Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 2003. / CD-ROM reproduces dissertation in PDF format; Adobe Acrobat required. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Kentucky, 2003. / Title from document title page. Document formatted into pages; contains x, 192p. : ill. Includes abstract. Includes bibliographical references (p. 165-189).

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 159-193).

Thesis (Ph. D.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ56223.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 140-172).

Neural crest cells (NCC) arise from the neural tube (NT) and migrate through given regions of embryos, where they generate most of the peripheral nervous system (PNS), facial skeleton and pigment cells. Defective NCC development gives rises to malformations in multiple NCC-derived structures, collectively known as neurocristopathies. NCC from the NT vagal and trunk levels express Hoxb5 plus a number of other Hox proteins. Hoxb5 is a member of Hox transcription factors family that binds to specific target nucleotide sequences in the genome via their DNA-binding domain, where they regulate gene expressions. Vagal NCC migrate to the intestine and generate the enteric nervous system (ENS). To test the Hoxb5 function in vagal NCC, we made use a transgenic mouse line (enb5) and showed that perturbation of Hoxb5 signaling in NCC resulted in down-regulation of Ret and defective ENS, indicating that normal Hoxb5 function was required for the development of vagal NCC. Current project aims to investigate the function of Hoxb5 in trunk NCC development. Transgenic mouse enb5 can be induced by Cre recombinase to express a hybrid protein namely engrailed-Hoxb5 (enb5), in which the transactivation domain of the mouse Hoxb5 is replaced with a repressor domain of the Drosophila engrailed (en) protein. With the intact DNA-binding domain, enb5 binds to target genes of Hoxb5, repressing the expression of target genes instead of induction. Therefore, enb5 produces a dominant negative effect on the developmental pathways that normally require Hoxb5. In this study, enb5 mice were crossed to Wnt1-Cre mice to express enb5 in NCC that arose from the entire length of NT. Wnt1-Cre/enb5 mutants displayed apoptosis of NCC, skin hypopigmentation and PNS defects (hypoplastic dorsal root ganglion and defective ENS). Expression of Sox9, Foxd3 and Ret was down-regulated in Wnt1-Cre/enb5 embryos. Conditional deletion of Sox9 and Foxd3 by Wnt1-Cre, or conventional deletion of Ret in mice produced NCC phenoptypes similar to those of Wnt1-Cre/enb5. Taken all these prompted me to further investigate if Hoxb5 functioned in the same pathway as Sox9 and Foxd3 for NCC development using multiple experimental approaches. In ovo electroporation of enb5 in chick embryos induced apoptosis of NT, and co-electroporation of Hoxb5, Ret, Sox9 or Foxd3 rescued enb5-induced cell death. By bioinformatics analysis, Hoxb5 binding sites were identified in SOX9 and FOXD3 promoter sequences. Binding of Hoxb5 protein onto these binding sites of SOX9 and FOXD3 promoters was revealed by electro-mobility shift assay and further confirmed by chromatin immuno-precipitation assay. In addition, enb5 was also shown to bind to the same regions of SOX9 and FOXD3 promoters as Hoxb5. Using dual luciferase reporter assay, Hoxb5 was shown to induce transcription from SOX9 and FOXD3 promoters, and enb5 blocked the induction. Taken all these indicate that (i) Hoxb5 binds and induces transcriptions from SOX9 and FOXD3 promoters, (ii) enb5 blocks the induction. In summary, Hoxb5 regulates NCC development by controlling the expression of Sox9, Foxd3 and Ret, and perturbation of Hoxb5 signaling results in NCC death and neurocristopathies. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Iroquois genes encode a family of highly conserved TALE homeodomain transcription factors that are involved in multiple developmental processes. Physiological tests indicated that Irx3 and Irx5 mutant mice displayed hearing impairment. However, the functions of these two genes during inner ear development are not known. The aim of this study is to characterize the roles of Irx3 and Irx5 during mammalian inner ear development using mouse models, in order to reveal the underlying mechanism for the hearing abnormality in the mutants. Two mouse mutants, Irx3tauLacZ and Irx3flox5EGFP with β-gal and EGFP reporters, were analyzed to examine the expression of these two genes in the otic vesicle and cochlear epithelium. In the otocyst, both Irx3 and Irx5 were expressed in the ventral-medial region. Irx5 expression was restricted to the non-sensory domain of the cochlear epithelia, while Irx3 was widely expressed, including the auditory sensory organ, the organ of Corti. The overlapping expression patterns of Irx3 and Irx5 suggest that they may share redundant functions. To investigate the roles of Irx3 and Irx5 during inner ear development, phenotypic analysis was performed on Irx3-/-, Irx5-/- and Irx3/5-/- mutant