OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness
Jerome E. Roger, … , Bo Chang, Anand Swaroop
Jerome E. Roger, … , Bo Chang, Anand Swaroop
Published January 2, 2014
Citation Information: J Clin Invest. 2014;124(2):631-643. https://doi.org/10.1172/JCI72722.
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OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness

Abstract

Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip). We determined that CrxRip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the CrxRip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in CrxRip retinas. Expression of NRL partially restored rod development in CrxRip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in CrxRip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.

Authors

Jerome E. Roger, Avinash Hiriyanna, Norimoto Gotoh, Hong Hao, Debbie F. Cheng, Rinki Ratnapriya, Marie-Audrey I. Kautzmann, Bo Chang, Anand Swaroop

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Figure 5

The CRXRip mutant protein is functionally null in vitro.

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The CRXRip mutant protein is functionally null in vitro. (A) Lack of t...
(A) Lack of transactivation by CRXRip mutant protein. HEK293 cells were cotransfected with constructs containing bovine Rho or mouse Opn1sw promoters driving firefly Luciferase reporter gene simultaneously with NRL, NR2E3, or RORβ, respectively. In both sets of experiments, different amounts of CRXWT, CRXRip, and/or CRX1–254 (0.15–0.55 μg) were cotransfected. Fold change is relative to the luciferase activity in presence of NRL and NR2E3 only. *P < 0.05. (B) CRXRip protein does not bind DNA. Autoradiograms of EMSA using nuclear extracts from HEK293T cells transfected with pcDNA4c, Xpress-tagged CRXWT, CRXRip, and CRX1–254 were performed using oligonucleotides encompassing CRX binding sites in Rho and Opn1sw promoters. 100 times more unlabeled specific probes were used for competition. Oligonucleotide supershift assays were performed with anti-Xpress antibody. Arrows indicate the CRX-shifted probe. (C) ChIP-qPCR with anti-CRX antibody from P21 WT, CrxRip/Rip, and Crx–/– retinas. Normal IgG was used control. Fold enrichment represents the fold change of qPCR amplification signals for the different genes tested between CRX ChIP DNA and IgG control ChIP DNA.