Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 83-89).

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

The aim of his study was to investigate the role of Dctn2 in mouse preimplantation embryonic development, specifically its effect on the first cell fate decision, when the number of cells increases from eight to sixteen.

Fetal growth restriction (FGR) is the failure of a fetus to reach its pre-determined genetic growth potential during development. FGR is associated both with poor outcome in the neonatal period, and the onset of major adult diseases such as diabetes, hypertension and obesity. Therefore understanding what causes restricted fetal growth is important both for improving neonatal health, and for the minimization of major worldwide healthcare burdens. Described here are two mutant mouse lines, each with a distinct mutation in the Mediator complex gene Med31. These mutations result in reduced fetal growth, allowing for the investigation of the role of Med31 in the proper control of growth during development. The first mutant mouse line (Med31 Null) carries a C/T point mutation in exon 4 of Med31. Homozygous mutant embryos display reduced growth during development, characterized by their reduced size and smaller forelimbs compared to their heterozygous littermate controls. The second mutant mouse line (Med31 Y57C) carries a T/C point mutation in exon 3 of Med31. Similarly, homozygous mutant embryos display reduced fetal growth with reductions in forelimb length compared to their heterozygous littermate controls. In both mutant lines whole embryo growth and endochondral ossification within the limbs is perturbed. This is due to defects in cellular proliferation and the misexpression of the cell cycle genes Ccnb1 and Mtor within the mutant embryos. Additionally, the Med31 Null line is embryonic lethal by E18.5 and displays morphological defects of the placenta compared to heterozygous littermate controls. These morphological differences are suggestive of defects in the function of the placenta, and are proposed as the cause of embryonic lethality. In support of this the Med31 Y57C line is viable with no defects in placental development. New roles for Med31 in embryonic growth, cellular proliferation and placental development are identified. Moreover the two mutant lines constitue an allelic series of Med31, and the two mutations provide insights into the various ways Med31 is able to regulate transcription during development.

No description available.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

The unique pluripotent character of human embryonic stem cells (hESCs) places them in the forefront of scientific research, especially as they hold great promise for application in regenerative medicine, as well as drug discovery and toxicity analyses. Conventionally hESCs are cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) that are derived from E13.5 mouse embryos. One of the biggest challenges in the hESC field is the development of a reproducible and defined hESC culture system that would eliminate batch-to-batch variability of the MEFs as well as exposure to feeder cells that makes hESCs less applicable for clinical use. Previous studies have shown that maintenance of pluripotency can be achieved using Matrigel, a mixture of ECM components, or ECM derived from MEFs or human fibroblasts (Xu, et al., 2001, Klimanskaya, et al., 2005). Other groups have succeeded in culturing feeder-free hESCs by using extracellular matrix (ECM) proteins, such as fibronectin, vitronectin or laminin, as substrates for hESC culture in the absence of feeders, confirming that ECM plays a key role in maintaining hESC growth (Amit, et al., 2004, Braam, et al., 2008, Baxter, et al., 2009, Rodin, et al., 2010).The aim of this work was to investigate the ECM deposited by MEF feeder cells and to isolate and identify proteins in the ECM that support undifferentiated growth of hESCs in the absence of feeders. We have investigated whether matrices derived from different passage feeders differ in their ability to support pluripotency. I also assessed the integrin receptor profile of hESCs in order to define the mechanisms of ECM engagement. ECM was extracted from two strains of feeder cells, CD1 x CD1 and MF1 x CD1, at passages 4 (early passage), 9 and 14 (late passage), and assessed for its ability to support hESC self-renewal over at least 3 passages. Tandem mass spectrometry was used to analyse the ECM composition of each MEF line, thereby allowing a comparison between different passages and different cell lines. More than 100 proteins were identified for each sample, the majority of which were ECM proteins and shared between different passage feeders. As predicted, fibronectin, which is known to support hESC self-renewal was the most prevalent species in all MEF-derived matrices. Furthermore a proteomic analysis of matrix derived from hESCs cultured in feeder-free conditions on fibronectin coating substratum revealed a number of proteins shared between supportive MEF populations and hESC, suggesting other potential candidates that may either assist or interfere with the maintenance of pluripotent hESCs. Of the proteins identified fibrillin-1, perlecan, fibulin-2 were tested as substrates for culturing hESCs in the absence of feeders, with the prospect of developing an optimised feeder-free culturing system that uses a combination defined animal-free substrates. Finally this study sought to dissect the interaction between ECM and growth factors and how these extrinsic factors may affect self-renewal and maintenance of pluripotency-associated gene expression. Interruption of hESC attachment, as well as removal of growth factors appeared to affect transcript levels of pluripotency genes, OCT4 and NANOG, suggesting that the microenvironment can influence hESC fate.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

Embryos of oviparous reptiles develop on the surface of a large mass of yolk, which they metabolize to become relatively large hatchlings. Access to the yolk is provided by tissues growing outward from the embryo to cover the surface of the yolk. A key feature of yolk sac development is a dedicated blood vascular system to communicate with the embryo. The best known model for yolk sac development and function of oviparous amniotes is based on numerous studies of birds, primarily domestic chickens. In this model, the vascular yolk sac forms the perimeter of the large yolk mass and is lined by a specialized epithelium, which takes up, processes and transports yolk nutrients to the yolk sac blood vessels. Studies of lizard yolk sac development, dating to more than 100 years ago, report characteristics inconsistent with this model. We compared development of the yolk sac from oviposition to near hatching in embryonic series of three species of oviparous scincid lizards to consider congruence with the pattern described for birds. Our findings reinforce results of prior studies indicating that squamate reptiles mobilize and metabolize the large yolk reserves in their eggs through a process unknown in other amniotes. Development of the yolk sac of lizards differs from birds in four primary characteristics, migration of mesoderm, proliferation of endoderm, vascular development and cellular diversity within the yolk sac cavity. Notably, all of the yolk is incorporated into cells relatively early in development and endodermal cells within the yolk sac cavity align along blood vessels which course throughout the yolk sac cavity. The pattern of uptake of yolk by endodermal cells indicates that the mechanism of yolk metabolism differs between lizards and birds and that the evolution of a fundamental characteristic of embryonic nutrition diverged in these two lineages. Attributes of the yolk sac of squamates reveal the existence of phylogenetic diversity among amniote lineages and raise new questions concerning the evolution of the amniotic egg. J. Morphol. 278:574–591, 2017. © 2017 Wiley Periodicals, Inc.