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 February 3, 2014
Citation Information: J Clin Invest. 2014;124(2):631-643. doi:10.1172/JCI72722.
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Categories: Research Article Ophthalmology

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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Dominant congenital blindness in the Rip mutant is caused by a 1-bp dele...

Figure 1

Dominant congenital blindness in the Rip mutant is caused by a 1-bp deletion in Crx.

(A) Dark- and light-adapted ERG recording in 1-month-old WT and Rip mutant. (B) Linkage cross analysis. 75 backcross progenies from the (Rip mutant X C3A.BLiAPde6b+/J) F1 X C3A.BLiA-Pde6b+/J were phenotyped by retinal fundus examination and genotyped for the indicated microsatellite markers. The black boxes represent heterozygosity for Rip-derived allele, and white boxes represent homozygosity for C3A.BLiA-Pde6b+/J–derived alleles. The number of chromosomes sharing the corresponding haplotype is indicated. Genetic map of chromosome 7 in the Rip region. (C) Identification of 1-bp deletion in Crx, visualized by Integrative Genome Viewer displaying sequence reads generated by exome capture sequencing. (D) Sanger sequencing of Crx+/+, CrxRip/+, and CrxRip/Rip mice showing deletion of a G nucleotide in exon 4 in Rip mutant but not WT mice. (E) Schematic of the CRX protein, indicating the position of 1-bp deletion upstream of Otx-like domain. Colored boxes show the functional domains. (F) Alignment of mouse CRX and CRXRip mutant protein predicts that the frameshift mutation would lead to the addition of 88 amino acids, starting at residue 299. Conserved amino acids are indicated in red. (G) Immunoblot analysis of retinal extracts from 1-month-old Crx+/+, CrxRip/+, and CrxRip/Rip mice. Anti-CRX antibody identifies 2 CRX bands in CrxRip/+ retinas. The lower band (34 kDa) corresponds to CRXWT, whereas the 44-kDa isoforms correspond to CRXRip protein. Anti-actin antibody was used as a loading control.