William Cresko - US grants

The synthesis of developmental and evolutionary biology has become a major focus of research over the pas several years. However, the role of allele frequency changes at regulatory loci during short-term evolution is poorly understood. In this project I will exploit a special opportunity to investigate these issues afforded by the rapid and extensive morphological evolution among populations of threespine stickleback. When this boney fish has invaded freshwater habitats it has often evolved divergent body, head and jaw shapes in order to exploit different resources, and concurrently lost boney plates and spines. I will construct a genetic map or stickleback and identify quantitative trait loci (QTLs) by using a cross between ancestral marine and derived freshwater forms. Comparative genomics with the well studied zebrafish will allow me to define the molecular basis of the development-pathways leading to divergent morphologies. In situ expression analyses will then be performed to link changes in adult phenotype to changes in expression patterns during embryogenesis. The synthesis of zebrafish and stickleback research will greatly increase our understanding of the microevolution of development. A fuller understanding of the microevolution of development promises significant gains for human health. Higher rates of genetic disorders in some human populations can be better understood using models of the evolution of development, and population specific treatments can be devised using insight gained from these models. Additionally, more predictive models of the developmental evolution of infectious diseases will allow targeted treatments that will decrease the probability of epidemics of drug resistant pathogens.

DESCRIPTION (provided by applicant): Threespine stickleback fish is an emerging model system for studying the developmental genetic basis of quantitative traits. Researchers within the community agree that tools to leverage and enhance the recently completed genomic sequence are essential. We propose to help investigators understand the underlying genetics of the naturally varing traits seen in stickleback by providing to the community a microarray-based system for efficient and cost effective genotyping. The high-throughput genotyping system that we have developed, RAD mapping, will enable the rapid identification of the genes controlling complex traits ranging from behavior to bone shape. In order to facilitate the use of this tool, simple web-accessible array analysis tools and a hands-on course to train researchers new to genomics will be provided. Stickleback are a complementary model system for many laboratory-based organisms, in that the numerous isolated and divergent natural populations have evolved adult traits that are a difficult or impossible to study by traditional mutagenesis approaches. Importantly, many of these traits are quantitative, influenced by alleles at genetic diseases, and stickleback are a unique vertebrate model for examining these quantitiative traits. The genotyping tools that we propose here will greatly accelerate this important research. Investigators from other research communities such as Drosophila, zebrafish, and mouse have expressed enthusiasm for initiating work in stickleback if these proposed tools were available. Thus, our genomic tools will increase the rate of discoveries of the genetic basis quantitative variation that underlies most common human diseases both by assisting present stickleback researchers;and by drawing additional researchers to work on this emerging model organism. Relevance: Most common human diseases, such as heart disease, high blood pressure, and osteoarthritis, are the result of complex interactions between an individual's genes and her or his environment. Threespine stickleback fish are an excellent model for these kinds of traits, and have numerous traits that are similarly controlled by many genes and the particular environment in which they live. We propose to create research tools to enable the rapid discovery of the genes controlling these complex traits, and help further the emergence of stickleback as an important model for common human traits specifically, and complex traits in general.

Biologists have made great progress in understanding the genetic basis of simple traits, from the study of induced mutations in model organisms. However, most traits are complex, and their development is directed by many genes that are influenced by environmental conditions. The Cresko laboratory will examine natural populations of threespine stickleback fish to understand the developmental genetic basis of variation in complex head and jaw traits. These structures vary tremendously among individuals, populations and species. Despite this diversity, the development of head and jaw structures occurs through conserved genetic interactions and will prove highly informative about the proper development of similar structures in other vertebrates such as humans. This research will provide a much better understanding of the genetic basis of complex traits, be they characters important for stickleback, or the most common types of human diseases that afflict tens of millions of people. Dr. Cresko's group has an outstanding of outreach to elementary students and also of undergraduate training. They are also active contributors to the resources of the research community.

