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.

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

Thesis supervisor: Robert A. Cornell. Includes bibliographical references (p. 121-142).

Thesis (D. Min.)--New Orleans Baptist Theological Seminary, 1996. / Includes abstract and vita. Includes bibliographical references (leaves 168-174).

Thesis (Ph. D.)--Ohio State University, 2008.

Abstract Secreted signalling molecules are important for the regulation of developmental cell responses. In the developing kidney, signalling occurs between epithelial ureteric bud and metanephric mesenchyme and in between their derivatives. Wnt6 gene activity was localized to the ureteric bud and newly formed branches of the ureteric tree during early stages of kidney development. In a classic organ culture system, Wnt6 signalling induced the activation of marker genes for early nephrogenesis. The metanephric mesenchymes isolated from the Wnt4 deficient embryos were also induced, and the Wnt4 gene became activated in the presence of a Wnt6 signalling source. We propose that Wnt-6 is involved as a metanephric inducer and controls nephrogenesis. Wnt4 is essential for nephrogenesis in mouse and we indicate an additional role for Wnt4 in the control of periureteric stromal differentiation. A failure in vascular development was also found. Bmp4 expression in the medullar stroma of the Wnt4-deficient kidneys was absent concomitantly with a loss of expression of the smooth muscle marker, α-SMA. In vitro Wnt4 signalling induced Bmp4 expression and local α-SMA production. Hence, we conclude that lack of Wnt4 signalling leads to a loss of the periureteric smooth muscle cells, and Wnt4 may locally regulate this cell population in normal kidneys via regulation of Bmp4 signalling. The pluripotent neural crest cells are proposed to play regulatory roles in the early metanephros. Here, the use of transgenic animals allowed visualisation of the lumbo-sacral neural crest (NC) cells in close proximity to the early metanephros. The NC cells, however, disappeared in most part of the kidney by E12.5. The Splotch embryos lack the NCs from the early urogenital region. A developmental defect in the kidneys of Splotch embryos was not observed in vivo or in vitro. The results suggest that the neural crest is not essential for early embryonic kidney development. In sum, the work presented indicates an important role for Wnt6 in the induction of kidney tubules in vitro, for Wnt4 in the specification of kidney smooth muscle cells and for endothelial development in kidney. The neural crest cells apparently have no active morphogenetic role in early kidney development.

Abstract Reduction of morbidity in osseous reconstruction of cranio-maxillofacial bony defects could come from development of less invasive bone graft harvesting techniques or by elimination of bone graft donor sites using a bone graft substitute. This work studies outcomes and morbidity associated with these two approaches. A power-driven trephine was used to harvest bone from the anterior iliac crest using a minimally invasive surgical technique. Initially the safety of the technique was evaluated in a cadaver model. Twenty-five freshly preserved adult cadavers had a total of 250 cancellous cores of bone harvested from 50 anterior iliac crest sites. Twenty intentional perforations were made to the maximum depth possible with the instrumentation tested. No encroachment upon the peritoneum was found. A total of 84 patients had 333 cores of cancellous bone harvested using the same approach with a complication rate of 3.6% and a patient satisfaction rate of 98.8%. In a further 76 patients the motorized trephine method was compared to traditional open iliac crest corticocancellous block harvesting. The trephine group ambulated earlier, required fewer days of hospital stay and had significantly lower pain scores than the open iliac crest group. Coral-derived granules were used as a xenograft bone graft substitute to treat bony defects in the cranio-maxillofacial skeletons of 36 patients with 54 sites and followed for 12 to 36 months. The augmentations produced satisfactory results with the following complications noted: overt wound infection 1.8%, wound irritation 3.8% and clinically evident resorption in 9.3% of augmented sites. Coral-derived granules were then used to treat 48 dento-alveolar defects in 21 growing patients with trauma induced tooth-loss in the anterior maxilla and elective ankylosed tooth removal in the posterior maxilla and mandible. Coral granules were significantly more efficacious in reconstructing alveolar defects in the posterior maxilla or mandible (93.5%), than the anterior maxilla (17.6%). The minimally invasive technique using a power driven trephine was successful at reducing morbidity from bone graft harvesting at the anterior iliac crest. Coral-derived granules can be used in selected situations as a bone graft substitute and minimize post surgical morbidity by eliminating the bone graft donor site.