Thesis (Ph. D.)--Ohio State University, 2002. / Title from first page of PDF file. Document formatted into pages; contains xxiii, 216 p. Includes abstract and vita. Robert W. Curley, College of Pharmacy. Includes bibliographical references (p. 201-216).

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published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

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Matthew-Wood syndrome is a rare human birth defect condition defined by the phenotypic constellation of clinical anophthalmia, diaphragmatic hernia, pulmonary hypoplasia and cardiac defects. Matthew-Wood syndrome has a high mortality rate, with most patients dying due to respiratory insufficiency as a consequence of pulmonary hypoplasia, within the first year of life. Mutations within STRA6 are causative for Matthew-Wood syndrome. STRA6 acts as a retinol transporter for retinol bound to its physiological carrier RBP4 allowing regulated entry of retinol into the cell. A mammalian model for Matthew-Wood syndrome was not found within the literature; however a morpholino knockdown of stra6 in the zebrafish did show phenotypic features consistent with those observed in human patients. The desire to create a mammalian model of Matthew-Wood syndrome drove the work contained within this thesis. Stra6-/- mice do not represent a model for Matthew-Wood syndrome with homozygous animals being viable, found in the expected ratio and demonstrating none of the developmental abnormalities observed in human patients. Retinal defects, cataracts and persistent hyperplastic primary vitereous affect the microphthalmic eye of Stra6-/- offspring of Stra6-/- mothers fed a retinoid-free diet from plug to birth indicating that Stra6 is required for normal eye development under low-retinoid stress. The disparity in phenotype between human Matthew-Wood patients and Stra6-/- mice may be the result of functional redundancy in the mouse between Stra6 and its paralogue, Stra6.2. Stra6.2 is well conserved through evolution and is found in diverse species, including the basal eumetazoan Trichoplax. STRA6.2 has become split across its resident chromosome with an associated break in gene synteny, in humans and great apes, causing most of the gene to no longer be transcribed. However a small portion of the gene, representing the final transmembrane domain and the C-terminal intracellular tail of the protein, remains expressed in human. stra6.2 is required for normal development in the zebrafish with stra6.2 morphants being phenotypically distinguishable from control injected embryos from the 10-somite stage by a larger head-tail distance indicating an axial extension defect. stra6.2 morphants also display microphthalmia, jaw malformation, shortened and curved body axis and retinal lamination defects. stra6.2 was found to be required to prevent an excess of retinoic acid resulting in an upregulation of retinoic acid-dependent gene expression through an increase in RA synthesis by Raldh enzymes in morphants. Stra6.2-/- mice are viable and fertile and phenotypically normal, even under retinoid-stress, supporting the notion of functional redundancy. In compound knockouts, normal development and postnatal survival can be maintained by a single copy of Stra6 in Stra6+/-;Stra6.2-/- animals. Stra6.2 is less able to support normal development and survival with ~50% of Stra6-/-;Stra6.2+/- animals dying before weaning or showing reduced growth although the remaining animals are indistinguishable from their littermates. Stra6 and Stra6.2 are functionally redundant for development under normal dietary conditions in the mouse and a single copy of either is able to support development in at least 50% of animals. Stra6-/-;Stra6.2-/- mice were therefore hypothesised to be the logical mouse model of Matthew-Wood syndrome, however these mice die early in gestation between E7.5-E9.5. The early embryonic lethality in Stra6-/-;Stra6.2-/- mouse embryos compared to postnatal survival in human Matthew-Wood patients, to which they are the comparable genetic model, could be attributed to the shortened STRA6.2 remaining within the human genome. The equivalent portion of Stra6 has validated signalling motifs, which may still be active in STRA6.2, allowing development to proceed in human ‘STRA6-/-’ embryos.

