Diana J. Laird, Ph.D. - US grants

DESCRIPTION (Provided by the applicant) Abstract: Competition between cells within organisms has been recognized in social amoebae, Drosophila, and the inception and metastasis of cancer. "Winner" cells exhibit advantages in growth, adhesion or survival, putatively increasing the overall fitness of the individual organism. However, only competition among germ cells results in direct inheritance of "winner" traits. As gametes are established during early embryonic development from primordial germ cells (PGCs), heritable genetic mutations that undergo selection and drive evolution of species must occur in this cell lineage. I previously demonstrated germline stem cell competition in a basal chordate and recent work hints at a parallel phenomenon in mammals. Our goal is to determine the gene regulatory and genetic bases for germ cell competition in mice. We will study competition among PGCs in the mouse embryo in three aspects of their development: the allocation of pluripotent epiblast cells to the germline, the migration of PGCs to the gonadal ridges, and the expansion of PGCs in the embryonic gonad as they commence sex-specific differentiation. To model in vivo competition among isogenic PGCs, we will employ genetic marking strategies;comparison of "winning" PGC clones to unpurified or non-dominant ones will reveal gene expression and epigenetic differences that potentially bestow an advantage to developing germ cells. To model competitive advantage by genetic alteration, we will employ 7 different PGC mutants that we previously identified in a forward genetic screen of mouse embryos. We will use transplantation approaches to pit these PGC depletion, overabundance and migration phenotypes against one another in vivo and ask how a particular genetic mutation impacts fitness to enter the germline, migrate, and contribute to gamete biogenesis. We hope to gain a molecular understanding of how PGCs interact with their niches and provide insight into basic regulatory and genetic mechanisms of cellular selection that underlie evolution and tumorogenesis. Public Health Relevance: Since the origin and progression of cancer occurs by cells gaining a selective advantage in survival, proliferation or migration, study of the mechanism of cell competition will identify genetic alterations, regulatory or epigenetic changes that become dysregulated in tumor development and could be useful in cancer diagnosis or targeted for therapy. In assisted reproductive technologies, where the prospect of differentiating gametes from pluripotent stem cells looms on the horizon, understanding germ cell competition will be instrumental to informing the potential risks and biological consequences of short circuiting in vivo germ cell development.

DESCRIPTION (provided by applicant): Recent studies suggest that in utero exposure to environmental toxins can produce phenotypic changes over several successive generations. Although initial work suggests that epimutations propagate these transgenerational phenotypes, the cells that must carry this information to the next generation have not been examined. Primordial germ cells (PGCs), the embryonic founders of the egg or sperm, undergo rapid epigenetic reprogramming during mid-gestation in a timeframe that overlaps with the observed sensitivity to transgenerational effects following exposure to certain toxins. The long-term goal of our research is to elucidate genetic and epigenetic mechanisms underlying PGC development. The objective of this application is to assess the impact of one class of environmental chemical, phthalates, on mouse PGC development and epigenetic reprogramming in the exposed individual (F1) and subsequent generations (F3). Our central hypothesis is that biologically relevant doses of phthalates disrupt DNA methylation changes involved in reprogramming of PGCs during development, leading to gene expression changes; in order to be heritable through generations, it follows that mismethylation of regions associated with the regulation of reprogramming in PGCs disrupts expression of critical reprogramming machinery in the germline. This hypothesis is based upon prior reports of transgenerational phenotypes produced by a mixture of plasticizers BPA and phthalates as well as the demonstration that phthalates alter DNA methylation at the imprinted loci in humans and mice. Our hypothesis will be tested with the following two specific aims: (1) to establish phenotypic consequences of phthalate exposure on F1 and F3 generation mouse PGCs and (2) to determine direct and transgenerational effects of phthalates on the mouse PGC epigenome and transcriptome. In the first aim, we will interrogate PGC phenotypes in the F1 and F3 generation following in utero exposure to a range of environmentally relevant doses of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP). Doses producing phenotypes may be informative for the second aim, in which we will survey epigenetic reprogramming in F1 and F3 PGCs by examining histone modifications and global methylation semi-quantitatively and by genome-wide methylation analysis. Resulting changes in the F1 and F3 PGC transcriptome, together with the epigenetic data, will reveal the consequences of observed dysregulation at the level of DNA methylation, enable prediction of phenotypes in other tissues, and could identify candidate epigenetic regulators responsible for propagating observed epimutations. The proposed work is innovative as it uses advanced, genome-wide approaches and, apart from previous work, will directly examine epigenetic changes and potential consequences in the very cell responsible for trangenerational transmission. The proposed work is significant as it will determine the extent to which phthalate exposure alters reprogramming in PGCs and potentially identify candidate mechanisms of transgenerational epigenetic inheritance.

