Date of Award

12-1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Anatomy and Neurobiology

Research Advisor

Daniel Goldowitz, Ph.D.

Committee

John Cox, Ph.D. Eldon E. Geisert, Jr., Ph.D. D. J. Surmeier, Ph.D. Robert Williams, Ph.D.

Abstract

Development of the eye is controlled by a network of genes, often conserved, that regulate the timing and location of cellular differentiation. One approach to understanding this network of genes and their interaction is to focus on mutations, spontaneous or induced, that predictably disrupt the proper function of such networks, and by examining the effect of such disruption on the function of other genes.

The Belly spot and tail (Bst) semi-dominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5, and measured retinal size, cell density, cell death, mitotic index and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation, and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation.

In an effort to determine the role of Bst within the network of genes controlling eye development, we examined the effects of Bst in relation to two genes believed to regulate eye development during the same developmental period (E9-13): the boundary gene Pax2, which plays a role during optic stalk fissure fusion, and the proneural gene Hes1, which has been shown to regulate retinal ganglion cell differentiation. We cross-mated hemizygous Bst/+ mutant mice with hemizygous Krd/+ (kidney and retinal defects, in which Pax2 is deleted) mice and Hes1 +/- knockout mice to produce Bst/+ Krd/+ and Bst/+ Hes1 +/- compound heterozygous mutants. We find that there is marked ectopic expression of Pax2 protein in the Bst/+ retina, and a potentiation of the retinal defects in Bst/+ Krd/+ compound mutant offspring compared to the parental mutant phenotypes; which indicates that the Bst mutation could have resulted in a loss of positional cues for subsequent morphogenetic events, such as those dependent upon Pax2 expression. In contrast, the level of Hes1 mRNA in the Bst/+ mutant appears normal. In the Bst/+ Hes1 +/- compound mutant offspring, there are fewer instances of ocular defects compared to those of Bst/+ Hes1 +/+ littermates. Given that Hes1 is believed to be an inhibitor of cellular differentiation, the partially improved Bst/+ Hes1 +/- retinal phenotype is consistent with the hypothesis that the effects of the two mutant alleles are antagonistic.

Taken together, our results suggest that the Bst locus is involved in the regulation of cellular differentiation during early eye development.

DOI

10.21007/etd.cghs.1999.0314

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