The homeo box is a 180 base pair DNA sequence that is found in some genes of Drosophila which are crucial to its embryogenesis. Hence, this sequence has been used extensively as a probe to search for genes which may play a role in the development of vertebrates. Sequences with homology to Drosophila homeo boxes have been found in a number of vertebrate species including mouse, man and frog. The purpose of this thesis was to clone homeo box sequences from the Mexican axolotl, an animal which has had a distinguished history in the study of development. Clones cross-hybridizing with the Drosophila Antennapedia homeo box were obtained from a partial genomic library enriched for such sequences. One clone (pAhox1) contained a homeo box sequence (Ahox1) which is 66% homologous to the Antp sequence, and is most closely related to the mouse Hox-1.6 homeo box (84% identity). Total RNA from several embryonic stages was probed with a 560 bp KpnI fragment of pAhox1 containing the homeo box (HB-Kpn). This showed that transcripts hybridizing to this sequence were detected only in neurula, and tail bud axolotl embryos, suggesting that this sequence is developmentally regulated. Tissue specific expression of Ahox1 was examined in dissected neurula and tail bud embryos. In the neurula, transcripts hybridizing to Ahox1 were found to be restricted to the posterior two thirds of the neural plate and in tail bud embryos from the anterior margin of the gill bulge to the pronephric duct. Another clone (pAhox2) obtained from this library contained a partial homeo box sequence, and its homology to the Antp sequence was found to reside in the last 60 nucleotides of the homeo box, a region thought to be responsible for DNA binding. One powerful method to study gene function in vertebrates has been the introduction of cloned DNA sequences into developing organisms by microinjection. Expression of exogenous sequences in fertilized axolotl eggs were therefore examined, by using constructs consisting of a eukaryotic promoter, fused with the E. coli lacZ coding sequence which could be detected by histochemical staining. Expression from several eukaryotic promoters was observed, and the strongest expression observed was with the mouse hsp68 promoter. In order to examine the possible role of Ahox1, a construct was prepared with HB-Kpn fused to the mouse hsp68 promoter. When this construct was microinjected into fertilized axolotl eggs, defects in the anterior neural tissues were observed. These were first observed in the neurula stage of development, when Ahox1 expression is first observed. The abnormalities observed in these embryos were in neural tissues not normally expressing Ahox1.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/5936 |
| Date | January 1990 |
| Creators | Whiteley, Mary. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 178 p. |
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