Geoffrey D. Findlay, PhD - US grants

DESCRIPTION (provided by applicant): Internal fertilization is a complex biological process that requires interactions between proteins provided by both males and females. In addition to proteins found on the sperm and the egg, additional accessory proteins from seminal fluid and the female reproductive tract are required for successful reproduction. Because the functions of these proteins directly impact reproductive success, reproductive proteins experience strong evolutionary selective pressures, and interacting proteins are predicted to coevolve with one another. However, while reproductive proteins from both sexes have been readily identified, how these proteins interact is a relatively open question. This proposal seeks to investigate these issues in the genetically tractable Drosophila melanogaster (fruit fly) model system, which bears important parallels to human and mammalian reproductive systems. The seminal fluid proteins (Sfps) found in Drosophila males belong to the same functional classes as those identified in mammalian taxa, and many Drosophila male and female reproductive proteins show the same evolutionary dynamics as those from other systems. Thus, the methods used to study the functions of Drosophila proteins and the types of interactions involving these proteins may generate useful techniques and hypotheses for studying reproductive proteins and infertility in other systems. In order to address reproductive protein function, interaction and coevolution, this proposal will investigate as a case study a network of Sfps that together regulate mated females'egg production, sperm storage and mating behavior. Particular focus will be paid to proteins that interact molecularly with a well-characterized Sfp, the sex peptide (SP), to mediate its association with and release from sperm. The research will use two complementary methods to identify other Sfps, sperm proteins, and female proteins that interact with SP and other components of this network. The first method is a novel phylogenetic approach that uses correlated evolutionary rates to predict protein-protein interactions. The second involves biochemical pull-down assays to identify interacting sperm and female proteins that act at critical steps in the pathway. Candidate interacting proteins identified from either method will be tested molecularly and genetically to determine how they interact with and regulate SP and, thereby, mediate female post-mating responses. These experiments will offer important insights into basic reproductive biology and the study of protein-protein interactions, while providing critical and novel training to the applicant in areas of genetics, biochemistry and evolutionary biology. PUBLIC HEALTH RELEVANCE: Interactions between male and female proteins are necessary for successful reproduction and fertility. The goal of this project is to develop genetic, genomic and biochemical methods to identify interacting reproductive proteins and then to use genetic and biochemical methods in the Drosophila model system to dissect their interactions. Insights from the proposed studies may help guide research toward diagnosis, and perhaps treatment, of infertility cases in human couples who have incompatible versions of a male reproductive protein and its female partner.

All species share many genes in common with other organisms, but each species has a handful of new genes that came into existence only recently and are thus either unique to that species or shared only with its closest relatives. In a variety of animal and plant species, many such newly evolved genes are thought to impact the male reproductive system. The goal of this project is to determine how newly evolved genes influence male reproductive success by focusing on those that affect sperm function in the fruit fly model system, Drosophila melanogaster. This research will generate important basic science knowledge about how new genes change reproductive systems to the benefit of their carriers. Its broader implications relate to the potential development of strategies that inhibit the reproduction of insect species that are agricultural pests or that transmit human diseases, such as the mosquitoes that carry malaria and the Zika virus. By establishing the importance of lineage-specific genes for male reproduction, this research may aid in the development of smart pesticides, or genetic manipulation strategies that target genes found only in the problematic insect species and not other beneficial insects in the surrounding environment. This project will also benefit society by increasing the participation of undergraduates in original research and supporting innovative mentoring programs that encourage the persistence of students from groups that are underrepresented in STEM disciplines. The research will be conducted at an exclusively undergraduate institution and will be carried out, in part, by students in an intermediate-level Genetics course and students conducting independent research in a faculty member's lab. The project will also support a program in which first-year college students from underrepresented groups join research labs immediately upon their arrival on campus, providing these students with a sense of community and introducing them to scientific research as they begin college. Finally, the project will support a student-run organization that pairs college students with girls from underserved public high schools for weekly mentoring sessions that include assistance with math and science skills.

This project focuses specifically on so-called "de novo" genes that have recently evolved from non-coding DNA sequence. Previous research from a variety of taxa has focused on identifying these genes and studying their emergence within populations, but little is known about their specific molecular and cellular functions. Working in the safe and genetically tractable Drosophila melanogaster model system, this project will begin by using RNA interference to screen all de novo genes expressed in the testes for effects on male fertility. Genes whose expression is required for full fertility will become targets for functional characterization, which will be facilitated by the development of tagged transgenes, antibodies, and CRISPR/Cas9-mediated knockout mutant lines. These tools will be used in cytological experiments to investigate how each gene influences the process of spermatogenesis and/or the function of mature sperm after they are transferred to females. In parallel with these functional genetic analyses, the evolutionary history of each gene will be examined across related Drosophila species in order to understand how the gene arose from non-coding DNA sequence and how the protein it encodes has evolved since its emergence. For de novo genes that show signatures of rapid divergence between species, further genetic experiments will be conducted to determine the functional consequences of divergence. Taken together, these experiments will provide a comprehensive view of how the process of new gene creation can modify reproductive phenotypes.

Newly evolved genes, which are non-essential at first, can acquire essential roles over time, but how they do so is currently unclear. This project will use the tractable fruit fly model system to investigate how three young genes, which have gained essential functions in male fertility, evolved novel capabilities, including physical binding partners and complex expression patterns. Ultimately, these findings will shed light on the kinds of genetic changes a new gene requires to gain functionality. This project will also enhance the research capacity of an exclusively undergraduate institution, thus providing opportunities for students to conduct original research. The PI’s lab will participate in an immersive, paid summer research program for second- and third-year students and a cohort-based mentoring program for first-year students designed to bolster the persistence of first-year students from backgrounds historically underrepresented in STEM fields. The host department’s bioinformatic curriculum will be expanded through the development of a lab-based genomics course that will provide a course-based research experience to undergraduates who may not otherwise participate in original research.<br/><br/>This project will explore how newly evolved genes that likely arose from non-protein-coding DNA sequences acquire essential functions. It will use as case studies of three genes shown previously to be essential for spermatogenesis in Drosophila melanogaster. First, an evolutionary approach will be used to estimate when each gene became essential for reproduction and to identify key amino acids stretches important for the function of the encoded protein. Specifically, the ability of both extant orthologous protein sequences and inferred ancestral sequences to rescue D. melanogaster flies lacking an endogenous copy of the gene will be measured. These experiments will generate testable hypotheses about which sequence features are required for these proteins’ essential functions in extant D. melanogaster flies. Second, to investigate how newly evolved proteins evolve functions in existing cellular networks and gain interactions with other molecules, a combination of biochemical and genetic approaches will be used to identify interacting partners of each focal protein. Third, this project will investigate how newly evolved genes gain post-transcriptional mechanisms of gene regulation and thus evolve spatiotemporal patterns of expression. These experiments will compare transcript and protein localization patterns and investigate genetically the role of different upstream and downstream regulatory regions on new gene expression.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.