embryos. As shown by paint-filling analysis, Irx3/5-/- displayed shortened cochlear duct, enlarged cochlear lumen with fused sensory organ. Whole-mount phalloidin staining of hair cell bundles showed that Irx3-/- displayed occasional ectopic inner hair cells. Moreover, only supernumerary vestibular hair cell-like cells were developed in Irx3/5-/- mutant. These results suggest that Irx3 and Irx5 are required for inner ear morphogenesis and the formation of organ of Corti. To understand the effect of Irx3 and Irx5 in the cellular patterning of the cochlea, mutant cochleae were analyzed with markers for different regions of the cochlear epithelia. Altered expression domain of MyoVIIa, Sox2 and Gata2 in Irx3/5-/- cochlea revealed that the boundary between the Kolliker’s organ and the organ of Corti was lost and the location of sensory and non-sensory region was shifted. These results imply that Irx3 and Irx5 function in the establishment of the sensory/non-sensory boundary. It is known that p27kip1 regulates the wave of cell cycle exit in the developing organ of Corti and Sox2 takes part in prosensory specification. To explore the underlying reason for the patterning defects in Irx3/5-/- mutant, cochlear duct from prosensory stages were analyzed. Irx3/5-/- showed altered Sox2 and p27kip1 expression, with expanded prosensory domain and disrupted cell cycle exit. Ectopic prosensory proliferation was detected in the middle turn of the cochlear duct at E13.5 by BrdU incorporation assay. Therefore, Irx3 and Irx5 may participate in the subdivision of sensory territory in developing cochlea by controlling prosensory proliferation. In summary, this study demonstrates that Irx3 and Irx5 cooperate in multiple aspects of inner ear development: an early role to regulate prosensory proliferation and cell cycle exit; a second role to regulate cellular patterning of the cochlear duct by controlling the setting of sensory/non-sensory boundaries in the cochlea; a later role to regulate inner ear morphogenesis. This study supports the idea that Irx3 and Irx5 act as patterning genes during vertebrate evolution. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Friedreich ataxia (FRDA) is the most common inherited ataxia disorder, caused by a GAA repeat expansion mutation within the first intron of the FXN gene. The subsequent deficiency of frataxin protein leads to neurological disability, increased risk of diabetes mellitus, cardiomyopathy and premature death. The exact FRDA disease mechanism is not yet clear, despite some understanding of epigenetic, transcriptional and DNA repair system effects that lead to frataxin reduction. Previous studies have shown that mismatch repair (MMR) genes can affect other trinucleotide repeat disorders by destabilisation of the repeats. Furthermore, it has been proposed that frataxin deficiency might lead to cell malignancy by an as yet undefined mode of action. Therefore, the principle aim of this thesis was to use human and genetically altered mouse cells and tissues to understand the effects of MMR proteins on GAA repeat instability and FXN transcription, and also to identify potential changes in MMR transcription that might cause malignancy in FXN-defective human cells. Firstly, by using FXN and MMR genetically altered mice, MMR proteins were shown to be involved in both intergenerational and somatic GAA repeat instability, although their effects in the two systems were different. Thus, Msh2 or Msh3 were both found to protect against intergenerational transmission of GAA contractions, while loss of Msh2 or Msh3 reduced somatic GAA repeat expansions and increased levels of FXN transcription in brain and cerebellum tissues. Loss of Msh6 induced both intergenerational GAA repeat expansions and contractions, while the frequency of somatic GAA repeat expansions was reduced. Curiously, the level of FXN transcription was also reduced in Msh6-deficient brain and cerebellum tissues. On the other hand, Pms2 was found to protect against both intergenerational and somatic GAA repeat expansions, with loss of Pms2 causing increased GAA repeat expansions and decreased levels of FXN transcription in brain and cerebellum tissues. Finally, loss of Mlh1 led to a reduced frequency of both intergenerational and somatic GAA repeat expansions, but the level of FXN transcription was also reduced in brain and cerebellum tissues. Furthermore, upregulation of MMR mRNA expression was detected in human FRDA fibroblast cells, but downregulation was seen in FRDA cerebellum tissues, suggesting tissue-dependent control of FXN and MMR expression. In summary, these studies indicate that the MMR system can affect GAA repeat expansion instability and FXN transcription through different mechanisms of action. Furthermore, frataxin deficiency can also affect the levels of MMR mRNA expression in a tissue-dependent manner. These findings will assist future investigations aimed at identifying novel FRDA therapies.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences