Early embryogenesis is highlighted by the emergence of several embryonic end extraembryonic lineages. One such lineage is the primitive endoderm, which will eventually give rise to the yolk sac. Once believed to be a vestigial structure, the yolk sac is now believed to play a more prominent role in embryogenesis as it provides nutrients to the preimplantation embryo. The endoderm may also interact with the trophectoderm lineage, as they develop in close contact within the embryo. The efficiency of developing primitive endoderm in vitro is considerably low, leading to a lapse in our understanding of its development and function in cattle and other ruminants. The goal of the first study was to establish a protocol for developing primitive endoderm cultures and characterizing these cells. Bovine embryos were produced in vitro, and primitive endoderm outgrowths were created with fibroblast growth factor 2 (FGF2) supplementation. These cells can be produced in culture with 80.3 5.6% efficiency. Furthermore, outgrowths can be maintained in culture for 6-8 weeks before reaching a quiescent state. A true bovine primitive endoderm cell line does not currently exist, however, these cells hold potential in improving the current understanding of early lineage specification in cattle. A second set of studies was performed to examine the effects of maternal obesity on the preimplantation conceptus and endometrium. Exposure to maternal obesity in utero affects offspring development at the postnatal, adolescent, and adult stages of development; however, its impacts on early embryogenesis are not well studied. This work utilized an obese ewe model. Once the obese phenotype was established, ewes were bred. Conceptus and endometrial tissue were collected at D 14 of pregnancy, and samples were processed for RNA-sequencing analysis. There were no differences in pregnancy rate, ovulation rate, or pregnancies/ovulation between obese and lean animals. At an RPKM threshold of 0.2, fold-change 2, and FDR 0.05, 669 and 21 differentially expressed genes (DEGs) were identified between obese- and lean-derived endometrial samples and conceptus samples, respectively. Likewise, 137 DEGs were identified between male and female conceptuses. The PANTHER GO-Slim Biological Process system identified several biological processes affected by obesity in both the endometrium and conceptus tissue. GO terms do not currently exist for "placenta" and "trophoblast", so a literature search was conducted to identify DEGs involved in implantation and placentation. This revealed 125 placentation DEGs in the endometrium, and 4 DEGs in conceptuses between obese and lean groups. A follow-up study was conducted to examine the abundance of transcripts with regulatory roles in embryogenesis. Conceptuses exhibited differential expression of DNA methyltransferase 1 (DNMT1) based on obesity exposure, fibroblast growth factor receptor 2 (FGFR2) in a sex*obesity interaction, and peroxisome proliferator-activated receptor gamma (PPARG) and prostaglandin-endoperoxide synthase 2 (PTGS2) in a sex-specific manner. Collectively these results identify the preimplantation period as a susceptible time to maternal obesity in both conceptus and endometrial tissue. Together, these studies aim to provide a better understanding of the events controlling early embryogenesis, and insight into the implication of insults during this time. These findings will prove to be beneficial in establishing the link between maternal health, endometrial function, and subsequent offspring outcomes, with the hope of promoting a more viable embryo and thus healthier offspring. / Ph. D. / Early embryogenesis in cattle is afflicted with high embryonic loss, costing the dairy and beef industries a fair portion of their profits. The mechanisms behind these losses are not well understood, however, cellular miscommunications during lineage specification are likely to blame. Of particular interest is the endoderm lineage, which gives rise to the embryonic yolk sac. Initially believed to be a transient structure, we now believe the yolk sac is indispensable in embryogenesis as it provides nutrients to the preimplantation embryo. Our current understanding of primitive endoderm and the resulting yolk sac in cattle is severely lacking because few primitive endoderm in vitro models exist. A portion of the following work is focused on developing a protocol for producing primitive endoderm cell lines in vitro. This work improved the rate of producing primitive endoderm cells in vitro and characterized those cells. These cells will be used as a tool to better understand the mechanisms involved in early embryogenesis. Furthermore, they may help identify