Browsing by Author "Bouma, Gerrit J., committee member"
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Item Open Access Biomarkers of disease progression and chemotherapeutic resistance in canine osteosarcoma(Colorado State University. Libraries, 2011) O'Donoghue, Liza E., author; Duval, Dawn L., advisor; Thamm, Douglas H., committee member; Bouma, Gerrit J., committee member; Weil, Michael, committee memberOsteosarcoma is the most common primary bone malignancy in both humans and dogs. Over 10,000 canine patients develop this highly aggressive cancer annually and many succumb to metastatic disease in less than a year. In recent years, canine osteosarcoma has been increasingly recognized as an excellent model for the disease in humans, especially with regard to the molecular biology of the disease. Thus, research targeted at canine osteosarcoma benefits not only dogs but the field of human oncology as well. Research into the genetic and molecular derangements of osteosarcoma in both species has identified a number of oncogenes and tumor suppressor genes that may contribute to tumorigenesis. Additionally, some mediators of invasion and metastasis have been recognized (e.g. Ezrin, matrix metallopeptidases). Despite this, only a limited number of studies have been performed that examine the molecular genetics of osteosarcoma in the context of patient outcome. Thus, with the aim of identifying new target genes and pathways that contribute to disease progression and chemoresistance in osteosarcoma, we first performed transcriptomic and genomic analyses of primary tumors from dogs that had experienced good or poor outcomes following definitive treatment for osteosarcoma. These broad survey experiments yielded a selection of targets for future investigation. To further focus in on the genes that were most deranged from "normal" expression patterns, we compared gene expression patterns from tumors to those of normal bone. This study provided valuable perspective on genes that were identified in the outcome-based experiments, allowing selection of four promising gene targets to pursue. We next set out to validate in vitro models of canine osteosarcoma so that mechanistic studies could be pursued. Assays to test species and short tandem repeat identity were adapted to cell lines in use in our facility and presumed osteosarcoma cell lines were verified to be bone-derived via PCR testing of a bone-specific marker. Additionally, four anti-human antibodies were validated for use in canine samples. Two genes whose expression progressively altered with increased tumor aggressiveness where chosen for further study: insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and n-Myc downstream regulated gene 2 (NDRG2). IGF2BP1 has been identified as an oncofetal protein and its mRNA was strongly overexpressed in patients with the worst outcome while it was virtually undetectable in normal bone. We identified one possible mechanism for dysregulation of this gene in OSA and we also discovered that knock down of this gene in a canine osteosarcoma cell line inhibited cell invasion. NDRG2 has been dubbed a tumor suppressor in a number of different tumor types yet had not been previously investigated in osteosarcoma. We found NDRG2 mRNA to be underexpressed in all tumors relative to normal bone; patients with poor outcomes had the lowest expression levels. Multiple isoforms of the gene were found to be expressed in canine samples: these were cloned and transfected into a low-NDRG2-expressing cell line. Exogenous expression of NDRG2 in this in vitro system enhanced sensitivity to doxorubicin, one of the drugs most commonly used to treat osteosarcoma. Additionally, three possible mechanisms of dysregulation of this gene were identified. The studies presented herein progress from fact-finding surveys to in-depth functional examination of two genes that likely contribute to osteosarcoma invasion and chemoresistance. Furthermore, additional genes identified in our survey experiments offer promise for future studies into molecular mechanisms of osteosarcoma metastases and chemotherapeutic resistance. Finally, these studies have laid the groundwork for the development of gene-expression-based prognostic screens for dogs with osteosarcoma.Item Open Access Characterization of selected gene expression patterns as potential markers