Biologists are just beginning to understand the genetic basis of variable aspects of or-ganisms such as size, shape and color, to name a few. However, most traits are com-plex, their development directed by many genes whose effects are influenced by environmental conditions. These combined effects on trait variation must be mediated through the identity and behavior of cells such that, for example, some cells proliferate more to make larger cartilages, or excrete additional matrix to make stronger bones. However, little is presently known about the precise mechanisms of cellular integration, and filling this gap in knowledge is a fundamental problem in biology and is the primary focus of this project. Natural variation in threespine stickleback fish provides an excel-lent opportunity to address this question. The goal of this research is to understand how cells integrate genetic and environmental information and lead to variation in complex head and jaw traits among populations of stickleback. These structures vary tremen-dously among individuals, populations and species. Despite this diversity, all vertebrates share conserved genetic interactions for the development of head and jaw struc-tures. Thus, research on these traits is useful for understanding the situation in stickle-backs, and is also highly informative about the proper development of similar structures in other vertebrates such as humans. These conditions are the product of many genes and their interactions with the envi-ronment, and lead to variations in cellular identities or behaviors (i.e. uncontrolled cell growth in cancer). This research will provide a much better understanding of the genetic and cellular basis of complex traits, be they characters important for stickleback, or the most common types of human diseases. In addition, Alaskan stickleback populations are studied in collaboration with laboratories at the University of Alaska Anchorage, which has a large Alaska Native student population, and the unique opportunity exists to include individuals in this underrepresented group into research and educational activi-ties. Furthermore, an educational website is maintained that uses stickleback to provide educators and researchers around the world with knowledge and skills necessary to learn about and perform research with stickleback.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Biologists are making significant progress in understanding how changes in single genes allow organisms to cope with changing environments. However, it is still unknown how entire genomes - the total genetic information encoded in an organism?s DNA - respond to novel conditions. In particular, when independent populations adapt to similar environments, it is unclear what proportion of genomic changes occur in the same way. Studies of threespine stickleback fish provide an excellent opportunity to address this problem. Repeatedly, the ocean form of stickleback has invaded and become isolated in freshwater habitats, which has resulted in replicated patterns of divergence in traits such as bone size, body coloration and mating behavior. The goal of this research is to analyze patterns of divergence across entire genomes of multiple populations of Alaskan oceanic stickleback that colonized freshwater ponds formed during a massive earthquake in 1964. Using cutting-edge sequencing technology and newly developed techniques for evaluating genome-wide patterns of genetic variation, this project will evaluate the similarity of genomic responses of each population to their new freshwater habitats. This research provides a case study of the genomic changes that occur during the responses of organisms to both natural and human-caused environmental changes. In addition, the results of this work will provide a better understanding of the genomic basis of how organisms respond to climate change, another environmental perturbation that could stimulate rapid evolution. Graduate students and undergraduates including Alaska Natives will be part of the research, and a website provides elementary-school level materials about the project.

A significant challenge for biologists is understanding the genetic basis of variation among organisms. Meeting this challenge will require numerous organisms to become "genomically enabled", that is having a large number of genetic markers spread evenly throughout the genome that can be assembled into a genetic map. This will allow researchers to move very quickly from roughly locating a gene on a chromosome to identifying DNA sequences. In the past, developing these tools was time consuming and very expensive. The goal of this project is to develop a clear set of protocols and computational software to rapidly produce genomic analysis tools for just about any organism. After first testing the protocols on the well-developed model nematode worm, Caenorhabditis elegans, the techniques will be further refined by applying them to a non-model vertebrate organism, the pipefish Syngnathus scovelli. The research on these two organisms will serve as test cases from which an easily followed set of methods will be created and distributed widely, including the wet lab procedures for data generation and the software for the analysis of these data.

This research will have broad impacts by increasing the number of researchers who can genomically enable the organisms they study. This will lead to a more general set of answers to classic unanswered questions such as what genes and alleles are contributing to evolutionary change. In addition these tools will help biologists tackle practical problems such as what is the genomic basis of how organisms respond to climate change, a fundamental problem of our time.

