Date of Award

2004

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Biological Sciences

Keywords

Biology, Genetics.

Supervisor

Crawford, M.

Rights

info:eu-repo/semantics/openAccess

Abstract

Pitx homeodomain proteins are highly conserved regulatory proteins that were first discovered on the basis of their involvement in the transcriptional regulation of pituitary-specific genes. Recently, a third member of Pitx family, Pitx3, has been identified which expresses in the midbrain and eye. The main focus of this study was to elucidate the role of Xenopus Pitx3 during eye development. As a side project, we also studied the role of this gene in the left-right patterning and axis formation in Xenopus. Here, we report the cloning of a Xenopus homolog which encodes a conceptual protein of 292 amino acids and shows a high similarity in amino acid level to human and murine Pitx3. Early neurula expression is restricted to lateral prechordal mesoderm, anterior paraxial mesoderm, a crescent of anterior sensorial ectoderm, and a discrete spot which is fated to form Rathke's pouch. xPitx3 is expressed throughout lens induction in the presumptive lens ectoderm, lens placode, and later in differentiating lens. During tailbud stages, xPitx3 is also expressed symmetrically in the pituitary, lateral plate mesoderm, branchial arches, somites, and asymmetrically in looping gut. Ectopic expression assays suggest that xPitx3 directs development of the optic placode, and that without its influence; both the lens and retina fail to form. xPitx3 over-expression expands the early expression domains of Pax6 and Six3 and alters development of the lens, optic vesicle, optic nerve, and diencephalon. Expression of a xPitx3/engrailed repressor chimera alters early expression domains of Pax6, Rx, and Six3, and later inhibits lens development consequently abrogating retinal induction. This later inhibition of eye development is reflected by diminished expression of Pax6, Six3, Rx, betaB1-crystallin, Otx2, and Lens1. Similar effects are obtained using antisense morpholino oligonucleotide-mediated translation knockdown. Reciprocal grafting experiments using wildtype and morpholino treated tissues demonstrate that xPitx3 in the presumptive lens ectoderm is required for both lens and retina formation. Ectopic expression of xPitx3 results in cyclopia which is indicative of the midline defects. Consistent with this result, overexpression of xPitx3 or mutant constructs leads to profound visceral situs anomalies, suggesting that xPitx3 may be involved in midline specification. Lastly, our results indicate that xPitx3 may also play a role in somitogenesis through modulating Hox-1A and hairy2 genes that are involved in spatiotemporal specification of developing somites. Source: Dissertation Abstracts International, Volume: 66-02, Section: B, page: 0692. Adviser: Michael Crawford. Thesis (Ph.D.)--University of Windsor (Canada), 2004.

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