Dissertations / Theses on the topic 'Embryonic development'

The formation of the embryonic vasculature is essential for life. The components driving this process are well conserved across vertebrate species. At the core of vascular development is the specification of endothelial precursor cells from nascent mesoderm. Transcription factors of the ETS family are important regulators of endothelial specification. In this document we characterize the role of the ETS transcription factors, ETV2, during embryonic vascular development.Expression analysis shows that Etv2 is highly expressed in hematopoietic and endothelial precursor cells in the Xenopus embryo. In gain-of-function experiments, ETV2 is sufficient to activate ectopic expression of vascular endothelial markers. In addition, ETV2 activated expression of hematopoietic genes representing the myeloid but not the erythroid lineage. Loss-of-function studies indicate that ETV2 is required for expression of all endothelial markers examined. However, knockdown of ETV2 has no detectable effects on expression of either myeloid or erythroid markers. This contrasts with studies in mouse and zebrafish where ETV2 is required for development of the myeloid lineage. Our studies confirm an essential role for ETV2 in endothelial development, but also reveal important differences in hematopoietic development between organisms.Although ETV2 is a pivotal molecule in development it remains unidentified in the chicken genome. We hypothesize that chicken Etv2 is expressed in the early Gallus embryo, and is necessary for endothelial specification consistent with its role in other species. To test this hypothesis we attempted to amplify Etv2 transcripts from Gallus embryos using degenerate PCR. Disappointingly this strategy did not reveal a putative Etv2 candidate. However, some important findings were uncovered, including the cloning of a previously uncharacterized Gallus ETS protein, SPDEF. Additionally the identification of an annotation error mis-identifying Ets gene "Erf" as "Etv3" (also an Ets gene) provided details on gene arrangement previously unknown. The workflow described could be used in future studies for the identification of other members of gene families that exhibit gaps, keeping in mind the goal of the study and the limitations of each technology.

Successful mammalian development to term requires that embryonic and extra-embryonic tissues communicate and grow in coordination, to form the body. After implanting into the uterus, the mouse embryo is comprised of three cell lineages: first, the embryonic epiblast (EPI) that forms the embryo proper, second, the extra-embryonic ectoderm (ExE) which contributes to the foetal portion of the placenta, and third, the visceral endoderm (VE) that contributes to the yolk sac. These three tissues form a characteristic ‘egg-cylinder’ structure, which allows signals to be exchanged between them and sets the stage for body axis establishment and subsequent tissue patterning. The mechanisms underlying this process are difficult to study in vivo because a different genetically manipulated mouse line must be generated to investigate each factor involved. This difficulty has prompted efforts to model mammalian embryogenesis in vitro, using cell lines, which are more amenable to genetic manipulation. The pluripotent state of the EPI can be captured in vitro as mammalian embryonic stem cells (ESCs). Although mouse ESCs have been shown to contribute to all adult tissues in chimeric embryos, they cannot undertake embryogenesis when allowed to differentiate in culture. Previous studies have shown that ESCs formed into three-dimensional (3D) aggregates, called embryoid bodies, can become patterned and express genes associated with early tissue differentiation. However, embryoid bodies cannot recapitulate embryonic architecture and therefore may not accurately reflect what happens in the embryo. In this study, a new technique was developed to model early mouse development which is more faithful to the embryo. ESCs were co-cultured with stem cells derived from the ExE, termed trophoblast stem cells (TSCs), embedded within extracellular matrix (ECM). These culture conditions lead to the self-assembly of embryo-like structures with similar architecture to the mouse egg cylinder. They were comprised of an embryonic compartment derived from ESCs abutting an extra-embryonic compartment derived from TSCs, and hence were named ‘ETS-embryos’. These structures developed a continuous cavity at their centre, which formed via a similar sequence of events to those that lead to pro-amniotic cavity formation in the mouse embryo, and required active Nodal/Activin signalling. After cavitation, ‘ETS-embryos’ developed regionalised mesodermal tissue and primordial germ cell-like cells originating at the boundary between embryonic and extra-embryonic compartments. Inhibitor studies revealed that this occurred in response to endogenous Wnt and BMP signalling, pathways which also govern these tissue specification events in the early mouse embryo. To demonstrate that ‘ETS-embryos’ were comparable to mouse embryos at the global transcriptional level, RNA-sequencing was then performed on different tissue regions of ‘ETS-embryos’ and the resulting transcriptomes were compared to datasets from mouse embryos. These data showed that ‘ETS-embryos’ were highly similar to mouse embryos at post-implantation stages in their overall gene expression patterns. Taken together, these results indicate that ‘ETS-embryos’ are an accurate in vitro model of mammalian embryogenesis, which can be used to complement studies undertaken in vivo to investigate early development.

Branchial arches (BAs) are a series of transient structures that develop on the ventro-lateral surface of the head in vertebrate embryos. BAs initially appear as a series of similar segments; as development proceeds each BA will contribute to different structures. Here, it was investigated the transcriptional mechanisms that instruct the different fates of the BAs in development. Initially, each BA contains a blood vessel, known as aortic arch (AA) artery, that connects the dorsal aorta with the heart. Remodelling of the AAs is crucial to form the adult heart circulation. This process leads to regression of the anterior AAs, running though the first and second BAs (BA1 and BA2), and persistence of the AAs contained in more posterior BAs (PBA). To identify the mechanisms that control remodelling of the AAs, we compared the transcriptomes and epigenomic landscapes of different BAs. Using RNA-seq and H3K27Ac ChIP-seq, we uncovered the activation of a vascular smooth muscle cell (VSMC) differentiation transcriptional program exclusively in the PBAs (and not in BA1/BA2). In support of this finding, we show that VSMC differentiation occurs specifically in the PBAs, but not BA1-2 in mouse embryonic development. Despite the absence of VSMC differentiation in developing BA1-2, cells harvested from these tissues reveal a spontaneous tendency to differentiate towards VSMC fate when grown in vitro, and activate several VSMC-specific genes (Myocd, Acta2, Tagln, Jag1). Together, our results suggest that forming VSMCs is a key process for the persistence of AAs. We also showed that cells derived from all BAs have the potential to differentiate to VSMCs in vitro. However, only cells in the PBAs differentiate to VSMCs in vivo, resulting in the maintenance of posterior AAs. In this study, we also uncovered a novel transcriptional principle that specifies the fate of BA2. Using ChIP-seq, we found that binding of Meis transcription factors establish a ground pattern in the BAs. Hoxa2, which specifies BA2 identity, selects a subset of Meis-bound sites. Meis binding is strongly increased at these sites, which coincide with active enhancers, linked to genes highly expressed in the BA2 and regulated by Hoxa2. Thus, Hoxa2 modifies a ground state binding of Meis to instruct segment-specific transcriptional programs.