DESCRIPTION (provided by applicant): The recent dramatic increase and geographic differences in frequency of reproductive diseases are likely influenced by changes in the environment, including perchlorate exposure. Perchlorate (ClO4-) is a persistent, chlorinated water-soluble contaminant that is pervasive in the United States. As a toxicant, perchlorate poses a major risk to human health through ingestion of contaminated water, food, and breast milk. Perchlorate is a known endocrine disruptor that competitively inhibits iodide uptake at the Sodium-Iodide Symporter (NIS) in the thyroid, thus hindering thyroid hormone synthesis. Studies demonstrate, however, that perchlorate exposure masculinizes both female and male stickleback fish (Gasterosteus aculeatus), leading to hermaphroditic females and males with testicular hypertrophy, results that are not predicted by a simple, direct thyroid- disruption mechanism. The goal of this project is to reconcile the dominant paradigm of perchlorate action - exclusively by disruption of NIS in the thyroid - with masculinization of behavior, physiology, and morphology in stickleback. The project's goal is to identify previously unsuspected pathways by which perchlorate may impact human reproductive health. Our working hypothesis is that perchlorate disrupts gonadal development by acting independently of the thyroid. Aim 1 will determine whether all observed phenotypic responses to perchlorate exposure in stickleback are mediated by the thyroid by rescuing thyroid hormone levels in perchlorate-exposed fish. Aim 2 will define the functional roles of NIS and NIS-paralogs in disruption of gonadal development by perchlorate using in situ hybridization to localize mRNA (Aim 2a), loss-of-function experiments to knock down expression of NIS and NIS-paralogs with morpholino anti-sense oligonucleotides and induced mutations using zinc finger nucleases (Aim 2b), and gain-of-function experiments by over- expressing the NIS and NIS-paralogs (Aim 2c). Aim 3 will determine the mechanism by which perchlorate alters sex differentiation using whole genome transcription profiling to determine which genes are early responders to perchlorate exposure, which are likely to be downstream genes, and whether responding genes are related to thyroid or gonad development. Quantitative PCR (qPCR) and in situ hybridization will verify expression profiling results. Significance. The proposed experiments will identify molecular and physiological pathways by which perchlorate disrupts gonadal development, whether solely via NIS in the thyroid or by other mechanisms. Because perchlorate is a pervasive contaminant in the U.S., our proposed work has direct implications for human health, particularly regarding thyroid diseases and disorders of sexual development. PUBLIC HEALTH RELEVANCE: Perchlorate is a persistent, water-soluble contaminant that is pervasive in the United States and poses a major risk to human health through ingestion of contaminated water, food and milk. Perchlorate not only inhibits thyroid activity, but also alters sexual development in stickleback fish, a commonly used model organism in genetic studies. The recent dramatic increase and geographic differences in frequency of human reproductive disorders are likely due to changes in the environment, including perchlorate exposure. Proposed experiments will identify genes and gene functions that, under the insult of perchlorate contamination, disrupt normal development of male and female gonads. Experiments will also determine the hormonal mechanisms that translate genetic changes into developmental disorders of the gonads. This research will advance our understanding of human thyroid diseases and the recent epidemic of impaired human reproductive health.

DESCRIPTION (provided by applicant): Human disease often involves changes in the timing and level of gene expression, processes investigated by transcriptomics, the identification of each expressed gene and its transcriptional levels. Studies of the association of human variation with altered gene expression can lead to a deeper, mechanistic understanding of disease states and can suggest therapies. A promising route to discovery is the transcriptomic analysis of novel models of common human diseases in aquatic species for which few genomic resources presently exist. Breakthroughs in sequencing technologies have opened the door for transcriptomics in non-model organisms. As detailed at the Sept. 2010 workshop 'Realizing the Scientific Potential of Transcriptomics in Aquatic Models', several impediments currently hinder the successful use of these tools in aquatic model systems. The goal of this project is to overcome these barriers by developing the protocols and tools necessary for researchers to capitalize on recent advances in DNA sequencing technology to better perform transcriptome analyses on aquatic medical models. This project will develop optimal laboratory protocols, analytical theory and computational software for transcriptome analyses of aquatic models of human disease and will help researchers plan and analyze experimental results. Protocols and tools will be made available via the Galaxy web platform as easy-to-use interfaces for computational software and web-based tutorials. Because many researchers working with aquatic non-model organisms lack access to the latest sequencing facilities and computational expertise for the analysis of high throughput transcriptomics, mechanisms will be developed for aquatic model organism researchers to use the Univ. of Oregon High Throughput Sequencing Facility. Similarly, many researchers lack access to computer clusters sufficiently powerful to execute the computational demands of modern transcriptomics, so this project will increase the availability of computational pipelines running at the Univ. of Oregon. This project will therefore provide widely needed tools, raise barriers to progress, and improve methods and technologies for transcriptome analysis in aquatic medical models, thus advancing our understanding of the role of gene regulation in health and disease.

