Tag: William Cresko

Expression of FGF Signaling Genes during Threespine Stickleback Development

Presenter: Micah Woods – Environmental Science

Co-Presenter(s): Vithika Goyal

Faculty Mentor(s): Hope Healey, William Cresko

Session: (In-Person) Poster Presentation

The fibroblast growth factor (fgf) signaling pathway is essential to vertebrate craniofacial development. Alterations in fgf receptors and ligands can lead to craniofacial disorders. While deleterious effects are observed in response to pathway mutations in many vertebrates, syngnathid fishes (pipefishes, seahorses, seadragons) have lost several of these genes (fgf3, fgf4, and others). Syngnathids have also evolved unique craniofacial features, such as an elongated snout, important to suction feeding, and absence of teeth. Since fgf3 and fgf4 are involved in craniofacial development, it is possible that their loss in syngnathids is related to the family’s unique faces. Our lab is investigating the developmental impact of the loss of fgf3 and 4 in syngnathids. To establish the ancestral expression patterns of fgf genes, we studied stickleback fishes due to their recent divergence from syngnathids. Using in situ hybridization, we assessed the spatial localization of fgf expression in stickleback embryos through development. Embryos were imaged and fgf/fgfrs staining patterns were compared to zebrafish. We observed expression of fgfr1a and fgf3 in the pharyngeal arches of stickleback embryos, paralleling zebrafish. Understanding the ancestral expression patterns of genes in the fgf signaling pathway reveals the deep conservation of the fgf signaling pathway in stickleback and provides opportunities for better interpreting the impact of the losses of these genes in syngnathids.

Expression of FGF Signaling Genes during Threespine Stickleback Development

Presenter(s): Vithika Goyal — Marine Biology

Co-Presenter(s): Micah Woods

Faculty Mentor(s): Hope Healey, William Cresko

Session: (In-Person) Poster Presentation

The fibroblast growth factor (fgf) signaling pathway is essential to vertebrate craniofacial development. Alterations in fgf receptors and ligands can lead to craniofacial disorders. While deleterious effects are observed in response to pathway mutations in many vertebrates, syngnathid fishes (pipefishes, seahorses, seadragons) have lost several of these genes (fgf3, fgf4, and others). Syngnathids have also evolved unique craniofacial features, such as an elongated snout, important to suction feeding, and absence of teeth. Since fgf3 and fgf4 are involved in craniofacial development, it is possible that their loss in syngnathids is related to the family’s unique faces. Our lab is investigating the developmental impact of the loss of fgf3 and 4 in syngnathids. To establish the ancestral expression patterns of fgf genes, we studied stickleback fishes due to their recent divergence from syngnathids. Using in situ hybridization, we assessed the spatial localization of fgf expression in stickleback embryos through development. Embryos were imaged and fgf/fgfrs staining patterns were compared to zebrafish. We observed expression of fgfr1a and fgf3 in the pharyngeal arches of stickleback embryos, paralleling zebrafish. Understanding the ancestral expression patterns of genes in the fgf signaling pathway reveals the deep conservation of the fgf signaling pathway in stickleback and provides opportunities for better interpreting the impact of the losses of these genes in syngnathids.

Genetic Basis of Evolution of Armor Phenotypes in Threespine Stickleback

Presenter: Taylor Wilson, Biology

Poster: D-6

Mentor: William Cresko, Biology

Differences in physical traits among species arise first as variations within and among populations. Studying this intraspecific variation provides important new knowledge about the process that gives rise to biodiversity. To better understand this variation and its genetic and developmental basis I am studying a small fish, the threespine stickleback (Gasteroseus aculeatus). The threespine stickleback is undergoing rapid evolution in bodies of water recently formed by uplift and glacial melt, diverging into very different anadromous and freshwater life history forms. With each saltwater-to-freshwater evolutionary transition, there is a reduction in the stickleback’s bony armor plates and characteristic spines. In order to discover the genetic basis for the diversity of armor phenotypes, I am measuring variation in the size and number of lateral and pelvic armor structures of 148 stickleback collected from the McKenzie River in Springfield, OR. The genetic variation in these same individuals has been measured using high-throughput sequencing techniques.