Recurrent processes are a general feature of living systems, from the cell cycle to circadian day-night rhythms to hibernation and flowering cycles. During development and life, numerous recurrent processes are controlled by genetic oscillators, a specific class of genetic regulatory networks that generates oscillations in the level of gene products. A vital mechanism controlled by genetic oscillators is the rhythmic and sequential segmentation of the elongating body axis of vertebrate embryos. During this process, a large collection of coupled genetic oscillators gives rise to spatio-temporal wave patterns of oscillating gene expression at tissue level, forming a dynamic prepattern for the precursors of the vertebrae. While such systems of genetic oscillators have been studied extensively over the past years, many fundamental questions about their collective behavior remain unanswered. In this thesis, we study the behavior and the properties of genetic oscillators from the single oscillator scale to the complex pattern forming system involved in vertebrate segmentation. Genetic oscillators are subject to fluctuations because of the stochastic nature of gene expression. To study the effects of noisy biochemical coupling on genetic oscillators, we propose a theory in which both the internal dynamics of the oscillators as well as the coupling process are inherently stochastic. We find that stochastic coupling of oscillators profoundly affects their precision and synchronization properties, key features for their viability as biological pacemakers. Moreover, stochasticity introduces phenomena not known from deterministic systems, such as stochastic switching between different modes of synchrony. During vertebrate segmentation, genetic oscillators play a key role in establishing a segmental prepattern on tissue scale. We study the spatio-temporal patterns of oscillating gene expression using a continuum theory of coupled phase oscillators. We investigate the effects of different biologically relevant factors such as delayed coupling due to complex signaling processes, local tissue growth, and tissue shortening on pattern formation and segmentation. We find that the decreasing tissue length induces a Doppler effect that contributes to the rate of segment formation in a hitherto unanticipated way. Comparison of our theoretical findings with experimental data reveals the occurrence of such a Doppler effect in vivo. To this end, we develop quantification methods for the spatio-temporal patterns of gene expression in developing zebrafish embryos. On a cellular level, tissues have a discrete structure. To study the interplay of cellular processes like cell division and random cell movement with pattern formation, we go beyond the coarse-grained continuum theories and develop a three-dimensional cell-based model of vertebrate segmentation, in which the dynamics of the segmenting tissue emerges from the collective behavior of individual cells. We show that this model is able to describe tissue formation and segmentation in a self-organized way. It provides the first step of theoretically describing pattern formation and tissue dynamics during vertebrate segmentation in a unified framework involving a three-dimensional tissue with cells as distinct mechanical entities. Finally, we study the synchronization dynamics of generic oscillator systems whose coupling is subject to phase shifts and time delays. Such phase shifts and time delays are induced by complex signaling processes as found, e.g., between genetic oscillators. We show how phase shifts and coupling delays can alter the synchronization dynamics while leaving the collective frequency of the synchronized oscillators invariant. We find that in globally coupled systems, fastest synchronization occurs for non-vanishing coupling delays while in spatially extended systems, fastest synchronization can occur on length scales larger than the coupling range, giving rise to novel synchronization scenarios. Beyond their potential relevance for biological systems, these results have implications for general oscillator systems, e.g., in physics and engineering. In summary, we use discrete and continuous theories of genetic oscillators to study their dynamic behavior, comparing our theoretical results to experimental data where available. We cover a wide range of different topics, contributing to the general understanding of genetic oscillators and synchronization and revealing a hitherto unknown mechanism regulating the timing of embryonic pattern formation.

Adenosine 5'-triphosphate (ATP) has been shown to be an important extracellular signaling molecule that mediates various physiological activities via the P2 (P2X and P2Y) receptors. However, information on the expression patterns of the P2 receptors during mammalian embryogenesis is limited. We therefore examined the expression patterns of different P2 receptor subtypes in rat embryos. In the hindbrain neural tube, the P2X3 receptor was transiently expressed at embryonic day E11 in the cranial motor neurons and the outgrowing axons. ATP significantly inhibited neurite outgrowth from neural tube explants. P2X3 receptors were also prominently expressed in sensory ganglia at this early stage and were coexpressed with P2X2 receptors in El6.5 embryos. Other P2X receptor subtypes were observed in different brain regions such as subventricular zones, the site of postnatal neurogenesis. In addition, the P2Y receptor expression was detected in the somites and subsequently in the developing skeletal muscle but was downregulated as development proceeded. While the P2Y1 receptor was no longer expressed in the adult skeletal muscle, the expression of P2Y2 receptor was present, although restricted in the satellite cells and the P2Y4 receptor showed reduced expression in adult skeletal muscle. Likewise, the expression of the P2Y receptors was initially expressed throughout the myocardium (El2) but was gradually restricted to the trabeculated myocardium (El4-18). Studies on Ca2+ influx showed that particular P2 receptor subtypes of P2X2, P2X4, P2Y1, P2Y2, P2Y4 and P2Y6 receptors responded to nucleotides in E14 cardiomyocytes. P2X7 receptor expression was detected in developing pancreatic islet cells and later coexpressed with glucagon in ?-cells. In addition, transient expression of the P2X7 receptor in insulin-expressing cells was observed in the embryonic, but not in adult, islet cells. Together, the results indicated that widespread and dynamic expression of P2 receptors was found in the three-germ layer-derived embryonic tissues, particularly in some transient embryonic structures during development, which suggested they may be important in embryonic organogenesis.

Transcription factors are a class of proteins that function to regulate the expression of genes. During early development, their role is to provide the precise order of activation or suppression of genes that are required for the formation of an embryo. A major goal of a developing embryo is to establish a complete body plan that includes the development of all of the organ systems. Thus it is paramount that the correct genes are switched on or off to insure that all organ systems form.Our studies investigate the role of several transcription factors involved in coordinating the expression of genes that are essential for the development of skeletal muscle and blood vessels.In the formation of skeletal muscle, a class of transcription factors called the myogenic regulatory factors (MRFs) is known to promote the induction of the structural genes that comprise the skeletal muscle. In fact, the MRF family member, MyoD, has been termed the "master regulator" of skeletal muscle gene expression. However, a recently discovered transcription factor, MASTR, has been suggested to play a role in skeletal muscle development. Our studies of MASTR are the first to demonstrate that, in vivo, MASTR is necessary and sufficient to activate genes involved in the formation of skeletal muscle. Furthermore, MASTR cooperates with MRFs to induce skeletal muscle genes and therefore places MASTR among a group of transcription factors, such as the MRFs, that are essential regulators of skeletal muscle development.In vascular development, the Flk-1 gene is critical to the formation of blood vessels. Mice lacking Flk-1 do not produce angioblasts, the precursor cells that give rise to the endothelial cells that make up blood vessels. In our efforts to understand the regulation of this important vascular gene, we have discovered a new function of the Kruppel-like transcription factor 2 (KLF2) to activate Flk-1 expression. Moreover, we have identified a new Ets transcription factor (Etsrp) capable of inducing Flk-1 expression alone and in cooperation with KLF2. These findings uncover a novel mechanism by which KLF2 and Etsrp act to promote the expression of Flk-1 during embryonic vascular development.