How do organisms survive in and adapt to novel, stressful conditions? Genetic variation that is not exposed in a normal environment (called cryptic genetic variation) may nevertheless be exposed through phenotypic plasticity in novel environments. A key gap in our understanding of cryptic genetic variation is the identification of its molecular basis and evolution. When exposed to ecologically relevant stressful environments in the lab, the nematode Caenorhabditis remanei evolves rapidly in response to selection, and populations alter their response to alternative environments. The PIs will use next-generation sequencing technology to identify the key genetic and transcriptional changes that have occurred in these evolved lines, enabling the identification of genes and developmental pathways that expose cryptic variation in new environments.

Currently little is known about how the exposure of this cryptic genetic variation by a new environment can impact evolution in natural populations. As global climate change rapidly alters habitats and creates novel environments, many species of economic and conservation concern are faced with new environmental challenges. Thus it is becoming increasingly important to understand how environmental factors interact with genes in populations to direct evolutionary change. This research improves our understanding of this process.

Research Area III: Evolution of Host-Microbe Systems. We will advance the fundamental understanding of the evolution of host-microbe systems by analyzing how variations in host-microbe systems correlate with natural genetic variation among host populations.

Little is known about the genetic basis of evolution to novel environments in natural populations. Addressing this problem is of broad importance because populations of organisms increasingly need to either adapt to a changing environment or go extinct. Few studies have identified genes important for adaptation in natural populations. This research into the rapid evolution of the vertebrate head offers a unique opportunity to conduct a statistically high-powered study on individuals that have undergone adaptive evolution in less than 50 years. The results of this study could provide insight into the genetic and developmental mechanisms of rapid evolution.

The vertebrate head is an impressive structure that houses some of the most important organs. Across vertebrates head shapes are diverse, and much of this variation appears adaptive. In contrast the early programs of head shape development are highly conserved across vertebrates. This begs the question; where in the conserved genetic programs of head development does variation lie to direct such a diversity of head shapes? This question will be answered by identifying genes that underlie head shape variation in the threespine stickleback fish. New methods of visualizing and measuring head morphology will be used in conjunction with genomic data from a pedigreed threespine stickleback population to map genomic regions regions associated with head shape variation.

Evolution by natural selection is thought to often take thousands or millions of years. The environments in which species live can change drastically on much shorter timescales, however, and species must adapt quickly or risk extinction. Along the west coast of the US and Canada, a small fish species, the threespine stickleback, is known to adapt within decades to wildly different environments. This study will examine how stickleback achieve this feat by focusing on the influence a species' gene pool has on its ability to respond rapidly to a changing environment. Specifically, the proposed research will examine (1) how much of adaptive evolution is aided by genetic variation already existing in the stickleback gene pool, and (2) how the stickleback's history of adaptation has influenced the organization of genetic variation in its entire genome. This understanding of adaptive potential is crucial to our ability to predict how species and ecosystems will respond to future environmental shifts, and thus has implications for conservation and game management efforts, climate change mitigation, and agriculture.

Standing genetic variation is the pool of available genetic variants in a population available for evolutionary change. Recent research, facilitated by next-generation DNA sequencing technologies and new analytical techniques, has revitalized the debate over the relative influence of standing genetic variation versus new mutation on adaptive evolution and speciation. The research funded by this grant will use approaches based in coalescent theory, a mathematically-rigorous approach at the interface of phylogenetics and population genetics, to understand the prevalence and influence of standing variation in natural populations of threespine stickleback fish. By combining restriction site-associated DNA sequencing, a powerful method for genome-wide inference, with new long-read sequencing capabilities, the proposed research will generate data-rich genealogies at tens of thousands of loci and provide unprecedented detail into adaptation of natural populations.

