Abstract
Purpose:
Several BTB/POZ family proteins are known to function in distinct biological processes. While some critically regulate differentiation of specific cellular lineages in the immune system, others are involved in maintenance of stem cells, or when mutant, are associated with oncogenesis. However, none have so far been associated with vertebrate eye development. Here, we have identified a BTB/POZ domain zinc finger transcription factor Zbtb8b that functions in mouse lens development.
Methods:
Analysis of lens microarray gene expression data in iSyTE, a bioinformatics tool for cataract gene discovery, led to the hypothesis that Zbtb8b functions in lens development. qRT-PCR, whole mount and section in situ analyses were performed to detect Zbtb8b mRNA expression in mouse lens. Immunostaining was performed on mouse lens sections using a polyclonal antibody generated against a conserved amino acid region of the mouse Zbtb8b protein. Zbtb8b function was investigated by generating siRNA-mediated gene knock-down (KD) in a Pax6-ectodermal enhancer driven GFP reporter transgenic mouse embryonic lens explant culture system. The tissue was immunostained for lens markers.
Results:
To investigate new regulators in the lens, we analyzed genes encoding transcription factors in iSyTE and identified Zbtb8b among candidates that received the highest lens enrichment score - top 1% within all lens-expressed genes. Meta analyses on mouse embryonic and post-natal microarray datasets indicated that Zbtb8b expression is highly enriched in early lens development through adulthood. In situ hybridization confirmed Zbtb8b expression in the mouse lens and immunostaining demonstrated Zbtb8b protein to be predominantly nuclear and enriched in the anterior epithelium and transition zone. To gain insight into Zbtb8b function in lens development, we generated siRNA-mediated Zbtb8b-KD in mouse explant culture. We find that expression of the Pax6-GFP reporter as well as the lens transcription factor Foxe3 is downregulated in Zbtb8b-KD explants and the lens tissue is abnormal. Together, these findings indicate importance of this new regulatory protein in early stages of lens morphogenesis.
Conclusions:
We have investigated the function of a new zinc finger transcription factor gene Zbtb8b in mouse lens development ex vivo. These data indicate that Zbtb8b is highly enriched in the lens and regulates early lens morphogenesis.