Abstract
Purpose: :
Over the past 20 years, the discovery of genes critical to mammalian lens development has relied on forward and reverse genetics. The goal of the present research is to: 1) devise a novel systems-based strategy which can enable direct and efficient identification of genes that are relevant to mammalian lens development and disease; 2) validate the utility of this bioinformatics tool; and 3) demonstrate its use to identify and characterize novel genes in lens biology and disease.
Methods: :
We developed a bioinformatics tool called Systems Tool for Eye gene discovery (SyTE) that uses a developmental profile of the mouse lens transcriptome generated by microarray analysis of the lens as it transitions from the stage of placode invagination to that of vesicle formation. Using a novel in silico subtraction protocol with a developmentally matched microarray dataset representing the whole embryo minus ocular tissue, we can assign a lens enrichment p-values to individual probes. Ranking of probe datasets based on their lens enrichment p-values, in turn allows detection of genes that function in lens development.
Results: :
When SyTE was tested on 23 previously identified non-syndromic cataract genes, it identified the correct cataract gene in over 90% of cases as one of the top 5 candidate genes - from an average of 100 candidate genes per locus - in the originally mapped locus. In the present study, we highlight the identification of a novel zinc finger transcription factor gene, Zbtb8b. Whole mount and section in situ analysis revealed that Zbtb8b is expressed in the presumptive eye region of the E9.5 mouse embryonic head ectoderm and becomes progressively lens-specific at later stages, eventually being restricted to the lens anterior epithelium. Immunohistochemical analyses demonstrate that Zbtb8b expression is nuclear and can be detected in the early stages of lens induction in the lens placode. Analysis of Small eye mutants revealed that Zbtb8b expression in the developing eye region of Pax6 null embryos is uncompromised similar to Meis1, an upstream regulator of Pax6. In support of this hypothesis, a Zbtb8b binding motif was discovered in region upstream of the Meis1 gene and EMSA analysis revealed binding of Zbtb8b to this region. A transgenic mouse model to further assess Zbtb8b function is presently being analyzed.
Conclusions: :
We report a novel lens gene expression database, SyTE, which serves as an efficient tool to identify genes with function in lens biology. We demonstrate its use in identification of a novel transcription factor gene, Zbtb8b, which functions in the lens developmental program independent or upstream of Pax6.
Keywords: transcription factors • gene microarray • development