by Poon Lit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 97-113). / Title page --- p.i / Acknowledgements --- p.ii / Table of contents --- p.iii / List of tables --- p.vii / List of figures --- p.viii / Abbreviations --- p.x / Abstract --- p.xi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Sacral agenesis --- p.2 / Chapter 1.1.1 --- Skeletal anomalies --- p.3 / Chapter 1.1.2 --- Neurological anomalies --- p.4 / Chapter 1.1.3 --- Other anomalies --- p.5 / Chapter 1.1.4 --- Etiology --- p.5 / Chapter 1.1.5 --- Pathogenetic mechanism of SA --- p.7 / Chapter 1.2 --- Retinoids --- p.8 / Chapter 1.2.1 --- RA in embryonic development --- p.9 / Chapter 1.2.2 --- Teratogenic effect of RA in embryonic development --- p.10 / Chapter 1.3 --- Strategy of the thesis --- p.13 / Chapter Chapter 2: --- General Materials and Methods --- p.15 / Chapter 2.1 --- Mouse maintenance and mating method --- p.16 / Chapter 2.2 --- All-trans RA preparation and injection --- p.16 / Chapter 2.3 --- Dissection of embryos --- p.16 / Chapter 2.4 --- Preparation of histological sections --- p.17 / Chapter 2.4.1 --- Dehydration and embedding --- p.17 / Chapter 2.4.2 --- Sectioning --- p.18 / Chapter 2.4.3 --- Haematoxylin and eosin staining --- p.18 / Chapter 2.5 --- Plasmid preparation --- p.19 / Chapter 2.5.1 --- Competent cells preparation --- p.19 / Chapter 2.5.2 --- Bacterial transformation --- p.19 / Chapter 2.5.3 --- Mini-scale preparation of plasmid DNA --- p.20 / Chapter 2.6 --- In situ hybridization --- p.21 / Chapter 2.6.1 --- Sample preparation --- p.21 / Chapter 2.6.2 --- Probe synthesis --- p.21 / Chapter 2.6.3 --- Hybridization --- p.23 / Chapter 2.6.4 --- Post-hybridization wash and antibody labeling --- p.24 / Chapter 2.6.5 --- Post-antibody wash and colour development --- p.25 / Chapter 2.6.6 --- Embryo powder preparation --- p.25 / Chapter Chapter 3: --- Time and Dose Responses to RA --- p.26 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.1.1 --- Time response --- p.27 / Chapter 3.1.2 --- Dose response --- p.29 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Dose response --- p.31 / Chapter 3.2.2 --- Time response --- p.31 / Chapter 3.2.3 --- Skeletal preparations and staining --- p.31 / Chapter 3.2.4 --- Early teratogenic responses to RA --- p.32 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- Dose response --- p.33 / Chapter 3.3.1.1 --- Tail length --- p.33 / Chapter 3.3.1.2 --- Vertebral pattern --- p.33 / Chapter 3.3.2 --- Time response --- p.36 / Chapter 3.3.2.1 --- Tail length --- p.36 / Chapter 3.3.2.2 --- Vertebral pattern --- p.36 / Chapter 3.3.2.3 --- Other anomalies associated with human SA --- p.40 / Chapter 3.3.3 --- Early teratogenic responses in RA-treated embryos --- p.41 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4: --- RA-induced Cell Death in Tail Bud --- p.51 / Chapter 4.1. --- Introduction --- p.52 / Chapter 4.1.1 --- Cell death --- p.52 / Chapter 4.1.2 --- Methods in cell death identification --- p.54 / Chapter 4.2 --- Materials and methods --- p.57 / Chapter 4.2.1 --- Histology of tail bud region --- p.57 / Chapter 4.2.2 --- Detection of internucleosomal DNA fragmentation --- p.57 / Chapter 4.2.2.1 --- Sample collection --- p.57 / Chapter 4.2.2.2 --- DNA extraction --- p.58 / Chapter 4.2.2.3 --- Analysis of DNA fragmentation pattern by ethidium bromide staining --- p.59 / Chapter 4.2.2.4 --- Analysis of DNA fragmentation pattern by autoradiography --- p.59 / Chapter 4.2.3 --- TUNEL --- p.61 / Chapter 4.2.3.1 --- Sample collection / Chapter 57 4.2.3.2 --- In situ end-labeling --- p.62 / Chapter 4.2.4 --- In situ hybridization of Wnt-5A --- p.63 / Chapter 4.2.4.1 --- Sample collection --- p.63 / Chapter 4.2.4.2 --- Probe preparation --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Histological examination of cell death in tail bud --- p.65 / Chapter 4.3.2 --- Analysis of DNA fragmentation of tail bud cells --- p.66 / Chapter 4.3.3 --- TUNEL --- p.67 / Chapter 4.3.4 --- Wnt-5A gene expression --- p.68 / Chapter 4.4 --- Discussion --- p.71 / Chapter Chapter 5: --- Retinoic Acid Receptors (RARs) --- p.75 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.1.1 --- RARs and their isoforms --- p.76 / Chapter 5.1.2 --- Expression pattern of RARs during embryogenesis --- p.77 / Chapter 5.1.3 --- RAR mutants and functional redundancy of RARs --- p.78 / Chapter 5.1.4 --- RARs and SA --- p.79 / Chapter 5.2 --- Materials and methods --- p.80 / Chapter 5.2.1 --- In situ hybridization of RAR-α --- p.80 / Chapter 5.2.1.1 --- Sample collection --- p.80 / Chapter 5.2.1.2 --- Probe preparation --- p.80 / Chapter 5.2.2 --- In situ hybridization of RAR-β --- p.81 / Chapter 5.2.2.1 --- Sample collection --- p.81 / Chapter 5.2.2.2 --- Probe preparation --- p.81 / Chapter 5.2.3 --- In situ hybridization of RAR-γ --- p.82 / Chapter 5.2.3.1 --- Sample collection --- p.82 / Chapter 5.2.3.2 --- Probe preparation --- p.82 / Chapter 5.3 --- Results --- p.83 / Chapter 5.3.1 --- RAR-α gene expression --- p.83 / Chapter 5.3.2 --- RAR-β gene expression --- p.84 / Chapter 5.3.3 --- RAR-γ gene expression --- p.86 / Chapter 5.4 --- Discussion --- p.88 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.92 / Chapter 6.1 --- Conclusion and future perspectives --- p.93 / References --- p.97 / Appendices --- p.114 / Figures --- p.118

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 166-190).

Thesis (Ph. D.)--University of Hawaii at Manoa, 1991. / Includes bibliographical references (leaves 177-207) / Microfiche. / xiii, 207 leaves, bound ill. 29 cm

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 137-151).

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