Establishment of the biochemical and molecular nature of cardiac development is essential for us to understand the relationship between genetic and morphological aspects of heart formation. The molecular mechanisms that underly heart development are still not clearly defined. To address this issue we have used two approaches to identify genes involved in early chick cardiac development. Differential display previously conducted in our laboratory led to the identification of two gene fragments differentially expressed in the heart that are further described in this thesis. The full-length cDNA sequence of both eukaryotic translation initiation factor-2b (eIF-2b) and NADH cytochrome b5 reductase (b5R) were isolated using library screening. The upreglation of these genes during heart development is expected given the heart is the first functional organ to form in vertebrates and protein synthesis and cell metabolism at this stage of development is maximal. Limitations in the differential display approach led to the development and optimisation of a subtractive hybridisation approach for use with small amounts of cells or tissue. To focus on cardiac gene expression during the initial phases of heart development, subtractive hybridization was performed between the cardiogenic lateral plate mesoderm of Hamburger and Hamilton stage 4 embryos and the heart primordia of stage 9 embryos. Of the 87 independent clones identified by this procedure, 59 matched known sequences with high homology, 25 matched unknown expressed sequence tag (EST) sequences with high homology, and 3 did not match any known sequence on the database. Known genes isolated included those involved in transcription, translation, cell signalling, RNA processing, and energy production. Two of these genes, high mobility group phosphoprotein A2 (HMGA2) and C1-20C, an unknown gene, were chosen for further characterisation. The role of each gene in early chick heart development and indeed development in general, was addressed using techniques such as in situ hybridisation, transfection analysis, in ovo electroporation and RNAi. HMGA2 is a nuclear phosphoprotein commonly referred to as an architectural transcription factor due to its ability to modulate DNA conformation. In keeping with this function, HMGA2/GFP fusion protein was shown to localise to the nucleus and in particular, the nucleolus. In situ hybridisation analysis suggested a role for HMGA2 in heart and somite development. HMGA2 expression was first detected at HH stage 5 in the lateral plate mesoderm, a region synonymous with cells specified to the cardiac fate. HMGA2 was also strongly expressed in the presomitic segmental plate mesoderm and as somites developed from the segmental plate mesoderm, the expression of HMGA2 showed an increasingly more restricted domain corresponding to the level of maturation of the somite. Restriction of HMGA2 expression was first detected in the dorsal region of the epithelial somite, then the dorsomedial lip of the dermomyotome, and finally the migrating epaxial myotome cells. The novel intronless gene, C1-20C, predicts a protein of 148 amino acids containing a putative zinc finger binding domain and prenyl binding motif. Zinc binding assays showed that the zinc finger domain of C1-20C/MBP fusion protein bound over six times the quantity of zinc compared to MBP alone, although not in a 1:1 stoichiometric molar ratio. C1-20C/GFP fusion protein was shown to localise to as yet unidentified intracellular cytoplasmic vesicular compartments. These compartments did not colocalise with the endosome/lysosome pathway, aparently ruling out a role for C1-20C in protein trafficking, recycling or degradation. Expression of C1-20C in the chick embryo suggests a possible role in heart and notochord development and preliminary results using siRNA suggest that C1-20C is involved in normal heart looping.

The cblC form of combined methylmalonic aciduria and homocystinuria (OMIM 277400) is the most frequent inborn error of vitamin B12 (cobalamin, Cbl) metabolism, with over 500 patients identified worldwide. Due to mutations in the MMACHC gene, patients with this disease are unable to convert cobalamin into the two active forms, methylcobalamin and adenosylcobalamin, which are cofactors required by mammalian methionine synthase and methylmalonyl-CoA mutase, respectively. Clinical features of patients can include hematological, neurological, and ophthalmological findings, along with developmental delay. In this study, the requirement of Mmachc during mouse embryonic development is examined. The Mmachc gene was found to be expressed in head mesenchyme, dorsal root ganglia, heart, trachea, lung, esophagus, gut, mesonephric mesenchyme, and notochord during organogenesis in the mouse embryo. Mice containing a genetrap in intron 1 of the Mmachc gene were characterized. Mmachc heterozygous mice for the genetrap were shown to be fertile and viable, although a small number of heterozygous and wild type embryos were found to be phenotypically abnormal. Mice and embryos heterozygous for the MmachcGT1 allele were found in higher numbers than expected by Mendelian ratios. Embryos homozygous for the MmachcGT1 were only observed at embryonic day 3.5. These findings show that Mmachc is required during mouse embryonic development.
La forme cblC de l'acidurie méthylmalonique et l'homocystinurie combinée (OMIM 277400) est la maladie génétique la plus fréquente affectant le métabolisme de la vitamine B12 (cobalamine, Cbl), avec plus de 500 patients identifiés à travers le monde. En raison de mutations dans le gène MMACHC, les patients atteints de cette maladie sont incapables de convertir la cobalamine en les deux formes actives, soit la méthylcobalamine et l'adénosylcobalamine, qui sont des cofacteurs requis par les enzymes mammifères méthionine synthase et methylmalonyl-CoA mutase, respectivement. Les symptômes cliniques des patients peuvent être de l'ordre hématologique, neurologique, ophtalmologique ou d'un retard de développement. Dans cette étude, l'importance de MMACHC durant le développement embryonnaire de la souris est examinée. Le gène Mmachc est démontré comme étant exprimé dans le mésenchyme de la tête, les ganglions de la racine dorsale, le cœur, la trachée, les poumons, l'œsophage, l'intestin, mésenchyme mésonéphrique et la notochorde au cours de l'organogenèse chez l'embryon de souris. Une mutation de type « genetrap » a été insérée dans l'intron 1 du gène Mmachc, noté MmachcGT1, afin de bloquer la production d'une protéine fonctionnelle MMACHC. Les souris adultes hétérozygotes pour le « genetrap » Mmachc se sont avérées être fertiles et viables, même si un pourcentage d'embryons hétérozygotes possédait un phénotype anormal. Les souris et embryons hétérozygotes pour l'allèle MmachcGT1 ont été trouvés dans un nombre plus élevé que prévu par les ratios mendéliens. Les embryons homozygotes pour MmachcGT1 n'ont été observés qu'au jour embryonnaire 3.5. Ces résultats démontrent que le gène Mmachc est essentiel au cours du développement embryonnaire de la souris.

The basis of developmental biology lies in the idea of when and how cells decide to divide or to differentiate. Previous studies have established several signaling pathways that determine cell fate decisions, including Notch, Wingless, Hedgehog, Bone morphogenetic protein, and Fibroblast growth factor. Signaling converges on transcriptional factors that regulate gene expression. In mouse embryonic stem cells, I explored how pluripotency and differentiation are regulated through opposing actions of beta-catenin-mediated canonical Wnt signaling, and the mechanisms underlying Sonic hedgehog signaling in generating progenitor cells in the ventral neural tube.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references.
In the mouse, germ cells can undergo differentiation to become either oocytes or spermatozoa in response to sex of their gonadal environment. The nature of the germ cell-intrinsic aspects of this signaling have not been well studied. The earliest known sex-specific difference in germ cells is the initiation of meiosis in female, but not male, embryonic germ cells. Experiments were performed showing that germ cells of both sexes transit through a state, the meiosis competent germ cell, that is required for initiation of meiosis. Acquisition of this state requires the function of the germ cellspecific RNA binding protein DAZL. The sufficiency for the absence of meiosis to drive male germ cell differentiation was then tested by examining non-meiotic XX germ cells in the Dazl-deficient ovary. These cells did not exhibit male differentiation indicating that the absence of meiosis is not sufficient for male differentiation. XX Dazl-deficient germ cells also failed to exhibit normal female differentiation. In addition, XY Dazl-deficient germ cells do not display characteristics of either male or female germ cells. Taken together, these results indicate that germ cells must first undergo a sex non-specific differentiation step prior to acquiring sexual fate.
by Mark E. Gill.
Ph.D.

