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Analysis of the Role of Crim1 in Heart Development Swati Iyer B.Sc, M.Sc

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Analysis of the Role of Crim1 in Heart Development Swati Iyer B.Sc, M.Sc Analysis of the Role of Crim1 in Heart Development Swati Iyer B.Sc, M.Sc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Biomedical Sciences i Abstract To overcome the societal and financial burden of cardiovascular disease, a thorough understanding of the molecular and cellular programmes governing cardiac and coronary vascular development is vital. Cre-mediated genetic tracing in various models has provided valuable insight into cell-lineage contribution and inductive cues required for cardiac morphogenesis. Using mouse models, we have identified the transmembrane protein Cysteine-Rich Transmembrane BMP Regulator-1 (Crim1) as an essential protein for numerous aspects of heart development. The presence of six Cysteine-Rich Repeats (CRRs) in Crim1 render it to be a versatile molecule – these have been shown to bind a wide range of growth factors when Crim1 is co-expressed in the same cell as the growth factor (Wilkinson et al., 2003, Wilkinson et al., 2007). The main objective of this thesis is to examine the role that Crim1 plays both temporally and spatially during heart development, and how it functions in tandem with other factors to ensure the progression of this critical process. Crim1 is strongly expressed in the proepicardium (PE), epicardium, coronary vascular smooth muscle cells and, to a weaker extent, in coronary endothelial cells (Pennisi et al., 2007). Interestingly, Crim1 mutant homozygotes die perinatally on a C57BL6 background (Chiu et al., 2012). Loss of Crim1 function leads to a reduced ventricular size and hypoplastic ventricular compact myocardium, and abnormal epicardial morphology including blebbing and a loss of the regular, cobblestone appearance of the epicardium. Furthermore, epicardium-restricted deletion of Crim1 resulted in increased epicardial epithelial-to-mesenchymal transition (EMT) and invasion of the myocardium, while primary epicardial cells lacking Crim1 displayed an increased migration. Growth factors like TGFs are known to promote epicardial EMT; however, we observed a paradoxical reduction in epicardial TGF signalling in Crim1 mutant embryos. Interestingly, there appeared to be an accumulation of -catenin, a cell-adhesion molecule present at cadherin- dependent junctional complexes in epithelial cells, at epicardial cell-cell junctions, suggesting an interaction with Crim1 to promote stabilization of these junctions. There was also an increase in the level of phospho-ERK1/2 in the ventricular compact myocardium of mutants, though phospho-AKT levels remained unchanged. This indicates a cell-autonomous role for Crim1 in controlling epicardial migration, EMT and invasion, and a potential paracrine role in regulating myocardial development, likely through regulation of epicardium derived factors. Another notable defect observed in the absence of Crim1 is that the coronary vasculature also appears to be malformed. We found a reduction in coronary vascular endothelial cell endowment in ii both ventricles, in Crim1Δflox/Δflox homozygotes and in endothelium-restricted mutants, identifying a cell-autonomous role for Crim1. To further study Crim1 function in vitro, lentiviral small hairpin ribonucleic acid (shRNA) targeting the CRIM1 gene was transduced into Human Cardiac Microvascular Endothelial Cells (HCMVECs), with a knockdown of more than 70% achieved at the transcript level. Trypan Blue viability assays in both HCMVECs and Human Umbilical Vein Endothelial Cells (HUVECs) indicate reduced viability in the absence of CRIM1. Protein lysates obtained from both Crim1-knockdown HCMVECs and HUVECs indicate a reduction in phospho- SMAD1/5 levels, but no change in phospho-ERK1/2 and phospho-AKT levels. We also sought to understand the role of Crim1 in growth factor modulation in endothelial cells in vivo. There was a decrease in phospho-SMAD1/5 activity in endothelial cells from ventricles of Crim1Δflox/Δflox homozygotes, suggesting a dysregulation of TGFβ/BMP signalling in these cells, confirming our in vitro data. However, there was an increase in endothelial phospho-SMAD1/5 levels in ventricles of Crim1Flox/Flox; Tie2-Cre hearts, indicating a possible paracrine role for the other resident cells of the ventricular myocardium that