for oocyte quality in young versus old mares(Colorado State University. Libraries, 2010) Rodrigues, Bernardo de Lima, author; Clay, Colin M., advisor; Carnevale, Elaine M., advisor; Tjalkens, Ronald B., committee member; Bouma, Gerrit J., committee member; Bruemmer, Jason E., committee memberAs a female ages a series of alterations in normal physiology take place, during this process fecundity decreases. The reproductive system starts to shut down as a consequence of hormonal, histological and anatomical changes, and in the middle of this process, playing a critical role, is the oocyte. As in women, fertility decreases with aging in mares. Recently the mare has been suggested as a promising model to study age-related infertility in women due to similarities in the reproductive cycle and similar age-associated reproductive changes. During the last decades the use of assisted reproductive techniques (ART) in human and veterinary medicine to treat infertility has increased. Unfortunately, ART can only partially compensate for declining fertility- particularly the age related decline in fertility. Therefore, we need to understand the mechanisms involved in age-associated infertility and improve both the diagnostic tools and the techniques currently used in ART. In that regard the identification of reliable oocyte quality markers is of great interest, specifically of extrinsic markers in follicular cells and follicular fluid (FF). Follistatin (FS1) and anti-müllerian hormone (AMH) have been suggested as potential oocyte quality markers. In addition, the rate of apoptosis in follicular cells has also been suggested to be a good indicator of oocyte quality In this study, our goal was to use the young and old mare model to obtain competent and incompetent oocytes, respectively, to try and elucidate the involvement of apoptosis of follicular cells and/or of the oocyte in the determination of oocyte quality. Oocytes, follicular fluid, granulosa and cumulus cells were collected through transvaginal follicular aspirations from young ( 4-10 years) and old (>20 years) mares. Preovulatory follicles were aspirated 30-36 h post induction of follicular maturation, which was performed by administration (i.v) of a combination of deslorelin and hCG when the biggest follicle reached 35 mm. We used real time PCR to examine expression of pro-apoptotic ( CASP2, CASP 3) and pro-survival (XIAP) genes, as well as of FST and AMH expression in these cell types. In addition we measured androgens and estrogens in FF and calculated the androgens to estrogens ratio to assess follicular atresia. We also sought to determine FF concentrations of FST and AMH, and relate it to oocyte quality. There was no difference in CASP3 expression levels in granulosa and cumulus cells between the two age groups. In addition, there was no difference in CASP2 and CASP3 mRNA expression in oocytes from young and old mares. XIAP mRNA levels were expressed 3.3 fold higher in oocytes from young when compared to old mares, and there was a tendency for XIAP to be more highly expressed in granulosa cells of young mares. In contrast, the levels of XIAP mRNA in cumulus cells were 1.46 fold higher in old when compared to young mares. There was no difference in the expression levels of AMH in granulosa cells between young and old mares, but in cumulus cells there was a tendency for AMH to be higher expressed in cells from old vs. young mares. Unfortunately we were not able to analyze AMH FF concentrations. FST mRNA levels in oocytes were similar between the age groups, but FST concentrations in FF of preovulatory follicles from young mares (197 ± 16.7 ng/mL) were higher (p=0.02) than in FF from old (153.3 ± 22.7 ng/mL) mares. In both age groups FST FF concentrations in preovulatory follicles significantly decreased when compared to mid-estrus and post-deviation follicles. In conclusion, we believe that our data suggest that FST follicular fluid levels can be a non-invasive marker to assess oocyte quality in the horse, and that FST levels decrease in preovulatory follicles of the horse. In addition, expression levels of caspase-3 in follicular cells, and caspases 3 and 2 in the oocyte, does not seem to be involved in the mechanism of fertility loss in the old mare. Finally, XIAP mRNA levels may be important for oocyte quality in the horse.Item Open Access