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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The conventional view for corneal epithelial maintenance suggests that a stem cell population found in the limbus (at the rim of the cornea) produces daughter cells, called transient amplifying cells, which migrate centripetally. This limbal stem cell (LSC) hypothesis was recently questioned and the alternative model suggests that stem cells are present throughout the corneal epithelium. The main aims of this thesis were to investigate whether age and Pax6 genotype affect LSC function. Previous work with X-inactivation mosaics revealed radial stripes of β-galactosidase-expressing cells in the corneal epithelium (from about 5 weeks of age), which decreased with age and were reduced in Pax6+/- mice (a model for aniridia, a human eye disease). The reduction in Pax6+/- mice could be due to either reduced LSCs function or a more coarse-grained mosaicism caused by reduced cell mixing during development. Comparison of patch sizes in Pax6+/- and wild-type X-inactivation mosaics showed that patches were smaller in Pax6+/- cornea epithelia before the initiation of stripes (3 weeks of age). This implies that stripe-number reduction is not caused by reduced cell mixing, so an effect on LSC function remained a possibility. Thus, the numbers of label-retaining cells (putative stem cells) in Pax6+/- were compared to controls at 15 and 30 weeks old but they were not reduced at 30 weeks or in Pax6+/- mice, as had been predicted. The failure to demonstrate the predicted result suggests either that the hypothesis was incorrect or the experimental approach was inappropriate. Furthermore, it was discovered that mice expressing β-galactosidase under the keratin 5 promoter produced rare stripes in the corneal epithelium, which are likely to represent clonal lineages derived from individual stem cells. Older mice demonstrated a significantly lower frequency of stripes, a result compatible with the predicted reduction of active LSC with age. Pax6+/- corneas were highly abnormal and stripes were not formed properly, so direct comparison was not possible. Finally, pilot experiments with conditional expression of a reporter gene revealed the successful formation of a stripe, and hence provide a plausible alternative approach to compare stripe numbers reflecting active LSCs but the method has yet to be optimised. Overall, the results suggest that LSCs are reduced with age and support the limbal location of stem cells maintaining the corneal epithelium.

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The small size and the lack of recombination set the fourth chromosome of Drosophila melanogaster apart from the other chromosomes. I have shown that the Minute gene on chromosome 4, earlier named Minute-4, encodes the ribosomal protein RpS3A. Two Pax6 genes, eyeless (ey) and twin of eyeless (toy) are also located on chromosome 4. Pax6 genes are important in head and eye development in both mammals and Drosophila. I have focused much of the study on ey and toy. The first mutant of toy that was characterized showed a headless phenotype. This indicates that Toy is important for the development of both the eye and antennal discs. The phenotype of the null mutation in toy is temperature sensitive due to that transcription of ey is temperature dependent in the eye-antennal primordium in absence of Toy. This temperature dependence was used to find out that the phenocritical period for ey in the adult head development is during embryonic stage 12-16 when ey first is expressed in the eye-antennal primordium. I also conclude that ey is activated by Toy in the eye-antennal primordium. The strong eyD mutation was molecularly characterized and it was finally settled that it is an allele in the ey locus. I also show that eyD homozygotes have a headless phenotype, much stronger than the earlier ey mutations.

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Gene regulatory networks, underlying the molecular regulation of eye development are conserved across many animal phyla. Genes from the Pax family of transcription factors are one of the most conserved members through the evolution, regulating the development of crucial parts of eye, including the photoreceptor cells. Pax transcription factors are considered to be regulators of opsins, molecules providing the conversion of the light stimulus into the electrochemical signalisation in the photoreceptors cells. In this thesis, pax6 and pax2/5/8 transcription factors are investigated as potential regulators of eye development in Platynereis dumerilii. pax6 and pax2/5/8 transcription factors are tested as potential regulators of the r-opsin in Platynereis, based on the observed early expression onsets of these genes. Wild-type expression analysis of pax6 and pax2/5/8 using the whole mount RNA in-situ hybridization is provided, accompanied by the initial analysis of the Platynereis pax6 knockout line. pax6 heterozygote mutants are shown to be viable and able to reproduce, however, homozygote mutation of pax6 in Platynereis is lethal. Our data suggest that transcription factors pax2/5/8, otx and six3 are not regulated by the pax6 in Platynereis. Concerning the r-opsin present in the Platynereis eyes, pax6...