Investigation of homeodomain interacting protein kinase hipk2 in mouse lens development
Date
2020
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
While several key transcription regulators are known to function in lens development and their mutations are linked to cataract and other ocular defects, the present knowledge on their post-translational control is limited. Furthermore, the factors that may be involved in these potential post-translational control events are not characterized. To address these critical knowledge-gaps, we used the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery, https://research.bioinformatics.udel.edu/iSyTE) to identify Hipk2 (Homeodomain interacting kinase 2) as a high-priority candidate gene in mouse lens development. Hipk2 phosphorylates serine and threonine residues in its target substrates that include homeodomain transcription factors. Notably, in vitro studies in human cell lines and in vivo studies in Drosophila suggest that Hipk2 is involved in phosphorylation of the key eye homeodomain transcription factor Pax6. Further, expression of dominant negative kinase dead Hipk2 results in “small eye” defects in Drosophila. Together, these findings suggest that Hipk2 may have an important function in the eye and lens development. ☐ Therefore, in this M.S. Thesis research, I sought to characterize the potential function of Hipk2 in mouse eye and lens development. iSyTE analysis indicated that Hipk2 mRNA expression is upregulated in lens development starting from embryonic stage E12.5, which correlates with fiber cell differentiation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on embryonic and postnatal lenses to estimate Hipk2 mRNA expression levels in the lens. This analysis confirmed Hipk2 mRNA expression at embryonic and postnatal stages in the mouse lens. In situ hybridization assays using Hipk2-specific anti-sense RNA probes were performed to define the spatiotemporal localization of Hipk2 transcripts in the mouse lens. This assay showed Hipk2 mRNA to be localized in early lens epithelium, lens fiber cells and in the retina of the embryonic mouse eye. Further, Western blot analysis was performed using anti-Hipk2 antibody for quantitative expression of Hipk2 in mouse embryonic and postnatal lens tissue. This analysis validated Hipk2 protein expression in the lens at embryonic and postnatal stages. To gain insight into the spatiotemporal expression of Hipk2 protein in eye development, I performed immunofluorescence (IF) staining using anti-Hipk2 antibody on eye sections at various postnatal stages. In the lens, Hipk2 was found to be expressed in both the lens epithelium and fiber cells, similar to the findings on Hipk2 transcripts. In early postnatal stages, Hipk2 appeared to be uniformly expressed throughout the lens, but with age, its strong expression was restricted to cells in the transition zone and early differentiating fiber cells, although some signal was detected in a few cells of the lens epithelium and late differentiating fiber cells. Further, IF assays showed that Hipk2 protein is primarily localized in the nuclei. In the retina, Hipk2 expression was detected in the ganglionic cell layer, inner nuclear cell layer, outer nuclear cell layer and in the choroid and sclera. Together, these approaches validated iSyTE’s prediction and demonstrated robust expression of Hipk2, on both mRNA and protein levels, in lens development. ☐ To gain insight into the impact of Hipk2 deficiency on mouse eye and lens development, I initiated the generation of Hipk2 targeted knockout mouse models. I generated Hipk2 lens-specific conditional ready allele mice (FLPeR+ve: Hipk2 WT/cR; referred to as Hipk2cR) by crossing mice carrying Hipk2 FRT knockout first alleles with those carrying hemizygous flippase (FLPeR). As generation of lens-specific conditional deletion model using these mice is a long-term process, I simultaneously also generated Hipk2 lens-specific reporter (lacZ) tagged deletion allele mice (Hipk2floxlacZ/floxlacZ:Pax6GFPCre+/-; referred to as Hipk2cKOlacZ) by crossing mice carrying Hipk2 loxP knockout first alleles with those carrying a single Pax6GFPCre allele, which allowed Cre-expression in cells contributing to lens fate beginning at embryonic day (E) 9.5. I next undertook initial phenotypic characterization of Hipk2cKOlacZ mouse eye and lens by light microscopy and scanning electron microscopy (SEM). ☐ Hipk2cKOlacZ mice exhibit cataract and microphthalmia defects at variable penetrance at age 2 months and beyond. SEM demonstrated abnormal cortical lens fiber cell morphology in Hipk2cKOlacZ mice. ☐ Thus, this research led to the spatiotemporal characterization of Hipk2 expression in the lens and the retina. Further, this study led to initial examination of Hipk2 function in the mouse lens and the eye through development of a new lens-specific conditional knockout model for Hipk2. These findings demonstrate that Hipk2 deficiency results in severe eye and lens defects. This study also provides initial insights into the cellular nature of the lens defects, revealing that morphology of cortical lens fiber cells is abnormal in the Hipk2cKOlacZ mouse. Together, these findings uncover a key role for a post-translational regulatory factor, Hipk2, in lens and eye development by demonstrating its deficiency to be linked to microphthalmia and cataract. Further, these data open up new areas for future investigations to define the potential targets of Hipk2 in the lens, characterization of which will provide insights into the mechanism of Hipk2 function in lens cells.
Description
Keywords
Kinase HIPK2, Mouse lens development