Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 87-102).

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

Τα κύτταρα της νευρικής ακρολοφίας (Neural Crest Cells - NCCs) αποτελούν έναν πολυδύναμο, μεταναστευτικό πληθυσμός βλαστικών κυττάρων τα οποία δίνουν γένεση σε μια πληθώρα κυτταρικών τύπων κατά την ανάπτυξη των σπονδυλωτών, συμπεριλαμβανομένων των νευρώνων και των νευρογλοιακών κυττάρων του περιφερικού νευρικού συστήματος (ΠΝΣ). Η δημιουργία των κυττάρων της νευρικής ακρολοφίας πραγματοποιείται στο στάδιο της γαστριδίωσης μετά την επαγωγή της νευρικής πλάκας. Οι διαδικασίες αυτές επηρεάζονται από σηματοδοτικά μονοπάτια στα οποία εμπλέκονται τόσο μεταγραφικοί παράγοντες όσο και επιγενετικοί τροποποιητές . Το Εντερικό Νευρικό Σύστημα (ΕΝΣ), προέρχεται από τα κύτταρα της νευρικής ακρολοφίας της αυχενικής και της ιερής μοίρας και ελέγχει την ομαλή λειτουργία της γαστρεντερικής οδού. Τα κύτταρα της νευρικής ακρολοφίας της αυχενικής μοίρας αποικίζουν ολόκληρο τον εντερικό σωλήνα και δίνουν γένεση στο ΕΝΣ ξεκινώντας από την 9η εμβρυική ημέρα. Η δημιουργία ενός πλήρως λειτουργικού ΕΝΣ εξαρτάται από την ικανότητα μετανάστευσης, πολλαπλασιασμού και διαφοροποίησης των NCCs. Στόχος της παρούσας διπλωματικής εργασίας ήταν η μελέτη της έκφρασης γονιδίων που ελέγχουν τον πολλαπλασιασμό και τη μετανάστευση τόσο των πρόδρομων κυττάρων της νευρικής ακρολοφίας όσο και των NCCs που έχουν δεσμευτεί προς εντερική μοίρα σε έμβρυα μυός απουσία της πρωτεΐνης Geminin. Η Geminin είναι μια πρωτεΐνη που έχει δειχθεί να επηρεάζει την ισορροπία μεταξύ αυτο-ανανέωσης και διαφοροποίησης, μέσω της αλληλεπίδρασης της με μεταγραφικούς παράγοντες και πρωτεΐνες αναδιαμόρφωσης της χρωματίνης. Με σκοπό να διερευνήσουμε τον in vivo ρόλο της Geminin στα κύτταρα της νευρικής ακρολοφίας, δημιουργήσαμε διαγονιδιακούς μύες από τους οποίους αδρανοποιήσαμε το γονίδιο της Geminin ειδικά στα κύτταρα της νευρικής ακρολοφίας. Τα αποτελέσματα μας έδειξαν ότι η απουσία της Geminin από τα κύτταρα της νευρικής ακρολοφίας, οδηγεί στη δημιουργία εμβρύων με μορφολογικές αλλοιώσεις κατά τα πρώιμα στάδια της ανάπτυξης ενώ σε μεταγενέστερα αναπτυξιακά στάδια χαρακτηρίζονται από σοβαρές κρανιοπροσωπικές δυσμορφίες. Επιπλέον, η ιστοειδική αδρανοποίηση της Geminin οδήγησε σε απορρύθμιση των επιπέδων έκφρασης γονιδίων που επηρεάζουν τόσο την επαγωγή όσο και τη μετανάστευση των NCCs (mChd7, mSnail2, mTwist2,mFoxD3) κατά την 10.5η εμβρυική ημέρα. Μελέτη του κυτταρικού κύκλου των εντερικών κυττάρων νευρικής ακρολοφίας έδειξε ότι η αποσιώπηση της Geminin διαταράσσει το προφίλ του κυτταρικού τους κύκλου καθώς τα κύτταρα αυτά «μπλοκάρουν» κατά τη μετάβαση από την G2 στη Μ φάση (E9.5). Συμπερασματικά, τα αποτελέσματά μας δείχνουν ότι η Geminin συμμετέχει ενεργά στον ρύθμιση της έκφρασης γονιδίων που παίζουν σημαντικό ρόλο στην επαγωγή και τη μετανάστευση των NCCs κατά τα πρώιμα στάδια της εμβρυικής ανάπτυξης (Ε10.5). Τέλος, η Geminin διαδραματίζει σημαντικό ρόλο στον πολλαπλασιασμό των πρόδρομων κυττάρων του εντερικού νευρικού συστήματος (Ε9.5). / Neural Crest cells (NCCs) comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types during vertebrate development, including neurons and glial cells of Peripheral Nervous System (PNS). The induction of neural crest specification occurs at the end of gastrulation at the neural plate border initiate an epithelial-to-mesenchymal transition (EMT) that transforms these stationary cells into migratory cells. These processes are influenced by changes in the expression of transcription factors and epigenetic regulators. The Enteric Nervous System (ENS), is a subdivision of the PNS that controls the function of the gastrointestinal (GI) tract and is derived from the Vagal and Sacral NCCs. The formation of a fully functional ENS depends on the coordinated proliferation and differentiation decisions of NCCs. Vagal NCCs emigrate from the vagal region of the neural tube (somites 1-7), in a rostal to caudal direction and enter the foregut at embryonic day E9-9.5 (in mice), generating the ENS. The formation of a fully functional ENS depends on proliferation and differentiation decisions of NCCs. The aim of our work was to study the expression of genes that control self-renewal decisions and migration ability of the NCCs in mouse embryos in the absence of Geminin. Towards this direction we studied the in vivo role of Geminin in the early stages of NCCs development and in the developing ENS. Geminin is a Protein that has been shown to affect the balance between self-renewal and differentiation through multiple interactions with transcription factors and chromatin remodeling proteins. In order to further elucidate the in vivo role of Geminin in NCCs, we generated transgenic mice lacking Geminin expression specifically in NCCs. We have shown that the deletion of Geminin in NCCs, causes severe morphological and craniofacial malformation during embryonic development. The conditional inactivation of Geminin resulted in the deregulation of various genes that affect the induction and migration of NCCs (mChd7, mSnail2, mTwist2, mFoxD3) at E10.5. Examination of the cell cycle profile of enteric NCCs showed that the deletion of Geminin disrupted the proliferation of NCCs as cells are blocked at the transition from G2 to M phase (E9.5). In conclusion, our results highlight Geminin as an important molecule during the induction and migration of NCCs at the early stages of mouse embryonic development (10.5). Finally, Geminin plays an important role for the proliferation of ENS progenitor cells (E9.5).