DESCRIPTION (provided by applicant): The goal of this application is to obtain funds to support the Santa Cruz Developmental Biology Conference which will be held on the Santa Cruz Campus August 20-23, 2014. This bi-annual meeting has been held at the University of California, Santa Cruz, campus since 1992, and it is a major forum for bringing together a diverse spectrum of scientists to present and discuss recent advances in developmental biology. This meeting is unique in being a small (140-180 participants) conference, with an international reputation, and abstracts to attend the meeting will be accepted by all Developmental Biologists starting in February 2014. As detailed in our application, we have planned for considerable participation by graduate students and postdocs by including short talks, posters, and two workshops aimed at career issues. The meeting format is single platform sessions, with two non-overlapping abstract sessions so every participant is engaged in the same topic and activity for the duration of the meeting. The Santa Cruz campus offers a beautiful setting for this meeting, and the housing, meal, and session arrangements encourage significant participant interactions. The session topics will include, Translational Developmental Biology, Cell Polarity, Development and Disease, Regeneration and Stem Cells, Morphogenesis and Signaling, Evolution and Development and Neurogenesis and Patterning. Within each session, a range of model organisms used by contemporary Developmental Biologists to address major questions have been chosen. In conclusion, this is a grass roots meeting created de novo every two years by biologists for biologists, and we are thrilled to have the opportunity to plan the meeting entitled 'Frontiers of development: basic meets translational' for 2014.

PROJECT ABSTRACT The goal of this proposal is to establish how exposures to ubiquitous plastics and prenatal stress interact to confer epigenetic changes to developing germ cells, and how such changes lead to reproductive and metabolic phenotypes in later life and potentially in subsequent generations. Numerous studies involving consumer and agricultural chemicals demonstrate effects that appear in the progeny and even grandchildren of individuals exposed in utero. Correspondence between period of susceptibility and epigenetic reprogramming suggest that epigenetic changes to germ cells are involved. However, as the majority of studies utilize oral gavage for dosing, the effects of the chemical and prenatal stress are difficult to resolve. Phthalates are the major chemical in household dust and have been detected in 100% of pregnant women in California. Our preliminary studies in mice find that prenatal exposures to two different phthalates during the period of germ cell epigenetic programming leads in each case to decreased repression of retrotransposons and germ cell developmental defects; evidence from our lab suggests that this occurs through disruption of the piRNA pathway, which silences endogenous transposable elements through epigenetic and post-translational mechanisms. In this proposal, we will interrogate the combined and separate effects of biologically relevant doses of phthalate and early gestational stress on the epigenetic state of mouse germ cells and relate these changes to the extent of retrotransposon activity and reproductive phenotypes in the progeny. Studies in Aim 1 will determine how early gestational stress and phthalate exposure interact to alter the epigenetic state of fetal germ cells and the potential for inheritance of that state. This will disentangle the effects of stress and chemical exposure that accompany oral gavage in many prior studies with quantifiable, molecular readouts as well as testing potential mechanisms of direct and multigenerational epigenetic dysregulation. Aim 2 will examine phenotypic consequences of early gestational stress and phthalate exposure on germ cells and female reproductive aging in the next generation. By connecting adult reproductive phenotypes to fetal exposures, this will begin to disentangle the effects of plastics versus prenatal stress. Pharmacologic modulation of key nodes in the stress response pathway will potentially provide insight on appropriate therapies for women in high-risk pregnancies. Aim 3 will determine the extent of genetic disruptions via transposon insertions induced by early gestational stress and phthalates in directly exposed germ cells and subsequent generations. These studies will identify environmental factors that pose greatest risk to genomic integrity and potentially a drug therapy to mitigate those effects.