targets for manipulating early development to lessen the high rate of embryonic loss in cattle. The stage of early embryogenesis may also be particularly susceptible to intrauterine stressors, such as maternal obesity, because of the lineage segregation events occurring at this time. Insults to the earliest lineages can have lasting developmental effects, as these cell types will give rise to the embryo proper, yolk sac, and placenta. The effects of maternal obesity have been extensively studied in the postnatal, adolescent, and adult stages of development, however, insights into the effects on early embryogenesis are missing. The final studies of the following work are focused on the effects of maternal obesity on the preimplantation ovine conceptus and endometrium. This work utilized RNA-sequencing technology as well a qRT-PCR to identify differential gene and transcript expression in conceptus and endometrial samples collected from lean and obese ewes. Following analysis, we identified several crucial biological processes affected by maternal obesity. Of particular interest were those involved in implantation and placentation, indicating developmental programming events during early embryogenesis may be at fault for the abnormal offspring outcomes observed in previous studies. This work highlights the susceptibility of the preimplantation conceptus to maternal obesity and identifies the endometrium as a mediator between maternal nutrition and conceptus development. Additionally, this work identifies alterations in genes involved in placentation in both the conceptus and endometrium, indicating developmental programing events have occurred. As a whole, this work developed a new tool for examining early embryogenesis and the specification events that occur during that time. It also examines the embryonic impacts of maternal obesity during that critical window of development. These findings will prove to be invaluable in factors involved in early embryo development and function in ruminant species.

Hhex transcription factor is expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic effect of Hhex in the embryo and its dual role as a transcriptional repressor/activator suggest the presence of different interaction partners capable of modulating its activity and function. In the current study we identified two new Hhex protein interactors: SOX13 and c-Myc. We show that Hhex interacts directly with SOX13. By doing so, Hhex sequesters SOX13 from the SOX13•TCF1 complex, overturning SOX13-dependent repression of the Wnt pathway. On the other hand, Hhex induces proliferation of non-tumorigenic human fibroblast through a Myc-dependent mechanism. Hhex and c-Myc interact directly upregulating Cyclin D1, a c-Myc target gene involved in cell cycle progression and proliferation. Elevation of Cyclin D1 might be the final effector of Hhex capacity to regulate cell proliferation.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

ESC/iPSC-derive somatic cells may be ideal for treating disorders caused by cellular deficiency or dysfunction. To form a lineage-specific cell population, ESCs/iPSCs undergo a multi-step process that recapitulates embryonic development. ESC/iPSC differentiation protocols are hampered by the limitation of our understanding in development. Zebrafish embryos are fertilized and developed externally, a feature facilitates the observation and manipulation of embryonic development. To explore the zebrafish as a system to study cell lineage determination, in this thesis, I 1) identified an ortholog of the key pluripotency regulator Nanog in zebrafish and examined its role in early cell fate determination; 2) developed a high-throughput image-based chemical screening system in zebrafish blastomere cell culture that is very similar to, but much faster than, ESC/iPSC differentiation screens. Specifically, in an effort to examine the role of Nanog in vivo, I identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. I examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. The study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction. In the second part of the thesis, I developed a system that allows high-throughput image-based chemical screening using cultured zebrafish blastomere cells. To demonstrate its potential, this system is utilized to study skeletal muscle development. I screened 2,400 chemicals, finding 11 chemicals that block mature muscle cell differentiation and 17 chemicals that block skeletal muscle progenitor formation. The subsequent studies of these hits illustrate an RTK-PI3K-mTOR- GSK3 signaling cascade that is critical for skeletal muscle development. Preliminary data in mouse Skeletal Muscle Precursors (SMPs) suggest the pathway is conserved in murine adult muscle stem cells. This system, which can be modified for any cell lineage, promises to enhance our understanding of fundamental biology and to identify chemicals for cell-based therapies for many diseases.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