<p>Throughout vertebrate evolution, the cardiac outflow vasculature has changed from a branchial arch system to a systemic and pulmonary circulatory system. However, all vertebrate hearts and outflow tracts still develop from a single heart tube. In the chick and mouse, cardiac neural crest cells divide the single outflow tract into the aorta and pulmonary arteries. Additionally, cardiac neural crest cells provide the smooth muscle of the aortic arch arteries, help to remodel the aortic arch arteries into asymmetrical structures, and contribute cardiac ganglia. I review the major contributions of cardiac neural crest cells to the outflow vasculature of the chick and mouse and apply this information to study cardiac neural crest cell contributions to vertebrates that lack a divided circulatory system. I re-evaluate the role of cardiac neural crest cells in zebrafish vasculature and find that these cells do contribute to the gill arch arteries, the ventral aorta and cardiac ganglia, but they do not contribute to myocardium. I also study the outflow tract development of the turtle Trachemys scripta to understand the process of outflow septation in a vertebrate that has a divided outflow tract but an incomplete division of the ventricle. I compare the chick outflow tract to the turtle. The formation of the proximal versus distal cushions and the appearance of smooth muscle cells within the distal cushions of the turtle are very similar to the cushion position and cell types within the cushions of the chick. In the chick, the smooth muscle positive cells in the distal cushions are derived from cardiac neural crest cells. I hypothesize that cardiac neural crest cells are also responsible for the outflow tract septation of reptiles. These results demonstrate that the pattern of cardiac neural crest cell contribution to vertebrate vasculature remains predictable and consistent, enabling future studies to focus on changes in vascular patterning caused by cardiac neural crest cells among different vertebrate lineages.</p> / Dissertation

Neural crest cells go through an epithelial-mesenchymal transition (EMT) before they migrate. B1 integrins are necessary during these phases of neural crest development, but it is unclear if integrins are required for both EMT and neural crest migration. Chimeric integrin B1 subunit and mutant Rho GTPases are used in this study to assess function during neural crest emigration. Cultures of chick embryonic cells, transfected with these constructs, are used to confirm the effects and expression in conjunction with a green fluorescent protein (GFP) reporter. In control experiments targeting the neural ectoderm of the hindbrain by localized electroporation. GFP-expressing cells release from the neural tube, migrate along neural crest pathways and express the HNK-1 neural crest marker. Immunolabeling of Sox9 and Slug neural crest markers shortly after electroporation confirms transfection of prospective neural crest cells. Electroporation with a chimeric hemagglutinin-B1 integrin subunit inhibits release of transfected cells from the neural tube. Embryos electroporated with constitutively active RhoA have a few transfected cells outside the neural tube that express N-cadherin. but they fail to migrate to the branchial arch. Electroporation with constitutively active Rac1 results in numerous cells near the neural tube, none of which express N-cadherin. Embryos electroporated with Cdc42 mutants are not distinguishable from control embryos expressing GFP alone. In embryos co-electroporated with chimeric integrin and dominant negative RhoA together, co-transfected cells migrate along neural crest pathways. The conclusion is that integrin signaling, transduced through RhoA, is necessary for the EMT of cranial neural crest. Key to this investigation of neural crest emigration is the methodology of localized electroporation. This technique introduces transgenes to targeted patches of cells in the embryo. Localized electroporation employs a double-barreled suction electrode to deliver plasmid and produce an electric field. Parameters for localized electroporation are optimized for transfecting a range of cells in the chick embryo, and expansion of the technique to mammals is demonstrated. Localized electroporation has improved reliability and higher efficiency than existing in vivo transfection techniques.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 162-189). Also available in print.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 179-221). Also available on the Internet.