PROJECT ABSTRACT The goal of this proposal is to connect the events of fetal germ cell development to the origin and inheritance of mutations in the Fmr1 locus, which are associated with Fragile X diseases. Evidence suggests that Fmr1 trinucleotide repeat expansions occur during development of germ cells, however the precise timing of Fmr1 repeat expansions, mechanism for intergenerational transmission, and function of the Fmr1 protein (FMRP) in this lineage remains unclear, as few studies have examined the entire pool of gametes or their precursors. Mouse models of Fmr1 deficiency as well as Fmr1-PM provide an opportunity to elucidate these mechanisms in development. Our preliminary data support a role for Fmr1 in protecting the genomic integrity of fetal germ cells and suggest that the period of scheduled apoptosis of fetal germ cells acts in divergent ways in the testis versus the ovary. The studies proposed here will test the overall hypothesis that the development of fetal germ cells provides a window of opportunity for Fmr1-PM amplifications based on the function of Fmr1 in genomic integrity, but on the other hand development selects against the intergenerational transmission of germ cells with amplifications in the male germline as compared to the female germline. Fragile X syndrome, along with other trinucleotide repeat diseases, has been called a `double-edged sword' because the pathological repeat expansion occurs in a gene required for DNA repair. In Aim 1, we will investigate the function of FMRP in protecting the integrity of developing germ cells and identify its RNA targets. Given the evidence that pathological CGG amplification occurs during Fmr1 transcription, these studies will provide critical insight into the periods of vulnerability to Fmr1 repeat amplification during germ cell development. Although the size of inherited Fmr1 pre-mutations increases with maternal age, this information is derived from successfully used oocytes, whereas nothing is known about the entire gamete pool. In Aim 2, we will test the hypothesis that FMRP1 increases fidelity of meiosis I in fetal oogonia whereas Fmr1 CGG repeat expansions interfere with meiosis and compromise the quality of oocytes in the adult. These studies will elucidate the relationship between Fmr1 pre-mutation, oocyte quality, and the dynamics of meiotic entry in the fetal ovary. In Aim 3 we will use both Crispr-based imaging and next generation sequencing to reveal the impact of massive waves of apoptosis that occur during normal male and female germ cell development on the diversity of repeat expansions in Fmr1 pre-mutation mice. These studies will provide insight into the developmental origin of pathological Fmr1 repeat expansions and potentially other mutations.

ABSTRACT ? PILOT PROJECT PROGRAM The University of California, San Francisco (UCSF) Environmental Research and Translation for Health (EaRTH) Center offers the unique opportunity to support and grow an environmental health community at a premier multidisciplinary biomedical research institution with nearly 5,000 faculty members with expertise spanning basic, translational, clinical, and population arenas. Despite being the top NIH-funded public university, only 0.7% of NIH funding at UCSF currently comes from NIEHS. The EaRTH Center Pilot Project Program provides a mechanism to increase the amount of environmental health research at UCSF by tapping into the existing broad research base. By providing seed funds for exploratory studies, EaRTH Pilot Project awards will lower hurdles to initiating novel directions in environmental research and grow the pipeline of researchers and clinicians in environmental health. We will support the mission of the EaRTH Center by enabling cutting edge, exploratory research in areas directly related to environmental health. This goal is promoted by supporting basic, translational, epidemiological and clinical research projects through Core facilities as well as providing interaction with a community of researchers through the EaRTH Center. A Research Pilot Award will allow new or established faculty to rapidly transfer skills and developments from other research areas to the arena of environmental health research. A Mentored Scientist Award specifically for early-stage investigators will support innovative research projects and provide career and mentoring in environmental health research. A Mentored Clinician Award, administered jointly with the Community Engagement Core (CEC), for clinicians will provide dedicated time for a clinician to gain experience in environmental health. The Pilot Project Program will stimulate research that fills gaps in the area of environmental health in development and preconception at UCSF. To streamline the application process and to capture the largest number of high quality applications with innovative projects proposed from researchers outside of environmental health, we will conduct the RFA through the broadly used UCSF Resource Allocation Program (RAP). Evaluation metrics will be applied to the pilot projects to assess and promote success of projects and the overall program. The UCSF EaRTH Center engagement with the entire UCSF scientific community will enable discussions of the application of interdisciplinary approaches to environmental health research. This ensures our pilot grant recipients have full access to local expertise, which assists their entry into, and staying power in, environmental health-related areas.