Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


Transcriptional regulation of the collagen a1(IX) gene during eye development

Elena I. Frolova and David C. Beebe

Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, USA

We have shown that differentiation of the ciliary epithelium at stage 18 of chicken embryo development was accompanied by increased expression of mRNA for the long-isoform of collagen  a1(IX), LI-col(IX). Since the expression of this gene is very selective for the ciliary epithelium and occurs early in differentiation of this tissue study of its transcriptional regulation could provide information on transcriptional mechanisms responsible for targeting gene expression to the ciliary epithelium.

To determine the sites in the proximal promoter that were occupied by transcription factors in the differentiating ciliary epithelium we used an in vivo footprinting assay. The main conclusion from this experiment was that one or more DNA binding proteins in ciliary epithelium and retina occupies the proximal promoter of LI-col(IX). In addition, differences in the in vivo DMS footprinting patterns allowed us to conclude that there are different sets of proteins bound to this promoter in the ciliary epithelium and retina. Because LI-col(IX) is expressed in the ciliary epithelium, but not in the retina, these different complexes may be responsible for activating and repressing transcription in these two tissues. To map in more detail the fragments of the LI-col(IX) promoter that bind tissue-specific transcription factors, we used electrophoretic gel mobility retardation assays. Nuclear extracts from ciliary epithelium and neural retina of E7 embryos were analyzed for DNA binding with double-stranded fragme

Two fragments identified by mobility shift assay were used in a one-hybrid screen to identify prospective DNA-binding proteins. A cDNA library fused to the GAL4 activation domain (AD) in pACT2/Asc vector was synthesized from total RNA of the ciliary epithelium of day 7 chicken embryos. The size of the library was approximately 1.3x107 clones. At least 90% of clones contained inserts of 500 to 3,000 base pairs. Two potential DNA binding proteins were identified. One of the proteins (cZic2) showed high homology to mouse ZIC2 mRNA, a sequence recently shown to be expressed in the ciliary epithelium during mouse development.

To determine temporal and spatial pattern of cZic mRNA expression during development, we performed whole-mount in situ hybridization using chicken embryos. Overall expression in the chicken embryo was similar to expression of ZIC2 in mouse. In the eye, cZic is expressed throughout the inner layer of the optic cup at a low level at stage 14. At stage 18 expression is increased at the margin of optic cup, the differentiating ciliary epithelium. By stage 30 there is strong expression of cZic in the non-pigmented ciliary epithelium but not in the retina.

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