Establishment of the biochemical and molecular nature of cardiac development is essential for us to understand the relationship between genetic and morphological aspects of heart formation. The molecular mechanisms that underly heart development are still not clearly defined. To address this issue we have used two approaches to identify genes involved in early chick cardiac development. Differential display previously conducted in our laboratory led to the identification of two gene fragments differentially expressed in the heart that are further described in this thesis. The full-length cDNA sequence of both eukaryotic translation initiation factor-2b (eIF-2b) and NADH cytochrome b5 reductase (b5R) were isolated using library screening. The upreglation of these genes during heart development is expected given the heart is the first functional organ to form in vertebrates and protein synthesis and cell metabolism at this stage of development is maximal. Limitations in the differential display approach led to the development and optimisation of a subtractive hybridisation approach for use with small amounts of cells or tissue. To focus on cardiac gene expression during the initial phases of heart development, subtractive hybridization was performed between the cardiogenic lateral plate mesoderm of Hamburger and Hamilton stage 4 embryos and the heart primordia of stage 9 embryos. Of the 87 independent clones identified by this procedure, 59 matched known sequences with high homology, 25 matched unknown expressed sequence tag (EST) sequences with high homology, and 3 did not match any known sequence on the database. Known genes isolated included those involved in transcription, translation, cell signalling, RNA processing, and energy production. Two of these genes, high mobility group phosphoprotein A2 (HMGA2) and C1-20C, an unknown gene, were chosen for further characterisation. The role of each gene in early chick heart development and indeed development in general, was addressed using techniques such as in situ hybridisation, transfection analysis, in ovo electroporation and RNAi. HMGA2 is a nuclear phosphoprotein commonly referred to as an architectural transcription factor due to its ability to modulate DNA conformation. In keeping with this function, HMGA2/GFP fusion protein was shown to localise to the nucleus and in particular, the nucleolus. In situ hybridisation analysis suggested a role for HMGA2 in heart and somite development. HMGA2 expression was first detected at HH stage 5 in the lateral plate mesoderm, a region synonymous with cells specified to the cardiac fate. HMGA2 was also strongly expressed in the presomitic segmental plate mesoderm and as somites developed from the segmental plate mesoderm, the expression of HMGA2 showed an increasingly more restricted domain corresponding to the level of maturation of the somite. Restriction of HMGA2 expression was first detected in the dorsal region of the epithelial somite, then the dorsomedial lip of the dermomyotome, and finally the migrating epaxial myotome cells. The novel intronless gene, C1-20C, predicts a protein of 148 amino acids containing a putative zinc finger binding domain and prenyl binding motif. Zinc binding assays showed that the zinc finger domain of C1-20C/MBP fusion protein bound over six times the quantity of zinc compared to MBP alone, although not in a 1:1 stoichiometric molar ratio. C1-20C/GFP fusion protein was shown to localise to as yet unidentified intracellular cytoplasmic vesicular compartments. These compartments did not colocalise with the endosome/lysosome pathway, aparently ruling out a role for C1-20C in protein trafficking, recycling or degradation. Expression of C1-20C in the chick embryo suggests a possible role in heart and notochord development and preliminary results using siRNA suggest that C1-20C is involved in normal heart looping.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

The glycine cleavage system (GCS) is a multi-enzyme complex localised in the mitochondria and serves as the main catabolic pathway for glycine. It contributes to supply of one-carbon units into folate one-carbon metabolism (FOCM) which utilises them for vital processes such as purines and thymidylate biosynthesis and methylation reactions. This thesis focuses on the role of glycine decarboxylase (Gldc), a member of the GCS, in embryonic development of the brain. It utilises two loss-of-function mouse models for Gldc which were found to exhibit two distinct disease phenotypes: non-ketotic hyperglycinemia (NKH) and neural tube defects (NTDs). The aims of this project are to investigate what effects GCS deficiency has on FOCM, the developmental mechanisms underlying NTDs caused by loss of Gldc expression, and suitability of the Gldc mice models as animal models for classical NKH. NKH is a rare metabolic disease caused by mutations of GCS genes (mainly GLDC) and characterised by accumulation of glycine in body fluids, resulting in severe neurological dysfunction and poor survival. Gldc-deficient mice exhibited features of NKH including elevated glycine, early post-natal lethality, and hydrocephalus. Enlargement of the brain ventricles was found to already be present at late-foetal stage, while glycine levels in whole embryos were already elevated shortly after neurulation. Gldc-deficient embryos also displayed NTDs, a common birth defect of the central nervous system that result from failure of the neural tube to close. Gldc-deficient embryos displayed abnormal folate metabolism, growth retardation and reduced cell proliferation. Supplementation with one-carbon units through dietary means was able to normalise folate profiles, completely rescue the NTDs, and normalise proliferation and growth in Gldc-deficient embryos. Diet-induced folate deficiency and interactions with the Mthfr mutation (which results in a methylation defect) did not exacerbate the NTDs caused by the Gldc mutation. This study provides the first mouse model for classical NKH and suggests that the pathology of NKH begins earlier in development than suspected. It also suggests that Gldc deficiency causes NTDs by reducing the supply of glycine-derived, mitochondrial one-carbon units for FOCM reactions.

All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

The aims of this study were i) to characterise differences in the spatial and temporal expression of Pax6 isoforms during murine embryonic development ii) to analyse the expression of Pax6 isoforms in various Pax6 mutant mice during neurogenesis, and iii) to examine the effects of over-expression of the best understood isoforms, in a cell culture system. The overall aim was to elucidate the independent roles of Pax6 isoforms in organogenesis of the central nervous system. RNase protection assay was used to determine the ratio between Pax6 and Pax6+5a transcripts in a number of tissues of the central nervous system during neurogenesis. In most tissues studied, Pax6 is much more prevalent than Pax6+5a at embryonic day 12.5, but the ratio has fallen by embryonic day 18.5. This may be indicative of a change in the role of Pax6, from controlling proliferation to controlling neuronal differentiation. Pax6 protein expression was analysed in mice with 0, 1, 2, 8 and 14 functional copies of Pax6, in order to compare the levels of Pax6 expression between genotypes, and to determine if differential expression of one or more isoforms could be responsible for the mutant phenotypes. Most isoforms are down-regulated in the Pax6^Sey/+ eye, whilst their relative expression is more varied in the Pax6^Sey/+ brain. Most isoforms are significantly up-regulated in the brain and eye of mice with 8 or 14 copies of Pax6, but there are no differences between expression levels in the brain of the two genotypes, indicating that Pax6 is subject to autoregulation. Some Pax6 isoforms are observed in the brain of mice thought to lack functional Pax6.

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.

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

Historically, sperm has been considered merely as a carrier of genetic material at fertilisation. However, it is known that sperm supports embryonic development better than other cell types, suggesting that it might also have additional important, non-genetic contributions to embryonic development. The work described in this dissertation focuses on identifying the molecular determinants of developmental programming of sperm. First, the development of embryos derived from sperm and spermatids, immature precursors of sperm was compared. Sperm-derived embryos developed significantly better than spermatid-derived embryos. Further research aiming to identify the reasons for the developmental advantage of sperm led to the identification of proteins that are present specifically in sperm and not in spermatids. Moreover, egg factors which are preferentially incorporated into the sperm, but not into the spermatid chromatin were identified with the use of egg extracts, suggesting that the chromatin of sperm could be programmed to interact with the components of the egg. Subsequently, the reasons for developmental failure of spermatid-derived embryos were investigated. By comparing the sperm with spermatids it was shown that the programming of sperm to support efficient development is linked to its special ability to regulate expression of developmentally-important embryonic genes, and not to its ability to support DNA replication or rRNA production. Further characterisation of the sperm and spermatid chromatin with the use of genome-wide sequencing allowed me to link the correct regulation of gene expression in the embryo with a certain combination of epigenetic marks in the sperm, but not in the spermatid chromatin. Finally, it is shown that enzymatic removal of epigenetic modifications at fertilisation leads to misregulation of gene expression. This therefore suggests that epigenetic information contained in parental genomes at fertilisation is required for a proper regulation of embryonic transcription. My results support the hypothesis that the sperm is not only a carrier of genetic material, but also provides the embryo with epigenetic information for regulation of transcription after fertilisation. I believe that these findings advance our current understanding of the nature and mechanisms of sperm programming for embryonic development, and are important contributions to the emerging field of transgenerational inheritance of epigenetic traits in general.