We are correlating these genetic data with specific aspects of the armor variation, such as spine lengths and plate sizes. This is the first such study in stickleback, and is providing us with key information on which genes are responsible for the rapid evolution in some of the phenotypes of the threespine stickleback. My results will have general importance beyond stickleback by providing new knowledge about the genetic and genome basis of evolution in the wild.

Spatiotemporal Patterns of Inversion Allele Frequencies in Threespine Stickleback

Presenter :Erika Jackson

Mentor : William Cresko

Major : Biology

Poster 21

Chromosomal inversions have been linked to complex traits that facilitate adaptation in new environments in a small number of stud- ies. However, the generality of this pattern is still unclear. Studying the frequency of chromosomal inversions in threespine stickleback (Gasterosteus aculeatus) could provide an important case study to help us understand the role of chromosomal inversions in adaptive evolution. We focused on Alaskan and Oregon marine and freshwater stickleback populations to determine inversion frequencies between distant locations as well as between salinities in different bodies of water. We predicted that inversion allele frequency diver- gence between ocean and freshwater populations would occur if the inverted region contained genes important for adaptation to the alternative environments. We genotyped a large number of individuals for an inversion on Linkage Group XXI using polymerase chain reaction (PCR) designed to indicate alternate forms of the inversion. Our results showed that the inversion allele frequencies are highly divergent between Alaskan oceanic and freshwater populations. In addition, while Alaskan populations are geographically distant from Oregon populations, we found a similar pattern of divergence between Oregon populations in the two habitats. Our study provides ad- ditional evidence that chromosomal inversions may play an important role in adaptation to novel environments.

Effects of the Aquatic Contaminant Perchlorate on Expression of NIS Clade Genes in Divergent Populations of Threespine Stickleback

Presenter: Amanda Redmond

Mentor: William Cresko

Poster: 28

Major: Biology

Perchlorate is a known endocrine disruptor and a wide spread environmental contaminant that causes hypothyroidism in humans. Our previous work demonstrated that perchlorate results in a masculinizing effect in anadromous threespine stickleback (Gasterosteus aculeatus), but it is not known whether ecotypes of stickleback respond differently to this contaminant. Perchlorate has a known effect of suppressing thyroid hormone synthesis by competitively inhibiting the sodium-iodide symporter (slc5a5). The large slc5 gene family is associated with thyroid function and vitamin/mineral transport in all vertebrates. Using bioinformatic approaches we have identified that
there are several closely related genes to slc5a5 in stickleback including slc5a6a, slc5a6b, slc5a8a, and slc5a8b.
We investigate the effect of perchlorate on developmental gene expression in three genetically diverged populations of stickleback from Rabbit Slough (oceanic), Boot Lake (freshwater), and Riverbend (freshwater) ecosystems. We have developed RNA anti-sense probes for the NIS genes that we will be using for gene expression analysis using in situ hybridization to document the spatio-temporal expression of slc5a6a, slc5a6b, slc5a8a, and slc5a8b genes in each ecotype in response to perchlorate exposure. Because evolutionarily diverged populations of stickleback living in different habitats have different osmoregulatory and developmental nutrient needs, we hypothesize that a single chemical that affects solute transporters may have variable developmental effects among populations. In a preliminary study, we have mapped the temporal and spatial distribution of these genes in Rabbit slough (oceanic) fishes, and have found that they are expressed starting at 8-10 days in numerous tissues including the thyroid and the gonad. Our goal next is to identify gene expression patterns of the slc5 genes in the thyroid and gonad at 8, 14, and 30 days post fertilization (dpf) in the diverged populations, and to test for changes in spatial or temporal expression of the genes based on population, perchlorate treatement, and/or an interaction of both. We hypothesize that a gene by environment interaction (G-by-E) will lead to unique patterns of slc5a5 clade gene expression response to perchlorate in each of the three populations. Using cryosections of both control and 100ppm perchlorate treated fish we will determine the expression of these genes at 8, 14, and 30dpf. Our study will be relevant to understanding more about effects of toxin exposure between very genetically divergent populations of fish and other vertebrates, including humans.