Endocrine actions of IFNT during early ruminant pregnancy(Colorado State University. Libraries, 2013) Romero, Jared Jerome, author; Hansen, Thomas R., advisor; Campen, Hana Van, committee member; Bouma, Gerrit J., committee member; Nett, Terry M., committee member; Davis, John S., committee memberThe mechanisms of how conceptus-derived interferon tau (IFNT) induces interferon stimulated genes (ISGs) and cell survival genes through endocrine action that contributes to resistance of the Corpus Lutum (CL) to prostaglandin Fα (PGF) and the maintenance of early pregnancy in sheep were examined. Using microarray screens, several genes were identified in the CL to be significantly up-regulated [ISG15 and myxovirus (influenza virus) Resistance 1; MX1], maintained [interleukin 6; IL-6, Pentraxin, long 3; PTX3, luteinizing hormone receptor; LHR, and vascular endothelial growth factor; VEGF] or down-regulated [serpin peptidase inhibitor, clade E; SERPINE1, thrombospondin 1; THBS1] in response to pregnancy and luteolysis. These studies in the CL were expanded to other tissues (endometrium, liver, uterine vein tissue) and bodily fluids (blood and histotroph) to identify other endocrine actions of IFNT and other conceptus secretory products that could be used to explain mechanisms of establishment and maintenance of pregnancy and to be used as blood markers for pregnancy status. The extensive examination of key genes described herein that were differentially regulated during Days 12-15 of the estrous cycle and pregnancy in ewes is novel. The experiments are the first to examine extensive temporal regulation of key genes associated with luteolysis such as estrogen receptor (ESR1), oxytocin receptor (OXTR), prostaglandin transporter (SCLO2A1) and caspase 3 (CASP3); and luteotrophic-cell survival genes such as Phosphoinositide-3-Kinase/V-Akt Murine Thymoma Viral Oncogene (PI3K/AKT) and ISGS. To identify novel markers of pregnancy status and establishment of pregnancy, global mass spectroscopy was used for analysis of the proteome and metabolome. From this extensive analysis, 14 proteins/metabolites were more extensively examined: Acetyl-carnitine, Carnitine, Ecdysteroids, N-acetyldileucine, Valine, Collagen, Type I, Alpha 1 (COL1A1), Collagen, Type I, Alpha2, (COL1A2) Annexin A1, Annexin A2, Annexin A5, IFNT, Trophoblast Kunitz Domain Protein 1 (TDKP1) Profilin 1 (PFN1) and S100 Calcium-Binding Protein A11 (S100a11). The critical roles for these metabolites and proteins are discussed in context of the establishment and survival of a pregnancy. To confirm that IFNT has an endocrine role during early pregnancy, a highly specific [no cross-reaction with Interferon alpha, beta and gamma (IFNA, IFNB or IFNG)] and sensitive (23.95 pg/ml in serum) IFNT radioimmunoassay was validated herein. IFNT could be detected using this refined assay in ewes from Days 13-16 of pregnancy in histotroph, in uterine vein serum by Day 15-16 of pregnancy and as early as Day 19 of pregnancy in tail blood from pregnant dairy cows. Through these studies it was determined that Day 14 is a very pivotal day for the establishment of pregnancy in ewes. Detection of IFNT in tail blood of pregnant cows on Day 19 provides further evidence for the endocrine action and systemic release of IFNT during pregnancy in ruminants.Item Open Access Glial signaling mechanisms in the progression of neuroinflammatory injury(Colorado State University. Libraries, 2018) Popichak, Katriana A., author; Tjalkens, Ronald B., advisor; Bouma, Gerrit J., committee member; Goodrich, Laurie R., committee member; Legare, Marie E., committee member; McLean, Jennifer L., committee memberThe response of glial cells to foreign and endogenous stress signals is extensive. As a result, release of inflammatory factors as means of cellular communication and innate immune function, or neuroinflammation, can contribute to neurodegeneration and increased activation of surrounding glia, often associated with Parkinson's disease (PD). The identification of glial activation as an early event in the progression of neurodegenerative disease that precedes neuronal cell death presents an opportunity for better diagnostic markers, as well as new pathways that could be targeted therapeutically. The transcription factor, Nuclear Factor-kappa B (NF-κB), regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells but the signaling factors modulating glial-glial and glial-neuronal signaling during neurotoxic injury are poorly understood. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Recently, it was found that selected orphan nuclear receptors in the NR4A family (nerve growth factor-induced-β/NGFI-β), including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NF-κB but there are no approved drugs that target these receptors. In the current studies, we utilized several experimental approaches to target neuroinflammation in cellular models of PD and manganese neurotoxicity in primary glia and in animal models. One of these studies demonstrated that a novel ligand of NR4A1 and NR4A2, 1,1-bis (3'-indolyl) -1-(p-methoxyphenyl) methane (C-DIM5), suppressed NF-κB-dependent inflammatory gene expression in astrocytes following treatment with 1-methyl-4-phenyl-1, 2, 3,6-tetrahydropyridine (MPTP) and the inflammatory cytokines, IFN-γ and TNF-α. These data were further supported by previous studies from our laboratory, which examined efficacy of multiple C-DIM compounds in PD animal and cellular models, including one (C-DIM12) identified as a modulator of Nurr1 activity that also inhibited NF-kB-dependent gene expression in glial cells. Collectively, these data demonstrate that NR4A1/Nur77 and NR4A2/Nurr1 dynamically regulated inflammatory gene expression in glia by modulating the transcriptional activity of NF-κB. An additional study examined the role of NF-κB in manganese (Mn)-induced neurotoxicity by exposing purified microglia, astrocytes (from both wild-type and an astrocyte- specific NF-kB (IKK2) knock-out (KO) mouse) and mixed glial cultures to varying Mn concentrations and then treated neurons with the conditioned media (GCM) of each cell type. In doing so, we showed that mixed glial cultures exposed to Mn enhanced glial activation and neuronal death compared to microglia, wild type astrocytes or IKK2-knockout astrocytes alone or in mixed cultures suggesting that astrocytes are a critical mediator of Mn neurotoxicity through enhanced expression of inflammatory cytokines and chemokines, including those most associated with reactive phenotype such as C3 and CCL2. Thus, these studies elucidate key mechanisms associated with neuroinflammation and present potential therapeutic targets in glial cells that regulate the progression of neuroinflammatory injury.Item Open Access Influence of cardiolipin remodeling on mitochondrial respiratory function in the heart(Colorado State University. Libraries, 2013) Le, Catherine Hoang, author; Chicco, Adam J., advisor; Bouma, Gerrit J., committee member; Hamilton, Karyn L., committee member; Pagliassotti, Michael J., committee memberThe following investigation comprises a series of experiments with the overall aim of elucidating the role of cardiolipin acyl-chain remodeling on mitochondrial respiratory function in the mammalian heart. The experiments tested the general hypothesis that changes in the fatty acid composition of cardiolipin, a unique mitochondrial phospholipid, contribute to cardiac mitochondrial respiratory dysfunction, which is believed to be an underlying mechanism of myocardial hypertrophy and contractile dysfunction in several cardiac pathologies. The specific aims of each experimental series were to: 1) Determine the influence of cardiolipin compositional changes on cardiac mitochondrial respiratory function in models of aging, pressure overload hypertrophy and heart failure, and 2) Determine how defects in the cardiolipin remodeling process itself elicits cardiac mitochondrial respiratory dysfunction associated with a genetic childhood-onset cardiomyopathy, known as Barth syndrome. Studies in Aim 1 demonstrated that the distinct pattern of aberrant cardiolipin remodeling observed in the aged and failing heart resulted from increased metabolism of polyunsaturated fatty acids (PUFAs), which predominate in the cardiolipin molecular species. Pharmacological inhibition of delta-6 desaturase, the rate-limiting enzyme in the PUFA metabolism pathway, reversed cardiolipin remodeling, reduced myocardial hypertrophy and preserved contractile function in the rodent models of aging, pressure overload and hypertensive heart disease. However, in contrast to our hypothesis, reversal of these changes in cardiolipin composition did not have a significant effect