Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.712235
Title: Analysis of Pax6 expression in the developing vertebrate head
Author: Lin, Pei-Cheng David
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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Abstract:
Pax6 is a member of the Pax gene family - a family that encodes highly conserved and developmentally important transcription factors. Mutation of Pax6 leads to Small eye (Sey) in the mouse and aniridia in the human. Sey/Sey mice are characterized by craniofacial abnormalities, including absence of eye and nasal derivatives, formation of supernumerary upper incisor teeth and a rod-like cartilaginous structure in the premaxilla, as well as defects in the developing brain. This thesis aims to investigate Pax6 expression in the head region of normal and mutant vertebrate embryos in order to elucidate its role during development and understand the mutant phenotypes. Pax6 expression in the mouse is investigated by in situ hybridization, with particular emphasis on the higher centres of the visual and olfactory nervous system - areas that have not been previously investigated in detail. Pax6 expression is detected along the visual pathway, in the neural retina, optic nerve, pretectum, superior colliculus, pulvinar, dorsal and ventral lateral geniculate nucleus and other neural nuclei that are implicated in visual connections. Within the olfactory nervous system, Pax6 is expressed in the olfactory and vomeronasal epithelium, main and accessory olfactory bulb, anterior olfactory nucleus, precommisural hippocampus, piriform and endopiriform cortex, as well as olfactory and vomeronasal amygdaloid areas. The results demonstrate that Pax6 is extensively expressed throughout the visual and olfactory pathways and strongly indicate that Pax6 is involved in their development. It is also shown that Pax6 expression is developmentally restricted during mouse embryogenesis. The restriction is implicated in neuroblast proliferation, migration, connection and axonogenesis. Expression of Pax6 in the chick has not been previously reported for stages later than Hamburger- Hamilton stage 14. This study confirms formerly reported data before stage 14 and establishes that Pax6 is expressed in the olfactory epithelium, lens, corneal epithelium, and neural retina after stage 14. Furthermore, temporary Pax6 expression is found in Hensen's node before stage 9. In the hindbrain, in addition to the formerly reported expression in all the rhombomeres, Pax6 transcripts are also detected in cells flanking rhombomeres 3 and 5. The distribution of Pax6 transcripts in the head surface ectoderm appears to correspond to the previously reported areas that are capable of forming lens in culture. This suggests a crucial role for Pax6 in lens differentiation, which is supported by absence of lens in the Sey/Sey mice. The correlation between Pax6 expression and lens differentiation is investigated by culturing fragments of head surface ectoderm on millipore membranes. The fragments are then assessed for lens differentiation using an antibody against aB crystallin and the presence of Pax6 transcripts is checked by in situ hybridization. The results indicate that lens can differentiate from non-Pax6-expressing fragments of head surface ectoderm, whilst Pax6-expressing fragments do not always give lens. Whenever lens differentiates in culture, however, Pax6 is always expressed, suggesting that it is necessary for the process of lens differentiation. The phenotype of Sey/Sey mice suggests that Pax6 may play a role in tooth development and this possibility is preliminarily investigated in this study. The results demonstrate that Pax6 is not expressed in the premaxilla, suggesting that the supernumerary teeth are not directly caused by Pax6 mutation. To investigate whether the supernumerary teeth show normal development, the presence of two genes involved in tooth development, Msx-1 and Msx-2, are examined. The results show that the patterns of Msx-1 and Msx-2 expression in the Sey/Sey premaxilla are normal, although the area exhibits formerly reported abnormalities and is developmentally retarded. This suggests that Msx-1 and Msx-2 are not directly regulated by Pax6. Possible mechanisms that may cause the abnormalities with presumptive involvement of Pax6 regulation are discussed. One possible regulator of Pax6 is all-frans-retinoic acid (RA; a derivative of vitamin A). Maternal vitamin A deficiency causes a variety of abnormalities, including micropthalmia that is also seen in the Sey/Sey mice. In vitamin A-deficient quail embryos, the segmentation of rhombomeres 4-8, where Pax6 is expressed, is disturbed. Furthermore, expression of some Pax genes, for example, AmphiPaxl and Pax2, is affected by RA. This thesis investigates the effect of RA on Pax6 expression during gastrulation. Six groups of chick embryos at stage 4 are treated in vitro with RA at concentrations from 1 x 10⁻⁸ M to 5 x 10⁻⁵ M. Pax6 expression after culture for 30 hours is investigated by wholemount in situ hybridization. The results show dosage-dependent abnormalities in the head, heart, neural tube, and somites. Pax6 remains expressed in all the abnormal embryos investigated after RA treatment, indicating that Pax6 is not switched off by RA. In the head that is severely affected by RA treatment, most structures are condensed or distorted. In either cases, Pax6 expression patterns are altered. The alteration appears to be resulted from the gross malformations following RA treatment, although direct effect of RA on Pax6 expression can not be excluded.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.712235  DOI: Not available
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