gene expression patterns, wild-type and mutant tissues were processed on the same slides. Electroretinography (ERG) ERG was performed using the ESPION system (Diagnosys LLC, Littleton, MA) as previously described [32,33]. Briefly, mice that had been dark-adapted overnight were anaesthetized under safe light conditions using an intraperitoneal injection of avertin at 250 mg/kg. Eyes were dilated using both 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon Canada). To ensure constant body temperature during testing, mice were put on a warming source. Silver wire loop electrodes were placed on both corneas with a drop of 0.3% hypromellose (Novartis) to maintain corneal hydration. A gold minidisc reference electrode was placed on the tongue and a ground needle electrode was placed subcutaneously in the tail. The animal’s head was positioned under the center of the Ganzfeld dome. Single flash stimuli (4 ms duration) were presented at 11 increasing intensities ranging from 0.001 to 25 cd s/m2. Five ERG traces were obtained and averaged for each luminance step. The minimum negativity occurring between 10 and 40 ms post-stimulus was defined as the a-wave. The maximum positivity occurring between 40 and 80 ms poststimulus was defined as the b-wave [33]. Differences during ERG analyses were determined using ANOVA statistical analysis. LMO4 in Retinal Development Bhlhb5 promoter activity assay Using a mouse targeting vector Bhlhb5-lacZ [7] (gift of Dr. Lin Gan, University of Rochester, NY, USA), a 2,110 bp fragment containing sequence from 21,898 to +212 relative to the start site of transcription was amplified by PCR using sequence-specific oligonucleotides Bhlhb5-fwd 59-TTCTCGAGGGAGCCTCT- CATTAGC-39 and Bhlhb5-rev 59-ATAAGCTTCCAGCCGA- CGGTGCT-39. The PCR product was cloned between the XhoI and HindIII sites of the pGL3-basic vector and transfected into F11 neuronal cells using Lipofectamine 2000 as described previously [12] with control shRNA or shRNA specific to LMO4. LMO4shRNA (clone ID TRCN0000084375) and control scrambled non-silencing shRNA were purchased from Open- Biosystems. Transfection efficiency was normalized using a CMV enhancer-driven beta-galactosidase reporter plasmid as described [11]. Results LMO4 mice have developmental eye defects In phenotyping LMO4 mice, we identified several features of altered eye development including smaller eyes and the presence of a coloboma phenotype in ,40% of embryos (Figure 1). Although exencephaly was often associated with the eye phenotype, defective eye development was noted in some Figure 1. LMO4 mice have a partially penetrant neural tube closure defect and coloboma. (A) Wild type mouse at embryonic day 14.5 (E14.5) shows normal forebrain and eye development. (B) Three phenotypically divergent LMO4 (LMO4 KO) mouse embryos reveal exencephaly with relatively normal eye (left panel), exencephaly with a small eye and coloboma (middle panel), and non-exencephalic phenotype but with coloboma. (C & D) Close up views of the left and right eyes of wild type and LMO4 KO mice, respectively. doi:10.1371/journal.pone.0013232.g001 PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13232