Instructive signals, whether external or internal, play critical roles in regulating the developmental potency or ability to self-renew of stem cells. External signals may range from secreted growth factors to extracellular matrix proteins found in the stem cell niche. Internal signals include activated signalling cascades and the eventual transcriptional mechanisms they initiate. In either fashion, stem cells are regulated in a complex temporal and context specific manner in order to maintain or maximise their unique characteristics. Previous experiments suggest that Wnt3a plays a role in maintaining the pluripotent state of mouse embryonic stem (mES) cells. However, in the absence of leukemia inhibitory factor (LIF), Wnt signalling is unable to maintain ES cells in the undifferentiated state. This implies that maintaining the pluripotent state of mES cells is not the primary function of canonical Wnt signalling. To further characterize the role of Wnt3a in pluripotency and lineage specification undifferentiated and differentiated mES cells were induced with Wnt3a. Wnt3a induced the formation of a metastable primitive endoderm state and upon subsequent differentiation, the induction of large quantities of visceral endoderm. Furthermore, we determined that the ability of Wnt3a to induce a metastable primitive endoderm state was mediated by the T-box transcription factor Tbx3. Our data demonstrates a novel role for Wnt3a in promoting the interconversion of undifferentiated mES cells into a pluripotent primitive endoderm state. Aging of skeletal muscle tissue is accompanied by fibrosis, atrophy and remodeling all of which negatively affect muscle performance. Whether this reduction in skeletal muscle competency is directly attributed to a resident adult stem cell population called satellite cells remains largely unknown. Here, we undertook an investigation into how age affects the transcriptional profile of satellite cells and their repopulating ability following transplantation. We determined that as satellite cells age, both their regenerative capacity and ability to colonize the satellite cell niche is reduced. Additionally, we identified satellite cell specific transcriptional profiles that differed with respect to age. Therefore, we conclude that intrinsic factors are an important determinant of satellite cell regenerative capacity during the aging process.

Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques acinars en patologies exocrines (pancreatitis i càncer de pàncrees), la manca de models normals basats en cèl·lules ha limitat l’estudi de les alteracions que succeeixen en el programa de diferenciació pancreàtica. Hem demostrat prèviament que les cèl·lules mare embrionàries murines, que són pluripotents, poden adquirir un fenotip acinar in vitro. Això es va aconseguir, en part, amb una combinació de senyals que provenien del cultiu de pàncrees fetals que no era, però, específic del llinatge pancreàtic. L’objectiu d’aquest treball ha estat el de desenvolupar un protocol selectiu pel llinatge acinar basat en l’activació seqüencial de vies de senyalització que recapitulin el desenvolupament pancreàtic in vivo, a través de la formació definitiva de l’endoderm, l’especificació pancreàtica i acinar i l’expansió/diferenciació de progenitors acinars. El tractament de cossos embrionaris amb Activina A va promoure l’expressió de gens d’endoderm com està prèviament descrit. El tractament subsegüent amb àcid Retinoic, FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va resutar en la inducció dels marcadors de progenitors pancreàtics Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les cèl·lules van ser a continuació cultivades en Matrigel utilitzant un sistema de cultiu en 3D en presència de fol·listatina, dexametasona i KGF comportant una inducció significativa dels nivells de mRNA i proteïna de marcadors acinars i una disminució de l’expressió dels de marcadors acinars. A més, es va veure que Amyl es secretava en el medi. Aquestes dades indiquen que l’activació selectiva del programa de diferenciació acinar en cèl·lules mare embrionàries es pot dur a terme mitjançant una inducció esgraonada de vies de senyalització involucrades en el desenvolupament pancreàtic exocrí proporcionant una eina potencial per estudiar la diferenciació pancreàtica i malalties relacionades amb el pàncrees. / Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.