Die vorliegende Arbeit behandelt die Embryonalentwicklung des Rankenfußkrebses Elminius modestus (Thecostraca: Cirripedia). Der Entwicklungsprozess wurde mithilfe unterschiedlicher Methoden wie 4D Mikroskopie, in vivo Einzelzellmarkierungen, Fluoreszenzhistochemie und konfokaler Laserscanningmikroskopie in Verbindung mit 3D Rekonstruktionen untersucht. Die Furchung von E. modestus ist total, inequal in Bezug auf die Dotterzelle und asynchron mit einem anterior-posterioren Gradienten. Der gesamte Prozess folgt einem strengen Teilungsmuster mit nur sehr geringer Variabilität. Eine davon stellt das Auftreten spiegelbildlicher Embryonen ab dem 4-Zell. Die Keimblattdifferenzierung wurde vor allem mittels in vivo Zellmarkierungen untersucht. Die Trennung der endodermalen und endomesodemalen Keimblätter erfolgt nach der vierten Furchungsteilung, die Trennung des Ectomesoderm nach der sechsten Teilung. Die Urkeimzellen sind aller Wahrscheinlichkeit nach ein Produkt der siebten Furchungsteilung der Dotterzellen (3Da und 3Dp). Im Zuge der Untersuchung konnte die Zelllinie jedes Keimblattes rekonstruiert werden, die Zellschicksale der Abkömmlinge der Quadranten wurde bis zum 16-Zell Stadium beschrieben. Das Ectoderm entspringt allen vier Quadranten, ebenso das Ectomesoderm (die letzten identifizierten Mesectoblasten sind 3A, 3B, 3C, 1drp und 1dlp). Endoderm und Endomesoderm entwickeln sich aus einzelnen Vorläuferzellen im 16-Zell Stadium (2D bzw. 2d). Das Auftreten nur eines einzelnen Endoblasten stellt eine mögliche Apomorphie aller Ecdysozoa dar. Das Vorhandensein eines einzelnen Mesendoblasten wird als mögliches Merkmal des Grundmusters aller Protostomia in Betracht gezogen.
The present work is devoted to the embryonic development of the thoracican barnacle Elminius modestus (Thecostraca: Cirripedia). The developmental process was investigated by means of different techniques like 4D microscopy, in vivo labelling, fluorescent histochemistry, and confocal laser scanning microscopy combined with 3D reconstructions. The cleavage of E. modestus is total, unequal with regards to the yolky cell, and asynchronous with an anterior-posterior gradient. The entire process appears to follow a strict pattern of divisions with very little variability, one of which includes the occurrence of mirror image embryos from the 4-cell stage on. The germ layer differentiation was mainly studied by means of in vivo labelling. The segregation of the endodermal and the endomesodemal germ layers are shown to happen after the fourth division, whereas the ectomesoderm segregates after the sixth division. The primordial germ cells are suggested to be a product of the seventh cleavage division of the yolky cells (3Da and 3Dp). During the research the cell lineage of each germ layer was established, the fates of the quadrant descendants are described up to the 16-cell stage. The ectoderm originates from four quadrants, as does the ectomesoderm (the last identified mesectoblasts are 3A, 3B, 3C, 1drp, and 1dlp). The endoderm and the endomesoderm develop from single precursors at the 16-cell stage (2D and 2d, respectively). The presence of only a single endoblastic cell, might represent an apomorphy for the entire group of Ecdysozoa. A singular mesendoblast is suggested to be a possible feature in the developmental ground pattern of all Protostomia.

Objectives: The effects of estrogens on immune system formation and function are well documented. Diethylstilbestrol (DES), a synthetic estrogen, has been linked to neoplasia and immune cell dysfunction in humans and animals exposed in-utero. In-vitro effects of DES exposure of murine embryonic stem (ES) cells on the early embryonic immune system development and the expression of cellular surface markers associated with common hemangioblastic and hematopoietic precursors of the endothelial, lymphoid & myeloid lineages were investigated. Hypothesis: Early ES cell expression of CD45 a marker common to lymphoid lineage hematopoietic stem cells and differentiation of lymphoid lineage precursors are affected by in-vitro exposure to DES. Methods: Murine ES cells were cultured using a variety of techniques: an OP9 co-culture system, and formation of embryoid bodies (EBs) in a liquid medium and hanging drop system. The OP9 co-culture system did not appear to give rise to well differentiated lymphoid lineage cells during 12 days of differentiation. The hanging drop EB culture system, previously shown to promote differentiation of endothelial and lymphoid precursor cells, was chosen for further studies of ES cell differentiation. ES cells were harvested at five time points: undifferentiated (day 0), and differentiated (days 3, 8, 12 and 16). Differentiating ES cells were treated with DES beginning on day 3. The synthetic estrogen, DES, was chosen as a treatment because of its similar potency to 17β estradiol and documented association with neoplasia in women exposed in-utero. Surface marker expression, measured by real-time RT-PCR amplification, was recorded using fluorogenic TaqMan(R) probes designed specifically for the surface proteins of interest: oct4, c-Kit, Flk1, ERα, ERβ, CD45, Flt1, & VE-cadherin. Analysis & Results: Changes in surface marker gene expression between day 0 and day 16 of differentiation were analyzed using the RT-PCR threshold counts (CT) and the comparative threshhold cycle method. The expression of each target mRNA was normalized internally to a housekeeping gene (18s rRNA) and calculated relative to day 0. ANOVA (Type 3 fixed-effects analysis, SAS) was performed using the unexponentiated ΠΠCT values. The effects of DES, time, and the interaction between DES and time were evaluated for days 8, 12 and 16. Additionally, the effects of DES on the expression of each marker were evaluated for day 16. Expression of estrogen receptor receptor α & β (ERα & β) in the EBs was established, and did not appear to be affected at any time by treatment with DES. ERα was expressed in significant levels on day 16, while ERβ was expressed in low levels throughout the period of differentiation. The expression of the cell surface marker, c-Kit was significantly (P<0.0001) altered by the presence of DES between the three time points sampled. The expression of the VEGF receptor, Flt1, and the adhesion molecule, VE-cadherin, markers of endothelial cells, were also significantly (P<0.026) altered by treatment with DES on day 16 of differentiation. Treatment with DES appeared to have no effect on the expression of CD45, a marker common to lymphoid precursor cells. Conclusions: These results indicate the presence of estrogen receptors in differentiating ES cells as early as day three in-vitro (ERβ) until day 16 (ERα). DES alters expression of common hemangioblastic and hematopoietic precursor, as well as endothelial lineage markers, but has no effect on expression of the marker of lymphoid lineage development before day 16. These effects coincided with the expression of ERα. The enduring effects of DES exposure in-utero may not be manifest in this ES model, or may occur at later stages of differentiation or in selected subpopulations of CD45+ cells.
Master of Science

Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.