Perchlorate, Oxidative Stress and Thyroid Proliferation in Threespine Stickleback

Presenter: Connor Fitch

Mentor: William Cresko

Poster: 15

Major: Biology 

Perchlorate is a water contaminant that has been detected in drinking water across the U.S. Severe health effects result from exposure to high levels of perchlorate. Our lab has shown that perchlorate causes several abnormalities
in fish, including increased thyroid proliferation in stickleback and zebrafish. We aim to determine the molecular basis of perchlorate’s effects on thyroid development and cell proliferation. Two hypotheses for increased cell proliferation include activation of Wnt signaling pathways, and perchlorate reduced expression of miR-16, miR-24, and miR-195, a group of microRNAs that suppress cell proliferation. We hypothesize that perchlorate causes direct damage by increasing oxidative stress in threespine stickleback, subsequently activating Wnt pathways and leading to cell proliferation. In order to test for the presence of oxidative stress, we performed an assay to calorimetrically quantify the presence of malondialdehyde (MDA)—a known oxidative stress biomarker. Forty stickleback embryos (20 control, 20 chronically exposed to 100ppm perchlorate) between 100-150 days post fertilization were analyzed for MDA. Perchlorate significantly increased the presence of MDA by 0.34μM in the treated group, correlating to a 13% increase of MDA over the control group. This supports our hypothesis that perchlorate causes oxidative stress in fish. Perchlorate causes a multitude of abnormalities during development and our data demonstrate that oxidative stress may be one contributing factor. Currently we are performing qPCR analysis to determine the involvement of SOD, tp53, and axin2a/b in oxidative stress from perchlorate. In addition, we are using qPCR to examine if perchlorate decreases expression of miR-16, miR-24, and miR-195. These data will help us to determine a molecular connection between perchlorate and oxygen radicals on interruption of normal thyroid function.

Exposure to perchlorate affects differentiating germ cells in teleost fish

Presenter: Nathaniel Earp

Mentor: William Cresko

Poster: 37

Major: Human Physiology 

Perchlorate is a widespread contaminant in the environment. We have found that perchlorate has masculinizing effects on threespine stickleback, Gasterosteus aculeatus. The mechanism of this masculinization is unknown. Our previous studies found that during development female stickleback show increased proliferation of undifferentiated primordial germ cells (PGC) followed by a larger wave of apoptosis compared to male stickleback. We hypothesize that perchlorate treated stickleback will show a decrease in total number of PGCs compared to control fish. To test our hypothesis, stickleback of 15 and 20 days post fertilization (dpf) – a critical time for primary sexual differentiation– were grown from fertilization to the end of the experiment in 10 or 100 ppm perchlorate treated medium or control medium with no perchlorate. Stickleback were sectioned and stained with hematoxylin and eosin, and visualized by light microscope. Total number of PGCs were counted and characterized as either pre-meiotic or meiotic based on morphology of the PGC and nucleus. Perchlorate treated fish show a significant decrease (27.6%) in total number of PGCs compared to controls. Furthermore, control stickleback showed a decrease in total number of PGCs from 15 dpf to 20 dpf, presumably due to female germ cell apoptosis, while perchlorate treated stickleback did not show this decrease. These findings are consistent with perchlorate’s masculinizing effect. We are further investigating the effects of perchlorate on total PGC count in zebrafish, Danio rerio, as perchlorate has been shown to have a feminizing effect on zebrafish, giving us an opportunity to explore the basic mechanisms underlying the reproductive abnormalities caused by perchlorate. Our findings contribute to understanding mechanisms of perchlorate induced reproductive abnormalities in vertebrates, and to better understand the underlying process of sex determination in two divergent teleosts.

The Cellular Basis of Dermal Bone Evolution and Development in Threespine Stickleback Fish