This research will experimentally manipulate gene expression to understand how elongate fishes, such as pipefish, get their shape. Fishes, like other vertebrates, share a conserved set of genes that regulate development. Yet from this conserved set, fishes come in an amazing diversity of shapes. Pipefish are part of a group of nearly 300 species that also includes seahorses, pipehorses, and seadragons, that have remarkably diverse traits such as long snouts, tube-like bodies, and males that become pregnant. These fishes provide a unique opportunity to study trait evolution because of the breadth of characters absent in other fish lineages and the existence of powerful experimental tools based on the pipefish genomic. This project will involve the research training of an undergraduate from groups underrepresented in science. It will also contribute to public outreach in collaboration with The Museum of Natural and Cultural History at the University of Oregon.

This project will connect observed genetic and genomic changes with the morphological changes to the body plan in the Gulf pipefish (Syngnathus scovelli). This research focuses on a small subset of Hox transcription factors and their surrounding genomic content that are important in early cranial and axial skeleton development. Based on analysis of the Gulf pipefish genome, a few key Hox genes and nearby regulatory elements have been lost through the course of evolution in the Gulf pipefish lineage. Using advanced genomic and gene editing techniques, the researchers will perform functional tests in related teleost fish, the threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), to examine the morphological impact of the loss of genes and genomic content on the fish body plan. Possible effects of these losses on the evolution of the syngnathid body plan will be determined by creating mutations in these orthologous genes using the CRISPR technique in zebrafish and threespine stickleback fish models. This work will advance our understanding of how novel characters evolve in the context of the deeply conserved developmental toolkit.

The evolution of novel traits can change the way that organisms interact with their environments to survive, grow and reproduce. Deep knowledge of the underlying genes and developmental changes that underly most evolutionary innovations is sparse, as is understanding of the ecological consequences for both the organisms in which novel traits emerged and the organisms with which they interact in communities. A particular gap in understanding is how the evolution of novel traits influences the biodiversity of their associated microbial communities. This project will help fill this gap in our knowledge by studying a remarkable innovation ? male pregnancy in seahorses, pipefish and seadragons. This project will include the creation of new genome sequences and detailed studies of the developmental genetic underpinnings of the embryo brooding structures that make male pregnancy possible. The consequences of pouch evolution on the complexity and function of the community microbes in the pouch will also be studied, as well as how this unique host-associated microbiota can affect the fitness of embryos in the pouch. This project will provide research training to high school students, teachers, and undergraduates from underrepresented groups through immersive outreach and targeted support programs. The project will also support training of the next generation of scientists via education of Ph.D. students and postdoctoral scholars. Outreach to general public will be accomplished through public talks and through creation of a museum exhibit on syngnathid biology paired with web resources to support K-12 education.

Male pregnancy, accompanied by morphologically diverse embryo brooding structures, is a defining evolutionary innovation in syngnathid fishes. The goal of this project is to build an integrative understanding of the developmental genetic origin of this remarkable syngnathid novelty and its role in mediating multi-level ecological interactions with host-associated microbiota. This project will include production of 19 new annotated reference genomes strategically sampled across the syngnathid lineage, morphogenetic analysis and transcriptional/epigenetic profiling of the developing pouch in a comparative framework that leverages the repeated, independent evolution of complex brooding structures in the family, and analysis of brood pouch biocomplexity as a determinant of pouch-associated microbiome assembly. When complete, this project will provide novel insights into genome structural evolution in syngnathids, identify protein sequence and gene regulation changes involved in brood pouch development, and address whether the evolution of the brooding tissues created specialization in host regulation of microbiota with consequences for brooded progeny. The work will attract new researchers to syngnathids for studies of evolutionary innovation and diversification. The project will provide research training to high school students, teachers, and undergraduates from underrepresented groups, and will support education of graduate students and postdoctoral scholars, including the opportunity to take intense short courses to learn next generation sequencing, bioinformatics, complex statistical analyses, and genome editing. Educational outreach to general public will be accomplished through public lectures by the PIs, and through creation of a museum exhibit on syngnathid biology, which will be paired with an associated web resource directed toward K-12 education.

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.