Copper is an essential trace element serving as a cofactor for critical enzymes. Copper uptake occurs via two solute carriers; Copper transporter 1 (Ctr1) and Copper transporter 2 (Ctr2). The high affinity transporter, Ctr1, is crucial for embryonic development. Ctr1 null-mutant mice die in utero from a gastrulation defect. This inability to generate mesoderm has been phenocopied in in vitro differentiation cultures of Ctr1-/- embryonic stem (ES) cells. However, the role of copper transporters during development remains unknown. In this thesis we aim to elucidate the roles of Ctr1 and Ctr2 during murine development. In sperm, Ctr2 was found expressed on the acrosomal cap, whilst Ctr1 was expressed in the cytoplasmic droplet. In the preimplantation embryo, Ctr1+ particles coalesced during cleavage stages, whilst Ctr2 colocalised with Keratin-8+ trophectoderm on the surface of the morula and blastocyst. The effect of copper on development of the preimplantation embryo cultured in vitro. Addition of copper sulfate proved lethal at concentrations greater that 50 μM. Copper chelation with tetrathiomolybdate resulted developmental arrest at concentrations greater than 1 μM. To assess copper transport to the post-implantation embryo we assessed the extra-embryonic tissues, the yolk sac and the placenta. The placenta was the primary site of copper transport with Ctr2 expressed on the maternal and foetal vessels between E10.5 – E16.5, while Ctr1 was expressed at E14.5. In the yolk sac Ctr2 was expressed in the visceral endoderm from E8.5 – E18.5. We next assessed the role of Ctr1 in germ layer specification utilising in vitro differentiation of Ctr1-null embryonic stem (ES) cells. During neurectoderm differentiation, loss of Ctr1 resulted in a lineage bias towards surface ectoderm. Absence of Ctr1 did not affect the ability of Ctr1-/- ES cells to generate CXCR4+c-Kit+ definitive endoderm or PECAM- PDGRFα+ extra embryonic endoderm relative to Ctr1+/+ ES cells. In addition, the mitochondrial reactive oxygen species probe, NpFR2, revealed that decreased mitochondrial activity in the absence of Ctr1 inhibits mesoderm formation. These findings better elucidate expression of copper transporters, Ctr1 and Ctr2, in delivery mechanisms of copper to the developing embryo as well as characterise the role of Ctr1 during germ layer specification.

Malformations of the cardiovascular system are the most common type of birth defect in humans, affecting predominantly the formation of valves and septa. While many studies have addressed the role of specific genes during valve and septa formation, a global understanding is still largely incomplete. To address this deficit we have undertaken a genome-wide transcriptional profiling of the developing heart in the mouse. We generated and analyzed 19 Serial Analysis of Gene Expression (SAGE) libraries representing different regions of the mouse heart at multiple stages of embryonic development. We speculated that genes important for heart valve development would be differentially expressed in the valve forming regions, and have dynamic temporal expression patterns. We used our dataset to identify a novel list of valve enriched genes. Using k-means cluster analysis we also uncovered 14 distinct temporal gene expression patterns in the developing valves. Unique temporal expression patterns were found to be enriched for specific signalling pathway members and functional categories such as signal transduction, transcription factor activity, proliferation and apoptosis. The most highly expressed transcription factor within the developing valves was found to be Twist1. Analysis of gene expression changes in the Twist1 null developing valves revealed a novel phenotype consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. Our data suggests that TWIST1 directly activates valve specific and cell motility gene expression in the atrio-ventricular canal, while suppressing expression of valve maturation markers. This work provides the first comprehensive temporal and spatial gene expression dataset for heart development during formation of the heart valves. It is a valuable resource for the elucidation of the molecular mechanisms underlying heart development.

Glypican are a family of conserved cell surface Heparan Sulfate Proteoglycans (HSPGs) attached to the cell surface by glycosylphosphatidylinositol (GPI) anchor. Six members (Gpcl-Gpc6) have been identified in mammal so far. They are expressed in spatiotemporally regulated manner in developing and adult tissues which are known to be regulated by signalling growth factors pathways including Fgf, Bmps, Wnt and Shh. Genetic and embryological studies link glypicans to the regulation of signalling pathways during morphogenesis, pattering formation and adult physiology. We showed here that chicken Gallus-gallus glypicans (Gpcl-Gpc6) and No/urn a gene encoding a master regulator of glypican activity are differentially expressed during embryonic developmental stages and adult tissues. This finding suggested that glypicans act as modulator of developmental signalling pathways. Furthermore we show the expression of glypicans in adult chicken tissues and propose a role as regulator of stem cell homeostasis in brain and skeletal muscle. In developing embryo we highlight glypicans expression in some tissue like limb bud, feather bud and neural tube that are known as good models for tissue patterning sites. We provided novel data showing that the inhibition of the signalling pathways altered glypicans expression during early embryonic development. We demonstrated that Notum regulate the pattering of neural tube by regulating the activity and distribution of Shh in the neural tube and alters in the dorsal and ventral boundaries. We concluded that Notum regulate the activity of Shh during neural tube pattering via regulation of glypicans.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2006.
Includes bibliographical references (v. 2, leaves 150-197).
Imprinting is a mammalian adaptation that results in the mono-allelic expression of a subset of genes depending on their parental origin. It is believed that DNA methylation marks are responsible for maintaining imprinted gene expression patterns. The 'parental conflict' hypothesis was proposed to explain the evolution of imprinting and is based on the assumption that mammals arose from an ancestor that was polyandrous (multiple fathers within one litter). According to this hypothesis, conflict between the male and female over the allocation of maternal resources to the offspring led to the evolution of imprinting. Consistent with this, many imprinted genes are involved in embryonic or placental growth by regulating mitogenic pathways or the cell cycle. Loss of imprinting (LOI) has been found at specific loci in cancers, raising the possibility that altered expression of imprinted genes may also contribute to tumorigenesis. To investigate the effect of global LOI on embryonic development and cancer formation, imprint free (IF) embryonic stem (ES) cells were generated using conditional inactivation/reactivation of the DNA methyltransferase Dnmtl. Tetraploid complementation and chimera experiments revealed that IF-embryos fail to develop beyond E11.5 and display an overgrowth phenotype.
(cont.) As developmental comparisons, parthenogenetic and androgenetic (AT) embryos were derived and found to develop to E9.5 and E7.5, respectively. Removing the imprinted methyl marks from AT-ES cells rescued embryonic development to E9.5-10.5 and restored pluripotency. However, IFAT embryos were not developmentally equivalent to biparentally-derived IF-embryos, suggesting that mechanisms other than DNA methylation may be involved in maintaining parent-specific gene expression patterns. To study the effect of LOI on cell growth, murine embryonic fibroblasts (MEFs) were derived from E13.5 chimeric IF-embryos and analyzed in vitro. IF-MEFs grew faster, were resistant to the cytostatic effects of TGFI3, and formed tumors in SCID mice. In addition, IF-MEFs were immortal and when exposed to H-Ras became fully transformed. Western blot analysis of IF-MEFs revealed abnormally low levels of p19Arf and p53, two critical regulators of growth arrest and potent tumor suppressors. Somatic contribution of IF-ES cells in chimeric adults led to highly penetrant tumor formation by 12 months of age, causing multiple cancer types derived from the IF cells. Taken together, these data are consistent with global LOI having a causal role in tumorigenesis by affecting the regulation of the p53-p19Ar pathway and predisposing IF cells to transformation.
by Teresa M. Holm.
Ph.D.

