The role of Cited2 in left-right patterning and placental development in the mouse. Stanley Troy Meredor Artap Bachelor of Science (Advanced) (First Class Honours) This thesis is submitted in fulfilment of the requirements of the degree of Doctor of Philosophy 2012 Developmental Biology Program, The Victor Chang Cardiac Research Institute, and St. Vincent’s Clinical School, Faculty of Medicine, University of New South Wales Australia i PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Artap First name: Stanley Troy Other name/s: Meredor Abbreviation for degree as given in the University calendar: PhD School: St. Vincent’s Clinical School Faculty: Medicine Title: The role of Cited2 in left-right patterning and placental development in the mouse. Abstract 350 words maximum: (PLEASE TYPE) Cited2 is a member of the CITED gene family of transcriptional co-factors widely expressed in the mouse conceptus. It interacts with various transcriptional factors to drive the proper development of many organs in the mouse. Territories of Cited2 expression in the mouse coincide with the tissues most affected in Cited2 null mice. Embryos deficient in Cited2 display complex heart defects that may have multiple origins. One possibility is that the establishment of the left-right body axis that provide morphological cues for developing organs, like the heart, is perturbed in these mutant embryos. Cited2 is expressed in the relevant left-right patterning tissues such as the node and the lateral plate mesoderm. Therefore, the biochemical role Cited2 plays in left-right patterning was explored. This part of the study provides insight into how Cited2 may function in left-right development and organogenesis. Cited2 is also very important in the developing mouse placenta, where it is expressed in many trophoblast subtypes and vascular cells. Cited2 deficient placentas are grossly small in size, with reduced trophoblast numbers and an expanded foetal vasculature. This part of the study further characterised the cellular expression of Cited2 in placental cells. It also further describes the vascular phenotype in Cited2 mutants, and biochemically relevant molecules to inform on the potential molecular workings of the phenotype. Furthermore, conditional deletion of Cited2 was also performed to understand which placental compartments Cited2 is primarily required. It was found that endothelial cell-specific deletion of Cited2 phenocopied much of the complete Cited2 null placental phenotype, indicating that Cited2 is necessary in endothelial cells of the mouse placenta. 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I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... “Mon esprit, prends garde. Pas de partis de salut violents. Exerce-toi ! - Ah ! la science ne va pas assez vite pour nous !” - “Une Saison en Enfer” by Arthur Rimbaud (April-August, 1873) v Acknowledgements To my supervisor Sally, you will forever be etched in my mind as the supervisor who had a song for every occasion. So in keeping with your habit to break into song I tried to find a tune that aptly described you. I could only find one that was the least inappropriate. So to quote Little Richard, “Long Tall Sally”, I thank you for your guidance and support throughout this candidature. Thank you for allowing me to discover the wonderful world that is developmental biology. I would also like to thank my other mentors Richard and Duncan who together provided great technical guidance, advice and words of encouragement over the course of this PhD. Thank you to you both. To the Dunwoodie lab, past and present, thank you for all your advice, assistance and friendship throughout the years. I would like to especially mention a couple of people who made the time in the lab very enjoyable and academically stimulating: Gavin, my pseudo-mentor (deny all you want but we both know this is true) thank you for your listening ear, your mentoring, for introducing me to the wonderfully eerie music of Sigur Ros, but most of all for your friendship; Wendy, my lab sister thank you for sharing your world of gastronomy, there was one less hungry PhD student throughout your adventures into the world of food and pastries, but most of all I cherish your friendship. A large number of postdocs, research assistants, student members and support staff, past and present, of the Developmental Biology Program at the Victor Chang Cardiac Research Institute and throughout the institute itself have helped to colour my time at the institute. Thank you to all for your words of encouragement, desk space (for I had many) and friendship, but among them I would like to especially mention a few. Danielle, my Thursdays will never be the same again thanks to you. Lady, by the way, my shoulder has knots, care to get rid of them? To my French “amis” Rom-boules and Mimi, thank you for the support, laughter and your French-ship [sic]. “Tout d'abord, Rom-boules, suggérer aux doctorants d'abandonner avant qu'il ne soit trop tard est une manière vi ingénieuse de provoquer la réaction inverse et au final de les motiver à terminer rageusement - Chapeau bas, monsieur”! Mimi, ma petite baleine, tu es la soeur que je n'ai jamais eue, merci de prendre soin de moi et de m'offrir ton amitié”. Megumi, “domo arigato” for introducing me to Japanese culture, your help in the lab and of course your friendship. Brendan, thank you very much for all your hard work and swift IT help, without you this thesis would never have seen the light of day. Thank you! I would also like to extend my thanks to my fellow SAVI students, PhD and Honours students, past and present, for sharing the scientific experience with me. Thank you for all the drinks, tales and late nights shared contemplating everything and anything. I would like to particularly thank Jacque and Michelle for coordinating the genesis of SAVI, which brought a collegial atmosphere to the Institute’s student body. Also, I would like to thank my SAVI co-chair, Leah, for the tremendous effort spent with me in trying to maintain the existence of SAVI. My science was all influenced by many scientists in the nearby Garvan Institute of Medical Research for which I am truly thankful. I would like to especially name Amanda, thank you for sharing your happy disposition that always cheered me up, your technical advice and your zeal for science that is so infectious. I would like to express my deepest and sincere thanks to my very good friend, Ting, who I shared many great and not-so-great moments in and outside of the lab.