evolution doi 10.1007/s00427-013-0463-7 published online January 10 (2014) PDF PubMed



Evolution of Germ Cell Migration Mechanisms

Germ cells in many animals undertake long and complex migration routes through the forming embryo in order to finally reach the somatic component of the gonads. Successful completion of this route is pivotal for reproduction and hence survival of the species. Germ cell migration is a classical topic of developmental biology and has been subject to intense investigation in model organisms like fruit flies and mice. Serving as a paradigm for cell migration, it is relevant for many kinds of processes including organ development, immune system function, wound healing or carcinogenesis, which also directly employ several of the genes known to function in germ cell migration. Among our laboratory model organisms, Parhyale (crustacean) and Oncopeltus (milkweed bug) germ cells undergo extensive migration, but in the cricket Gryllus germ cells migrate only a few cell diameters in order to occupy the primordial gonad. We are developing live imaging tools that will allow us to study the cellular behaviors and genetic mechanisms involved in germ cell migration in these emerging model organisms.

References

Linsler, F. Migration of the Primordial Germ Cells in the Embryonic Development of Parhyale hawaiensis. Diploma Thesis, Freie Universitat Berlin/Extavour lab, Harvard University. (2008)