on mitochondrial respiratory function, dissociating alterations in cardiolipin composition from cardiac mitochondrial respiratory dysfunction in these conditions. Studies in Aim 2 demonstrated a marked substrate-specific impairment of cardiac mitochondrial respiratory function in mice lacking the cardiolipin remodeling enzyme, tafazzin. Cardiac mitochondria from tafazzin-deficient mice demonstrated a selective impairment in carbohydrate and lipid oxidation and a dramatic decrease in pantothenic acid amounts. These data suggest a role of tafazzin in the transport, activation, and/or generation of reducing equivalents by the TCA cycle. Additionally, these data implicate impairment of tafazzin function and/or cardiolipin remodeling process itself, rather than alter cardiolipin composition per se, in mitochondrial dysfunction associated in Barth syndrome. Collectively, these findings challenge previous studies suggesting that alterations of the distinctly uniform acyl-chain composition of cardiolipin impair cardiac mitochondrial respiration. Rather, they instead show that the widely observed redistribution of cardiolipin PUFA content in chronic cardiac pathologies appears to reflect a global increase in PUFA metabolism that profoundly influences cardiac structure and function by mechanisms we are only beginning to understand. Interestingly, the cardiolipin remodeling process itself and/or tafazzin enzyme may play a more important role than previously thought in cardiac mitochondrial respiratory function and cardiomyopathy.Item Open Access Influence of FADS2 expression on cardiovascular risk: role of mitochondrial arachidonic acid(Colorado State University. Libraries, 2020) Li Puma, Lance Christopher, author; Chicco, Adam J., advisor; Bouma, Gerrit J., committee member; Amberg, Gregory C., committee member; Gentile, Christopher L., committee member; Legare, Marie E., committee memberLong-chain polyunsaturated fatty acids (LC-PUFA) are widely believed to influence cardiovascular health and disease in humans and can be supplied through the diet or endogenously synthesized from the essential PUFAs linoleic acid (LA; n6) and alpha-linolenic acid (ALA; n3). Redistribution of PUFAs in serum and tissue phospholipids has been associated with various pathologies, manifesting primarily as a proportional loss of the essential PUFA LA paralleled by reciprocal increases in its long-chain product arachidonic acid (AA; n6). Epidemiological studies have linked greater AA/LA ratios in serum phospholipids to multiple parameters of cardiometabolic disease (CMD), such as obesity, insulin resistance, hypertension, atherosclerosis, and coronary artery disease. Single nucleotide polymorphisms in the FADS2 gene are associated with haplotypes of greater expression of FADS2, which may increase the production of AA from LA and increase serum AA/LA ratios. FADS2 encodes delta-6 desaturase, the rate-limiting enzyme in endogenous LC-PUFA biosynthesis, which is believed to participate in the development of CMD by interacting with the high dietary LA content found in the modern Western diet to disproportionally produce AA over other LC-PUFAs. The overarching hypothesis of this dissertation is that greater expression of FADS2 promotes the development of cardiovascular risk parameters. To investigate this, we generated mice with global (CMV promoter) transgenic overexpression of Fads2 (Fads2TG); these mice exhibit classic serum shifts in PUFA distribution characteristic of human FADS2 polymorphisms. A series of three projects were undertaken: 1) Investigate the interaction between dietary essential fatty acid intakes and Fads2 expression on cardiovascular risk; 2) Establish methodology for simultaneous measurement of mitochondrial respiration and ROS release in vitro; 3) Investigate effects of Fads2 expression on cardiac mitochondrial responses to Ca++-overload. These studies discovered that greater Fads2 expression is sufficient to increase several aspects of cardiovascular risk that were independent of the dietary ratio of n6:n3 essential PUFAs. Further investigation demonstrated that cardiac cardiolipin AA content predicted ischemia-reperfusion (IR) injury, suggesting a mechanistic role of mitochondria in this phenotype. Gain- and loss-of-function approaches in mice established that greater Fads2 expression lowers mitochondrial