Characterization of two regulatory factors in lens development
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The lens is a transparent tissue that refracts light onto the retina to allow high-resolution vision. Loss of lens transparency, clinically termed cataract, can present as a birth defect (congenital cataract) or more commonly as an age-related disease, the latter being one of the leading causes of human blindness worldwide. An estimated half of the congenital cases are due to underlying genetic alterations, while cataract at later stages can result from age-associated changes in lens homeostasis. To understand the pathology of both types of cataract, it is important to uncover the molecular processes that control lens development and homeostasis. Here, I have focused on understanding the significance of two gene expression regulatory factors to lens development and age-related cataract in mice. ☐ So far, several signaling pathways and transcription factors that are involved in normal lens development and the maintenance of lens transparency have been well characterized. However, there is a limited knowledge on the epigenetic aspect of gene expression regulation during lens development and homeostasis. Methylation of DNA is among the major epigenetic modifications, which results in suppression of gene expression. DNA methyltransferases are enzymes responsible for methylation of cytosine into 5-methylcytosine within target DNA sites. In this study, I demonstrate that lens-specific conditional double knock-out of the de novo methyltransferase genes Dnmt3a and Dnmt3b in early stages of mouse lens development results in late onset cataract at about 13 months of age. I find that defects in fiber cell organization likely contribute to the cataracts in the Dnmt3a:Dnmt3b cKO (Dnmt3aflox/flox;Dnmt3bflox/flox; Le-Cre+/-) mice. I demonstrate this by examining lenses from Dnmt3a:Dnmt3b cKO and control using scanning electron microscopy (SEM). SEM analysis indicates that compared to control, Dnmt3a:Dnmt3b cKO lenses exhibit disorganization of fiber cell packing, lack of membrane protrusions, and overall severe disruption of cortical fiber cells. Together, these data suggest that de novo DNA methyltransferases may function in maintenance of lens transparency and homeostasis, thus identifying new regulatory factors impacting age-related cataract in mice. ☐ Identification of genes with key roles in early stages of lens development is necessary for uncovering the regulatory networks that control formation of lens structure and transparency, which is the first step toward understanding how defects in these processes cause early-onset or congenital cataract. Previously, the Lachke laboratory has developed the bioinformatics tool called iSyTE (integrated Systems Tool for Eye gene discovery) that is effective in identifying genes linked to lens development and cataract. Based on high lens-enriched expression, iSyTE has predicted a novel gene Zbtb8b (Zinc finger and BTB Domain Containing 8B), encoding a putative transcription factor (TF), to have a potential role in controlling lens gene expression. Indeed, in agreement with iSyTE, Zbtb8b mRNA is expressed in the mouse presumptive eye region at embryonic day (E) 9.5, becoming progressively lens-enriched and restricted to lens epithelium as development proceeds. In agreement with this mRNA expression pattern, immunostaining for Zbtb8b protein shows that it is localized to the nucleus and is robustly expressed in cells of the lens placode and in later stages of lens development, in the anterior epithelium and early differentiating fiber cells. Moreover, Zbtb8b mRNA expression is found to be unaltered in the presumptive region of eye formation in homozygous null mutants of the key eye development regulator Pax6. These findings are similar to the Pax6 upstream regulator and the TALE-homeodomain transcription factor, Meis 1. Interestingly, protein-binding microarrays identified a target cis-DNA binding motif for Zbtb8b and a bioinformatics analysis shows that this motif is conserved among multiple vertebrate species in a putative non-coding region of the Meis1 gene. These data together suggest that Zbtb8b may function upstream of Meis1 which itself is upstream of Pax6 in lens development. To examine Zbtb8b-downstream target genes, I used small interfering (si) RNA to achieve Zbtb8b-knock-down (Zbtb8b-KD) in an established mouse lens epithelium derived cell line, 21EM15. Because Zbtb8b is predominantly expressed in the anterior lens epithelium, this cell line represents a effective reagent to rapidly analyze candidate genes whose expression is perturbed due to Zbtb8b deficiency. I find that several key early lens developmental transcription factors, namely Pax6, Meis1, Sox2, Foxe3, Dkk3 and Jag1 exhibit reduced expression as demonstrated by RT-qPCR analysis in Zbtb8b-KD cells. Furthermore, immunostaining analysis confirms the reduced of these targets at the protein level in Zbtb8b-KD cells. These data suggests that Zbtb8b is involved is the control of expression of several key transcription factors in lens development. ☐ Thus, by characterizing Zbtb8b-KD cells and Dnmt3a:Dnmt3b cKO lenses using molecular or phenotypic analysis, this research has identified a new role for these regulators in lens development and homeostasis, in turn suggesting their relevance to congenital and age-related cataract, respectively.
Description
Keywords
Cataract, Development, Genetics, Lens, Mouse