Thesis (PhD(Agric))--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The ostrich industry experiences high rates of embryonic mortalities during artificial incubation of eggs. Studies have been carried out to investigate factors influencing hatchability, as well as determining genotypic effects for commercial production. Eggs from the combination of South African Black (SAB) male ostriches crossed with Zimbabwean Blue (ZB) female ostriches had embryonic losses of 45.7%. The embryonic mortality of eggs produced by pure bred SAB or ZB breeding birds subjected to pure breeding was similar at around 33 - 34%, but embryonic mortality was improved in eggs produced by ZB males and SAB female crosses (27%). Female age had a significant effect on the proportion of chicks pipped, as well as on early and late the embryonic mortalities. Chicks from eggs stored for intermediate periods, i.e. 3, 4 and 6 days prior to being set, were more likely to pip than chicks from those eggs set directly after collection without storage. Embryonic mortality was increased in eggs that were set directly (32.0%) or subjected to longer than 6 days of storage (43.5%). Chicks that pipped in the correct position had a higher probability of successfully hatching than those pipping in the incorrect position. Transfer of eggs between setters (i.e. disturbance of eggs) during incubation reduced the number of ostrich chicks pipping in the correct position. Incubated ostrich eggs with intermediate levels of water loss, i.e. between 9.0 and 19% of fresh egg weight, were more likely to pip in the correct position than those with higher or lower levels of water loss. Such eggs were also less likely to sustain early, late or overall embryonic mortalities. To optimise hatching success it is important to understand embryonic development. After 2 days of incubation the blastoderm area in eggs from the SAB x ZB crosses (104.5 mm) was lower (P < 0.05) compared to the pure SAB (141.0 mm), pure ZB (161.7 mm) and ZB x SAB crosses (166.1 mm). For embryos incubated for 7 to 42 days, both embryonic and leg growth during the 42 days of incubation was similar and approximately linear, more or less doubling in size up to 35 days of incubation. The embryo eye size increased more rapidly than beak length and reached full size of approximately 16.2 mm by 28 days of incubation, whereas the beak length continued to increase until the chick hatched at 42 days. Incubation position, vertical or horizontal, did not affect any of the measurements of the developing embryo throughout the 42-day incubation period. Air cell volume at 29 day of incubation for infertile eggs (19.3%) was significantly (P < 0.05) higher when compared to dead-in-shell eggs (14.3%) and eggs that hatched successfully (13.8%). Air cell volume was largely independent of strain (SAB or ZB) and whether chicks were assisted to hatch or not. After 41 days of incubation there was a significantly greater (P < 0.05) air cell volume in eggs that hatched normally compared to dead-in-shell eggs (28.3% vs. 21.7%, respectively, suggesting that insufficient water loss contributed to reduced survival. This study provides an insight into the complexity of embryo development and all the factors playing a role in successful hatching of ostrich eggs. Data from a pair-mated ostrich flock were used to estimate genetic parameters for egg weight (EWT), weight of day-old chicks (CWT), water loss to 21 (WL21) and 35 (WL35) days of incubation, and pipping time (PT). Single-trait estimates of heritability (h2) were high and significant (P < 0.05) at 0.46 for EWT, 0.34 for CWT, 0.34 for WL21, 0.27 for WL35 and 0.16 for pipping time. Genetic correlations with EWT amounted to -0.21 for WL21 and to -0.12 for WL35. Corresponding correlations of CWT with WL were highly significant (P < 0.05) at -0.43 and -0.54. Physical characteristics of the eggshell were found to affect water loss and hatchability. Estimates of genetic parameters of 14 146 ostrich eggs for eggshell traits showed that heritability was 0.42 for pore count (PC), 0.33 for shell thickness (ST) and 0.22 for permeability (PERM). PC was negatively correlated with average pore diameter (-0.58) and ST (-0.23), while PC was positively correlated with total pore area (0.58), WL21 (0.24) and WL35 (0.34). The correlations of PC with total pore area and PERM were high and significant. ST was negatively correlated to WL21 and WL35. Additive genetic parameters strongly indicate that it should be possible to alter evaporative water loss and eggshell quality of ostrich eggs through genetic selection. When assessed as a trait of the individual egg or chick, embryonic mortalities exhibited moderate levels of genetic variation both on the normal scale (h2 = 0.16 - 0.22) and the underlying liability scale (h2 = 0.21 - 0.31). Early embryonic survival and late embryonic survival was governed mostly by the same genes (rg = 0.78). Late embryonic survival was genetically correlated to WL35, at -0.22. It was concluded that embryonic survival could be improved by using husbandry measures, a knowledge of the stage when incubation mortalities occur, and by genetic selection, using an integrated approach. Findings from this study will help to understand the mechanisms involved in hatching from artificial incubation better to improve hatchability and also implement selective breeding programs.
AFRIKAANSE OPSOMMING: Die volstruisbedryf ondervind tans ‘n baie hoë voorkoms van embrionale mortaliteite tydens die kunsmatige uitbroei van eiers. Studies is uitgevoer om die faktore wat uitbroeibaarheid beinvloed te ondersoek en om genotipiese effekte te bepaal vir kommersiële produsente. Eiers van die kombinasie van Suid-Afrikaanse swart (SAB) mannetjie volstruise, met Zimbabwean blou (ZB) wyfies, het embrionale mortaliteite van 45.7% gehad. Embrionale mortaliteite van eiers gelê deur suiwer SAB of ZB volstruise was dieselfde op omtrent 33 - 34%, maar embrionale mortaliteite was laer vir eiers geproduseer deur SAB wyfies wat gekruis was met ZB mannetjies (27%). Wyfie ouderdom het ‘n betekenisvolle effek gehad op die proporsie van kuikens wat gepik het, asook die aantal vroeë- en laat embrionale mortaliteite. Kuikens vanuit eiers wat vir die periode 3, 4 dae en 6 dae voor pak in die broeikaste gestoor is, was meer geneig om te pik as kuikens vanaf eiers wat direk na kolleksie gepak is. Embrionale mortalitiete het verhoog vir eiers wat direk na kolleksie gepak was (32.0%) of vir eiers wat langer as 6 dae gestoor was (43.5%). Kuikens wat in die korrekte posisie pik het ‘n hoër kans op uitbroei gehad as kuikens wat in die verkeerde posisie gepik het. Die skuif van eiers tussen verskillende broeikaste (of enige steurnisse) gedurende die broeiproses het ‘n verlaging in die aantal kuikens wat in die korrekte posisie pik, gehad. Volstruiseiers met ‘n gemiddelde vogverlies van tussen 9.0 en 19% van die vars eier massa, was meer geneig om in die korrekte posisie te pik as eiers met laer of hoër vlakke van vogverlies. Sulke eiers was ook minder geneig tot vroeë, laat en totale embrionale mortaliteite. Vir optimale uitbroeisukses is dit belangrik om die ontwikkeling van die embrio te verstaan. Na 2 dae van broei was die blastoderm area in eiers van SAB x ZB kruisings (104.5 mm) kleiner (P < 0.05) as die blastoderm area van suiwer SAB (141.0 mm), suiwer ZB (161.7 mm) en ZB x SAB kruise (166.1 mm). Beide embrionale- en beengroei tydens die 42 dae broeiproses was dieselfde en nagenoeg lineêr, met ‘n verdubbeling in grootte tot en met 35 dae broei. Die embrio se oog vergroot vinniger as wat die snawel verleng en bereik reeds volle grootte van ongeveer 16.2 mm op 28 dae van broei, terwyl die snawel aanhou groei tot uitbroei van die kuiken op 42 dae. Nie die vertikale of horisontale broeiposisie het enige invloed op die metings van die ontwikkelende embrio tot op 42 dae gehad nie. Lugsakvolume vir geil eiers (19.3%) op 29 dae van broei was groter (P < 0.05) as beide die lugsakke van eiers wat dood-in-dop (14.3%) en eiers wat suksesvol uitgebroei het (13.8%). Die lugsakvolume was onafhanklik van beide genotype en of die kuiken met of sonder hulp uitgebroei het. Na 41 dae broei was lugsakvolume groter (P < 0.05) vir eiers wat uitgebroei het teenoor eiers wat dood-in-dop was (28.3% vs. 21.7%, onderskeidelik), wat impliseer dat onvoldoende vogverlies moontlik kan bydrae tot ‘n verlaging in embrionale oorlewing. Hierdie studie gee ‘n insig in die kompleksiteit van embrionale ontwikkeling en al die faktore wat ‘n rol speel in die suksesvolle uitbroei van volstruiseiers. Tydens die bepaling van genetiese parameters vir spesifieke uitbroei-eienskappe in volstruise, is data gebruik afkomend van ‘n teelkudde in ‘n enkelparing stelsel om genetiese waardes vir eiermassa (EWT), dagoud kuikenmassa (CWT), vogverlies tot 21 dae broei (WL21), vogverlies tot 35 dae broei (WL35) en piktyd (PT) gebruik. Enkeleienskap-beraming vir oorerflikheid (h2) was hoog en betekenisvol teen 0.46 vir EWT, 0.34 vir CWT, 0.34 vir WL21, 0.27 vir WL35 en 0.16 vir piktyd. Genetiese korrelasies met EWT was -0.21 vir WL21 en -0.12 vir WL35. Ooreenkomstig was korrelasies van CWT met WL21 en WL35 hoog (P < 0.05) met -0.43 en -0.54 onderskeidelik. Fisiese eienskappe van die eiers het beide vogverlies en uitbroeibaarheid beinvloed. Beramings van genetiese parameters vir 14 146 volstruiseiers se dopeienskappe het gewys dat oorerflikehid 0.42 was vir die aantal porieë (PC), 0.33 vir dopdikte (ST) en 0.22 vir deurlaatbaarheid (PERM). PC was negatief gekorreleerd met gemiddelde porieë deursnee (-0.58) en ST (-0.23), terwyl PC positief gekorreleerd was met totale porieë area (0.58), WL21 (0.24) en WL35 (0.34). Die korrelasie van PC met totale porieë area en deurlaatbaarheid was hoog en betekenisvol. ST was negatief gekorreleerd met WL21 en WL35. Additiewe genetiese parameters het sterk daarop gedui dat dit moontlik sou wees om vogverlies en eierkwaliteit (bv. dopkwaliteit en poreusiteit) van volstruiseiers te verander deur genetiese seleksie. Indien embrionale mortaliteit geevalueer word as ‘n kenmerk van die eier of kuiken, toon dit matige vlakke van genetiese variasie op beide die normale (h2 = 0.16 - 0.22) en die onderliggende skale (h2 = 0.21 - 0.31). Beide vroeë- en laat embrionale oorlewing word deur dieselfde stel gene beheer (rg = 0.78). Laat embrionale oorlewing was geneties gekorreleerd met WL35 teen -0.22. Die gevolgtrekking was dat embrionale oorlewing verbeter kan word deur verbeterde broeikamerpraktyke, kennis van op watter stadium van ontwikkelings embrionale mortaliteite plaasvind en deur genetiese seleksie. Bevindinge vanuit hierdie studies sal help om die meganismes betrokke by die kunsmatige uitbroei van volstruiskuikens beter te verstaan om sodoende uitbroeibaarheid te verbeter en ook suksesvolle seleksie programme te implementeer.