tolerance to Ca++-overload demonstrated by loss of OXPHOS-linked respiration, greater mitochondrial ROS release, and increased mitochondrial permeability transition. Furthermore, mitochondrial permeability transition by Ca++-overload could be attenuated by inhibition of AA release or metabolism. Collectively, the significance of these studies establish the influence of Fads2 on serum and tissue PUFA composition and the pathogenesis of IR injury through modulation of mitochondria membrane composition, thereby demonstrating Fads2 expression as an independent factor for cardiovascular risk.Item Open Access Serum exosome profile as related to early pregnancy status in the mare(Colorado State University. Libraries, 2011) Hergenreder, Joanna R., author; Bruemmer, Jason E., advisor; Bouma, Gerrit J., committee member; Veeramachaneni, D. N. Rao, committee member; Clay, Colin McKeown, committee member; Han, Hyungchul, committee memberDuring early pregnancy in the mare the conceptus and mare must communicate in order to establish and maintain pregnancy. This coordinate communication is most pronounced between days 12 and 16 post-ovulation, at which time the conceptus is highly mobile throughout the uterus preventing endometrial prostaglandin F2α release and subsequent luteolysis. The mechanism behind successful establishment and maintenance of pregnancy in the mare is currently unknown. Recently, cell-secreted vesicles, called exosomes, were detected in high amounts in serum of pregnant women. Exosomes are 50-100 nm vesicles containing bioactive materials such as mRNA, miRNA, and protein. Exosomes can mediate immune-responses through membrane protein interaction and delivery of bioactive products into cells. Interestingly, exosomes have been described in various body fluids, including urine, breast milk, and serum. We hypothesized that exosomes are present in serum in the mare and that their relative amount differs with pregnancy status. To test this hypothesis, we determined the presence and relative amount of exosomes in serum of pregnant and non-pregnant mares. Serum samples were obtained from mares in a cross-over design, with each mare serving as both a pregnant treatment and non-mated control (n=3/day). Blood samples were obtained by jugular venipuncture on days 12, 14, 16, and 18 post-ovulation. Serum was removed, snap frozen, and stored at -80°C. Exosome isolation, for flow-cytometry and transmission electron microscopy (TEM), was performed using ExoQuick (System Biosciences, Inc.), a precipitation solution designed to isolate exosomes from fluids. After exosome isolation, samples were analyzed using flow cytometry with 100 nm sized beads as an internal control and a counting bead standard for relative amount determination. Flow cytometry analysis revealed the presence of exosomes in serum of both pregnant and non-pregnant mares in variable amounts. Furthermore, analysis revealed the presence of two distinct size populations, one of smaller exosomes (< 100 nm) previously undescribed, which were more abundant in mare serum from day 12 of pregnancy, and the second of the expected 100 nm size at each day examined. TEM analysis validated the results from the flow cytometry as each population, determined by size and granularity, was visually characterized. Along with the 100 nm and slightly smaller sized vesicles, TEM also revealed the presence of vesicles slightly larger than 100 nm, with small amounts of vesicles ∼200 nm in size, indicating the presence of exosomes as well as microvesicles. Therefore, we conclude that exosomes are present in mare serum and further characterization of such populations can provide clues about the intercellular mode of communication in early pregnancy.Item Open Access Tcfap2c regulation of primordial germ cell development(Colorado State University. Libraries, 2011) Guttormsen, Jillian Bosick, author; Winger, Quinton A., advisor; Bouma, Gerrit J., committee member; Anthony, Russell V., committee member; Garrity, Deborah, committee memberThe development of germ cells during embryonic development is driven by a complex expression pattern of genes. The transcription factor Tcfap2c is expressed in germ cells throughout development from specification to adult sperm and oocytes. Tcfap2c expression is first seen in primordial germ cells around embryonic day (E)6.75 and has been