Presenter: Sophie Sichel

Mentor: William Cresko and Kristin Alligood, Biology

Poster: 58

Major: Biology 

In vertebrates the development of the cranial skeleton is imperative because it provides structure and support for a number of critical organs. Cranial structures vary immensely across vertebrates, but how did these different mechanisms of morphogenesis evolve at the developmental and cellular level? To elucidate the molecular mechanisms controlling variation in morphogenesis, I used the opercle bone of threespine stickleback fish as a model. Threespine stickleback are used as a model to investigate vertebrate evolution because of rapid changes between ancestral oceanic and derived freshwater forms of this fish. The opercle bone is a neural crest-derived dermal bone that is critical for respiration, foraging and communication in stickleback, undergoes morphogenesis during development, and varies among populations. Neural crest cells form the facial skeleton of vertebrates through intramembranous ossification. After the initial condensation, bone shaping is hypothesized to be dependent on the recruitment of new osteoblasts in a space and time dependent manner, and variation in bone morphogenesis would therefore be linked to differences in osteoblast recruitment. To test this hypothesis, I conducted in situ hybridizations using a probe to col10a1, which is a gene present in the cartilaginous precursor of mature dermal bone, on stickleback embryos at various times during embryonic development. This probe allowed me to visualize the developing opercle and a proliferation assay to visualize proliferating cells thought to be contributing to the developing bone. I quantified the number of proliferating cells and determined their distribution near the developing edge of the opercle in two different populations of stickleback: a population that exhibits an ancestral phenotype and a population representing the derived phenotype. I will present results describing how the number of cells recruited varies among populations. Determining the molecular and genetic factors that underlie opercle development and how they differ between ancestral and derived populations could provide evidence for how development and evolution interact on a larger scale.

Advancing threespine stickleback as an outbred immunogenetics model by pinpointing the onset of adaptive immunity

Presenter(s): Emily Niebergall

Faculty Mentor(s): Emily Beck & William Cresko

Poster 54

Session: Sciences

T-cell deficiencies cause a wide range of cell-mediated immunodeficiencies including Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and DiGeorge Syndrome. The genetics underlying these deficiencies is complex and the genetic basis of many cell- mediated deficiencies is poorly understood. Due to the invasive nature of prenatal tests used to study T-cell deficiencies in mammals, the development of an outbred immunogenetics model system is needed to understand how genetic variation impacts phenotypic variation of immune disease. Threespine stickleback fish (Gasterosteus aculeatus) provide just such a model. Stickleback are genetically tractable laboratory organisms with a well-annotated genome, and individuals from disparate populations show high levels of genetic variation. Additionally, stickleback provide an excellent system to study T-cell deficiencies, as they experience external fertilization, providing an amenable system to study immune development. To characterize the early development of adaptive immunity in threespine stickleback, we will analyze the expression of known early indicators of adaptive immunity maturation in marine and freshwater stickleback. These include recombination activating genes, rag1 and rag2, and T cell receptor genes, tcr-β and tcr-γ. To analyze gene expression, we will perform rtPCR on a developmental time series of fish. We can then implement in situ hybridization to detect when and where the genes are first expressed, followed by flow cytometry to detect phenotypic variation of T cell activity. Knowing when adaptive immunity onset occurs in threespine stickleback advances these fish as an outbred disease model in immunogenetics studies, allowing manipulative studies of immunological disease phenotypes in the context of genetic variation.

Advancing threespine stickleback as an outbred immunogenetics model by pinpointing the onset of adaptive immunity

Presenter(s): Emily Niebergall—Biology

Faculty Mentor(s): William Cresko, Emily Beck

Session 4: Earning your Stripes

Understanding when the onset of the adaptive immune system occurs is important for understanding host-microbe interactions and etiology of disease . While the onset of adaptive immunity has been studied in inbred animal models, i .e . mice and zebrafish, these laboratory models lack the genetic diversity found in humans and may not be appropriate for all studies . We are advancing threespine stickleback fish (Gasterosteus aculeatus) as a novel outbred immunogenetics model to elucidate the complexities of these interactions in the context of genetic variation . It is currently unknown when adaptive immunity is onset in threespine stickleback . To pinpoint the timing of onset of adaptive immunity, we looked at the expression of an early adaptive immune gene known to be involved in T-lymphocyte development throughout a developmental time series . T-lymphocytes are a primary adaptive immune cells able to recognize and elicit a response against pathogens . Early development of these cells utilizes two interconnected protein complexes: CD3 and TCR . The pre-TCR/CD3 supercomplex has been used to study the ontogeny of the immune system and has provided insight into the development of the adaptive immune system . In this study, we chose to focus on cd3d, a gene involved in the CD3 complex . Similar work determining the onset of adaptive immunity in other fish has produced a wide range of results, from 72 hours post fertilization to 20 days post hatching (dph) . We found that by 10 dph, cd3d was expressed in all individuals, with population level variation indicating some may exhibit expression earlier in development .