Comment[ArrayExpressAccession]	E-GEOD-52006
MAGE-TAB Version	1.1									
Public Release Date	2014-01-13
Investigation Title	OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness									
Comment[Submitted Name]	OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness
Experiment Description	Leber congenital amaurosis (LCA) includes congenital or early-onset blinding diseases, characterized by vision loss together with nystagmus and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA. While most LCA is recessive, mutations in the homeodomain transcription factor gene CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. Here, we report a new spontaneous mouse mutant carrying a frameshift mutation in Crx (CrxRip). We show that, unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet the photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype that can be rescued by Crx. RNA-Seq profiling reveals progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restores the rod phenotype in CrxRip/+ mice. We show that the binding of Otx2 to Nrl promoter is obliterated in CrxRip mutant, and ectopic Otx2 can rescue the rod phenotype. Therefore, Otx2 is required to maintain Nrl expression in developing rods to consolidate rod fate. Our studies provide the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design. Retinal samples were harvested from WT, CrxRip/+, CrxRip/Rip, Crx-/- and Nrl-/- retina at postnatal days 2 and 21 for whole transcriptome sequencing (RNAseq).  Each sample included 2 independent frozen retina and experiments were performed in duplicates. RNA-seq transcriptome libraries were constructed from 1 μg of total RNA.									
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl								
Person Last Name	Roger	Roger	Hiriyanna	Gotoh	Hao	Cheng	Ratnapriya	Kautzmann	Chang	Swaroop
Person First Name	Jerome	Jerome	Avinash	Norimoto	Hong	Debbie	Rinki	Marie-Audrey	Bo	Anand
Person Mid Initials	Eric	E				F		I		
Person Email	rogerj@nei.nih.gov									
Person Affiliation	National Institutes of Health									
Person Phone	301-443-5132									
Person Address	National Eye Institute, National Institutes of Health, Bldg 6, Rm 307, MSC0610 6 Center Drive, Bethesda, MD, USA									
Person Roles	submitter									
Protocol Name	P-GSE52006-2	P-GSE52006-1								
Protocol Description	Single read sequence-by-synthesis of 76 base length using TruSeq SBS v5 reagents was carried out on Genetic Analyzer IIx running SCS2.9 software (Illumina). RNA-seq reads passing chastity filter were analyzed using Genomatix Software Suite (Genomatix Software GmbH, Munich Germany). Sequences were aligned to NCBI mouse reference genome Build 37 on Genomatix Mining Station with Genomatix Mapper 3.5.1. Alignments were annotated on Genomatix Genome Analyzer using ElDorado 08-2011 database, and differential expression analysis was performed using DEseq Genome_build: NCBI build 37 Eldorado version 08-2011 Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample, fold change, adjusted p-value	Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries. RNA libraries were prepared for directional next-generation sequencing using a modified TruSeq RNA Sample Preparation Kit protocol as described (Brooks et al. 2012). The protocol utilizes the labeling of the second strand of cDNA with uracil, which allows the degradation of this strand prior to sequencing								
Protocol Type	normalization data transformation protocol	nucleic acid library construction protocol								
Experimental Factor Name	GENOTYPE	AGE								
Experimental Factor Type	genotype	age								
Comment[SecondaryAccession]	GSE52006									
Comment[GEOReleaseDate]	2014-01-13									
Comment[ArrayExpressSubmissionDate]	2013-11-01									
Comment[GEOLastUpdateDate]	2014-03-12									
Comment[AEExperimentType]	RNA-seq of coding RNA									
Comment[AdditionalFile:Data1]	GSE52006_P21_NE2_APV005_FC2.txt									
Comment[AdditionalFile:Data2]	GSE52006_P2_NE2_APV005_FC2.txt									
Comment[SecondaryAccession]	SRP032458									
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR1023063-SRR1023082									
SDRF File	E-GEOD-52006.sdrf.txt