The Hippo pathway is a master regulator of cell proliferation and organ size, namely through regulation of transcriptional co-activators YAP and TAZ which bind TEAD1-4 transcription factors. The Hippo effector YAP is dysregulated in many human solid tumours including rhabdomyosarcoma and oesophageal cancer. Additionally, persistent hyperactivity of YAP in activated but not quiescent satellite cells can give rise to embryonal rhabdomyosarcoma. However, the question of exactly how YAP acts as an oncogene and actively gives rise to tumour progression in these cancers remains unknown. In this thesis I characterised the mechanisms which determine the functional role of YAP in driving instability in the genome. Secondly, lentiviral mediated knockdown of YAP is performed to determine and investigate its effect on tumorigenesis. Thirdly, gene sets from constitutive YAP S127A induced mouse ERMS tumours subjected to array-CGH were further analysed. Finally, I cloned chicken Yap1, Tead1 and Fstl5 to identify its role during chick embryonic development, by the retroviral mediated loss of function approach. The results demonstrated that constitutive YAP S127A expression in-vitro as well as in-vivo induces chromosomal instability by increasing the rate of mitotic chromosome segregation errors and copy number alterations of oncogenes and other cancer related genes. Recurrent copy number gains of the p53 inhibitor Mdm2 were observed in YAP S127A-driven ERMS tumours. Moreover, lentiviral mediated YAP knockdown showed significant reduction in proliferation, migration and invasion as well as transformation potential in human cultured cancer cells. Moreover, retroviral YAP S127A expression during early stages of chick embryo development did not lead to an overt phenotype and showed poor survival. Additionally, I have cloned RCAS-RNAi vectors to study the loss of function effect on Hippo targets and Fstl5 during chicken embryo development. Collectively, my data provides insight into the mechanisms with which YAP could drive tumorigenesis and that YAP knockdown can be considered a potential therapeutic target to reduce cancer progression.

Embryogenesis in vertebrates is regulated by highly complicated signaling networks, which involve various signaling pathways and factors. Sox3 is known to have critical roles during the whole of embryonic development in vertebrates. In zebrafish, it has been shown that Sox3 restricts organizer formation as well as inhibiting Fgf signaling, which is required for the expression of organizer genes. On the other hand, SUMOylation has been suggested to be a regulator of the transcriptional activity of Sox3. The SUMOylaton has previously been demonstrated on mouse Sox3 and chick Sox3. In this study, I examined the role and regulation of Sox3 in early embryogenesis in zebrafish. In the first part of the study, I inspected the detailed expression of gsc and chd, in comparison to foxd3 expression. It was demonstrated that Sox3 and Fgf signaling could repress both gsc and chd independently from each other. Sox3 was also found to be able to directly act on the promoter region of gsc. In the second part of the study, it was shown that the SUMOylation of Sox3 appeared to occur in zebrafish embryos. The SUMOylation of Sox3 was also shown to correlate with the chromatin fraction, although there was a very small fraction of Sox3 SUMOylated. The biological effect of SUMOylation of Sox3 has also been analysed. It was shown that SUMOylation of Sox3 could enhance the transcriptional repressor activity of Sox3. The result of luciferase reporter assay on the promoter region of boz also suggested that SUMOylation eliminated the transcriptional activator activity of Sox3. These data raise the possibility that SUMOylation of Sox3 might act as the switch from a transcriptional activator to a repressor.

The evolution of a strikingly elongated and webbed FL in bats, which contrasts with a small, free-toed HL, has seen extensive research into bat wing development in an effort to determine the molecular mechanism driving limb development. A recent RNA-seq and ChIP-seq study carried out on M. natalensis showed differences in FL and HL activity for several genetic pathways known to be involved in bone formation during key bat development stages CS15-CS17. In this project the prediction made from the literature and the RNA-seq results was that the observed decreased Wnt/β-catenin signalling and increased BMP signalling in the bat FL may lead to elevated levels of Sox9 expression, and larger fields of mesenchymal condensations. This was tested by annotating Sox9 in the M. natalensis genome to further analyse the expression levels and associated ChIP-seq data. In addition the behaviour of condensing mesenchymal cells during bat and mouse limb development was observed by visualising the various stages of chondrogenesis, using H&E and PNA stains. In addition the RNA-seq study found 3000 genes to be differentially expressed. Thus, the project also set out to create an immortalised bat autopod cell line to facilitate future testing and predictions. The Sox9 gene was successfully annotated and revealed to not be differentially expressed between FL and HL as predicted. However downstream targets of Sox9 were further inspected as potential ideas for further investigation. The histological stains provided a set of data characterising mesenchymal condensation in both mouse and bat stages, revealing many interesting features such as the non-specific binding behaviours of PNA prior to digit formation. In addition, quantitative results demonstrated the bat FL digits are already longer than the HL digits at CS16. Cell line work established a working protocol for the storage, dissociation and plating of bat primary cells that retain their bat limb expression identity. Mouse cells were successfully immortalised and a cell line was established from a HL digit cell. This project has facilitated further studies in understanding extreme digit elongation in the bat FL autopod during development.