classified as a germ cell specification gene. This study implicates Tcfap2c as a potential key factor in germ cells during specification, proliferation, migration and differentiation. In order to investigate the role of Tcfap2c in germ cells, we utilized the Cre/loxP conditional gene mutation strategy. Cre/loxP allows us to overcome the early embryonic lethality that arises from loss of Tcfap2c in traditional knock-out mice by creating Tcfap2c null mutation in specifically-targeted tissues. We created Tcfap2c mutant mice using the epiblast-specific Sox2-Cre model. Mutant ovaries from this model failed to express both germ cell specific markers and meiotic markers at E12.5. Immunohistochemistry at E18.5 failed to detect the germ cell specific marker NOBOX or the meiotic protein SYCP3, which confirmed that Sox2-Cre, Tcfap2c mutant mice lacked germ cells at late embryonic stages. However, Sox2-Cre, Tcfap2c mutant mice die prior to or at birth preventing us from studying adult gonads from these mice. To this end we used tamoxifen inducible ERTM-Cre mice to create Tcfap2c mutation in adult animals. We assessed ERTM-Cre, Tcfap2c mutant animals for fertility and gametogenesis; surprisingly, fertility, spermatogenesis and oogenesis were not affected in Tcfap2c mutant gonads. These results show that Tcfap2c is not necessary for adult maturation of gonocytes to produce mature sperm and oocytes. However, Sox2-Cre, Tcfap2c mutants lack germ cells indicating that Tcfap2c is necessary during fetal germ cell differentiation. The Sox2-Cre model was limited because Tcfap2c was deleted in the entire embryo and the mutants died at birth. Prdm1-Cre was used to produce a mouse where Tcfap2c is only deleted in germ cells beginning around specification. Prdm1-Cre, Tcfap2c mutants initially specified germ cell-like cells at E7.5; however, by E8.5 the germ cell numbers were decreased and they had not initiated migration towards the genital ridges. By E9.5 few if any germ cells were observed in Prdm1-Cre, Tcfap2c mutants. At E12.5 no germ cells were seen in Prdm1-Cre, Tcfap2c mutant XX or XY gonads. Adult ovaries and testes from Prdm1-Cre, Tcfap2c mutant mice were noticeably smaller than littermate controls and showed no oogenesis or spermatogenesis. The Prdm1-Cre model showed that mutation of Tcfap2c results in loss of germ cells in embryos by E9.5 suggesting that Tcfap2c plays a role during germ cell specification, proliferation and migration. We identified Tcfap2c as an important factor during early germ cell development; however, Tcfap2c expression is observed in germ cells well past specification. We believe that Tcfap2c is present in germ cells during during fetal gonad differentiation because it plays a role in regulating the gene expression pathways necessary for this event. We show that Tcfap2c is expressed in germ cells during the period of fetal gonad differentiation. Gene expression analysis of gonads from E11.5-13.5 reveals Tcfap2c as the most highly expressed member of the Tcfap2 family member. Tcfap2c is a member of a transcription factor family that regulates gene expression by binding consensus sequences within target gene promoters. TCFAP2 binding sites are present in promoter regions of germ cell specific genes Cadherin1 (Cdh1) and Kit oncogene (Kit), as well as in the promoter regions of genes involved in regulating pluripotency High mobility group AT-hook 2 (Hmga2), Nanog homeobox (Nanog) and Lin28. Using chromatin immunoprecipitation we demonstrate that TCFAP2C binds the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28. The interaction between TCFAP2C and the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28 indicates that Tcfap2c likely plays a functional role in the regulation of these genes. These genes are necessary for germ cell survival, migration and pluripotency. In conclusion, our results provide a new understanding of the role of Tcfap2c during different stages of germ cell development from specification to differentiation.Item Open Access The role of LIN28 in the molecular regulation of placenta development and function(Colorado State University. Libraries, 2013) Seabrook, Jill L., author; Winger, Quinton A., advisor; Clay, Colin M., committee member; Bouma, Gerrit J., committee member; DeLuca, Jennifer G., committee memberTo view the abstract, please see the full text of the document.