retain Crim1 function. cDNA synthesised from HCMVECs that had lost Crim1 was then used in a human endothelial cell biology array. This revealed an upregulation of genes involved in increased inflammatory response and cell death, and, an increased probability of disease states including vascular lesions, infarction and heart failure, upon Ingenuity Pathway Analysis. Collectively, we illustrate a cell-autonomous role for Crim1 in the development of coronary vascular endothelial cells, in vivo and in vitro. We further report for the first time, that CRIM1 is able to bind Insulin-like Growth Factors (IGFs). This was shown by means of co-immunoprecipitation experiments, and reveals that the IGFBP domain of CRIM1, and CRRs 3-6 are responsible for this binding. Moreover, in HCMVECs lacking CRIM1, phosphorylation of the IGF-1 receptor was decreased, indicating the ability of CRIM1 to regulate IGF signalling. Our findings thus provide a platform for understanding how Crim1 functions in both the early and late stages of embryonic heart development in vivo. Many of these findings are mimicked in vitro, most significantly in the context of growth factor modulation in human cardiac microvascular endothelial cells, and aids in understanding the pathogenesis of disease states. This indicates translational relevance to humans, making the study of CRIM1 an exciting new avenue for future research in the field of cardiac development. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature 1. Crim1 has cell-autonomous and paracrine roles during embryonic heart development. Iyer S, Chou FY, Wang R, Chiu HS, Raju VK, Little MH, Thomas WG, Piper M, Pennisi DJ. Sci Rep. 2016 Jan 29; 6:19832. 2. Crim1 – a regulator of developmental organogenesis. Iyer S, Pennisi DJ, Piper M. Histol Histopathol. 2016 Apr 5:11766. Review. 3. CRIM1 is necessary for coronary vascular endothelial cell development and homeostasis. Iyer S, Chhabra Y, Harvey TJ, Wang R, Chiu HS, Smith AG, Thomas WG, Pennisi DJ, Piper M. Journal of Molecular Histology. 2016 Nov 1. Epub ahead of print. 4. Usp9x-deficiency disrupts the morphological development of the postnatal hippocampal dentate gyrus. Oishi S, Premarathne S, Harvey TJ, Iyer S, Dixon C, Alexander S, Burne TH, Wood SA, Piper M. Sci Rep. 2016 May 16; 6:25783. Conferences attended Oral presentations – Role of Crim1 in Epicardial EMT and Coronary Vasculature Development. Iyer S., Chiu H.S., Wang R., Chou F.Y., Chand R., Porter A. and Pennisi D.J. 5th International Postgraduate Symposium in Biomedical Sciences, Brisbane, Australia 2014 (Oral Presentation Award) Analysis of the role of Crim1 in heart development. Iyer S., Chiu H.S., Wang R. and Pennisi D.J. Molecular Pathways of Diseases Seminar, SBMS, Brisbane, Australia, 2014. v Poster presentations - Cell-autonomous and paracrine functions of Crim1 in epicardial EMT and coronary vascular development. Iyer S., Chiu H.S., Wang R., Porter A., Chand R., Chou F.Y. and Pennisi D.J. Brisbane Cell and Developmental Biology Meeting, Brisbane, Australia, 2014 (Best Poster Presentation Award) Cell-autonomous and paracrine functions of Crim1 in epicardial EMT and development of the coronary vasculature Iyer S., Chiu, H.S., Wang, R., Porter A., Chand, R., Chou, F.Y. and Pennisi, D.J. ComBio 2014, Canberra, Australia, 2014. The Role of Crim1 in Coronary Vasculature Development. Iyer S., Harvey T.J, Piper M., Pennisi D.J. 4th Meeting of the Australian Network of Cardiac and Vascular Developmental Biologists, Adelaide, Australia, 2015 Crim1 regulates EMT and myocardial development in a cell-autonomous and paracrine manner. Iyer S., Chiu H.S., Wang R., Chou F.Y., Harvey T.J., Piper M. and Pennisi D.J. Brisbane Cell and Developmental Biology Meeting, Brisbane, Australia, 2015. Crim1 regulates EMT and myocardial development in a cell-autonomous and paracrine manner. Iyer S., Chou F.Y., Chiu H.S., Wang R., Piper M. and Pennisi D.J. 6th International Postgraduate Symposium in Biomedical Sciences, Brisbane, Australia 2015 vi Publications included in this thesis 1. Crim1 has cell-autonomous and paracrine roles during embryonic heart development. Iyer S, Chou FY, Wang R, Chiu HS, Raju VK, Little MH, Thomas WG, Piper M, Pennisi DJ. Scientific Reports. 2016 Jan 29; 6:19832. Contributor Statement of contribution Swati Iyer (Candidate) Wrote the paper (80%) Designed experiments (50%) Performed experiments (50%) Edited the paper (25%) Dr.
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