Germ cells are the cells responsible for making eggs and sperm in all multicellular, sexually reproducing animals. Because they are one of the most important cell types, it is important to understand not only the genes that control their development, but also their evolutionary history. Early multicellular animals probably did not have a dedicated group of germ cells, but rather used stem cells to make eggs and sperm. This means that the development of a dedicated group of cells responsible for reproduction can be considered an evolutionary novelty. This problem will be examined with comparative developmental genetics. Probably the first genetic program used to specify germ cells was that used in mice, which involves signalling between cells. Fruit flies use a completely different genetic program, raising the question of whether or not the fruit fly program is representative of all arthropods (insects, crustaceans, millipedes, centipedes, spiders and scorpions), or is specific to flies. Genes will be isolated from both the mouse and the fly and their expression and function will be studied to determine whether the inductive signals used in mice are also used in crickets and spiders. This project will help to bridge a gap in our current understanding of the evolutionary origins of germ cells, as almost nothing is known about germ cell origin in arthropods besides fruit flies. It may also provide the first demonstration of an inductive mechanism for germ cell specification in invertebrate animals. The project will provide training for a postdoc, a graduate student and undergraduate students. The PI is committed to providing a positive role model for women and under-represented minorities in academic science.
The goal of this grant was to increase our understanding of both the functions and the evolutionary history of the genes that control germ cell specification. Germ cells are the special cell type that becomes eggs and sperm, and are found in all sexually reproducing animals. All other cells of the body, for example those of the nervous system, appendages, and internal organs, are important for the life of the organism, but none of those cells will be directly passed on to the next generation. Studies in traditional laboratory organisms, including fruit flies and mice, have taught us a lot about how germ cells develop and the genes that are required to specify germ cells. The animal in which these processes are best understood is the fruit fly Drosophila melanogaster, which belongs to the phylum Arthropoda. Arthropods are animals with an exoskeleton and jointed legs: they include insects, crustaceans, spiders and centipedes. Arthropods are the most speciose animal phylum, which means that the vast majority of all animals on earth are arthropods. However, the way that fruit flies develop is not comparable to the way other animals develop in many respects. Fruit flies use an unusual mechanism, called germ plasm, to form germ cells very early in development. In mice, germ cells are formed by the action of genes that act together to communicate signals from one cell to another, called the BMP signaling pathway. Our previous work had suggested that cell-cell signaling pathways might be important for specifying germ cells in most animals, and that signaling might have been an ancestral mechanism for specifying germ cells in animals. In this grant, we therefore proposed to determine how germ cells were specified in two arthropods, a cricket and a spider, whose development is more representative of what we think development would have been like in a last common ancestor of all arthropods. When we began our research the techniques available to study genes and their functions in crickets and spiders were very limited. For this reason, we first invested in creating large collections of gene sequences (called transcriptomes) from crickets and spiders, which would help us to quickly and easily identify many different relevant genes for future study. In addition, we created transcriptomes for two other arthropods, a milkweed bug and a crustacean, which we and others have found to be useful laboratory animals for doing this kind of comparative genetic research. We also created a publicly accessible database to store all of these sequences and make them available for other researchers to use. With these tools in hand, we were able to identify many germ cell genes from the cricket, the milkweed bug and the spider. We used these genes to identify germ cells in early embryos of all of these animals, and in this way determined when and where germ cells first form during embryonic development. We found that instead of arising right at the beginning of development as in fruit flies, germ cells in our arthropods first appeared much later in development, more similar to how mouse germ cells develop. When we tested whether these genes were required very early in development for germ cells to form (as in fruit flies), we found that they were not. Instead, in the milkweed bug and the cricket, they were required in adult males for spermatogenesis (making sperm), as in mice. Finally, we tested whether genes of the BMP signaling pathway were required for making cricket germ cells. Our preliminary results suggested that indeed, as in mice but unlike in fruit flies, cell-cell signaling is likely to play an important role in making sure that germ cells are specified during embryogenesis. In summary, the research we performed with this grant provided evidence in support of the hypothesis that the mechanism used by fruit flies to make their germ cells, which is so well understood and had been assumed for many years to be the mechanism used by most animals, is not used by arthropods that may be more representative of ancestral mechanisms. Instead, germ cell formation in these "basally branching" arthropods appears to be much more similar to germ cell formation in mice. It is possible that the genetic mechanisms of germ cell formation that are shared between mice and crickets, were also present in a last common ancestor of all animals. Graduate students, undergraduate students, and a postdoctoral researcher all participated in the research performed under this grant. The PI and her student and postdoctoral researchers also hosted middle school students in the lab, and the PI gave presentations on arthropod embryology and germ cell biology to high school students and to the general public at the Harvard Museum of Natural History.