Adult haematopoiesis occurs in the context of a supportive stromal cell niche. The bone marrow, spleen and thymus all contain specific, but relatively poorly defined, stromal cells, which are important for maintenance of quiescence and directed differentiation. Even less is known about the haematopoietic niche during haematopoietic development. The formation of red blood cells (erythropoiesis) occurs during haematopoiesis, and is also controlled by a variety of stromal cells. This thesis examined the visceral endoderm, a group of cells that surrounds the developing epiblast and is required for primitive erythropoiesis (early blood production). We attempted to determine which factors in the visceral endoderm were responsible for induction of primitive erythropoiesis, and whether they would be useful as blood induction factors in embryonic stem cell differentiation. Thus, I attempted to immortalise the visceral endoderm using an immortalising agent (SV40Tag), driven off of a previously identified visceral endoderm gene, Indian hedgehog. We modified a bacterial artificial chromosome so that SV40Tag was driven off of the Indian hedgehog gene. The modified bacterial artificial chromosome was used in both pronuclear injections of mouse blastocysts and the electroporation of embryonic stem cells. After neither attempt produced a visceral endoderm cell line, we examined a visceral endoderm-like cell line, END2, for the presence of the blood inducing factors. We demonstrated the ability of END2 conditioned media to apparently increase expression of blood transcripts in differentiating embryonic cells indicating the presence of blood inducing factors. Expression profiles of END2 cells were compared to a previously completed embryonic stem cell differentiation profile to identify enriched genes. Two genes, angiopoietin-like 7 and Bc064033, were tested for an ability to induce blood in differentiating embryonic stem cells. When neither protein was capable of inducing blood, the END2 cells were examined for the presence of other known blood inducing factors and similarity to in vivo visceral endoderm. The END2 cells were found to produce bone morphogenetic protein 4, a potent inducer of blood in embryonic stem cell differentiation, which complicated the search for additional factors. Examination of END2 cells also indicated a lack of visceral endoderm markers such as alpha fetoprotein, indicating that the END2 cells may not be as visceral endoderm-like as expected from the current literature. The previously identified Indian hedgehog gene was also examined for its blood induction abilities in vivo. Indian hedgehog knockout mice were examined for the effect gene removal had on both primitive and definitive erythropoiesis. Levels of primitive erythrocytes were unaffected in the Indian hedgehog knockout mice, but levels of definitive erythrocytes were found to be significantly decreased. Further examination of Indian hedgehog knockout fetal livers also showed that they had decreased numbers of haematopoietic stem cells. The haematopoietic stem cells were fully capable when cultured and generated appropriate numbers of progenitor cells, indicating a non-intrinsic cause for this defect. Levels of hedgehog target genes that are usually highest in the stromal compartment were also found to be most significantly decreased in Indian hedgehog knockout fetal livers. Another hedgehog gene, desert hedgehog, was also shown to be expressed in the fetal liver and may act with Indian hedgehog to regulate stromal production in the fetal liver.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Instructive signals, whether external or internal, play critical roles in regulating the developmental potency or ability to self-renew of stem cells. External signals may range from secreted growth factors to extracellular matrix proteins found in the stem cell niche. Internal signals include activated signalling cascades and the eventual transcriptional mechanisms they initiate. In either fashion, stem cells are regulated in a complex temporal and context specific manner in order to maintain or maximise their unique characteristics. Previous experiments suggest that Wnt3a plays a role in maintaining the pluripotent state of mouse embryonic stem (mES) cells. However, in the absence of leukemia inhibitory factor (LIF), Wnt signalling is unable to maintain ES cells in the undifferentiated state. This implies that maintaining the pluripotent state of mES cells is not the primary function of canonical Wnt signalling. To further characterize the role of Wnt3a in pluripotency and lineage specification undifferentiated and differentiated mES cells were induced with Wnt3a. Wnt3a induced the formation of a metastable primitive endoderm state and upon subsequent differentiation, the induction of large quantities of visceral endoderm. Furthermore, we determined that the ability of Wnt3a to induce a metastable primitive endoderm state was mediated by the T-box transcription factor Tbx3. Our data demonstrates a novel role for Wnt3a in promoting the interconversion of undifferentiated mES cells into a pluripotent primitive endoderm state. Aging of skeletal muscle tissue is accompanied by fibrosis, atrophy and remodeling all of which negatively affect muscle performance. Whether this reduction in skeletal muscle competency is directly attributed to a resident adult stem cell population called satellite cells remains largely unknown. Here, we undertook an investigation into how age affects the transcriptional profile of satellite cells and their repopulating ability following transplantation. We determined that as satellite cells age, both their regenerative capacity and ability to colonize the satellite cell niche is reduced. Additionally, we identified satellite cell specific transcriptional profiles that differed with respect to age. Therefore, we conclude that intrinsic factors are an important determinant of satellite cell regenerative capacity during the aging process.