LMO4 in Retinal Development Figure 2. Pattern of LMO4 expression during retinal development. (A) In situ hybridization reveals gradually elevated expression of LMO4 mRNA from E14.5 to postnatal day 0 (P0). (B) Sense control probe shows background staining to be compared to the antisense probe used in (A). (C) Close-up view of P0 wild type (WT) retina shows high LMO4 mRNA levels in the displaced amacrine and amacrine layers and lower diffuse expression in the retinal progenitors. Immunofluorescence with anti-LMO4 antibody shows associated pattern of LMO4 expression in a P0 wt retina section and background staining in the LMO4 a-Cre conditionally ablated retina (LMO4 cko). Scale bar, 100 mm. doi:10.1371/journal.pone.0013232.g002 LMO4 mice without exencephaly (Figure 1B, rightmost panel), indicating that these phenotypes are independent or only partially penetrant. LMO4 is expressed in the retina during development In situ hybridization analysis revealed that LMO4 mRNA is expressed in retinal progenitor cells at embryonic day 14.5 (E14.5) and its expression increases as the retina develops (Figure 2A). At E14.5, we were unable to detect LMO4 protein with the LMO4 antibody. However, at postnatal day 0 (P0), LMO4 mRNA and protein were found to be highly expressed in the amacrine cells at the inner nuclear layer, in displaced amacrine cells at the retinal ganglion cell layer and lower levels were detected in retinal progenitor cells (Figure 2C). Immunostaining at P0 showed that LMO4 is co- expressed with the pan-amacrine cell marker Pax6 at the inner nuclear layer (arrowheads, Figure 3) and in displaced amacrine cells at the retinal ganglion layer (arrows, Figure 3). LMO4 protein expression was again below detectable levels at P15 when the retinal layers and neuronal subtypes are fully developed (data not shown). Reduced Bhlhb5+ amacrine and bipolar cells in the peripheral retina of alpha-Cre/LMO4flox mice Retinal cell differentiation begins at around E12 in mouse and continues into the first 2 postnatal weeks [2]. Because LMO4 Figure 3. LMO4 is colocalized with Pax6 in amacrine and displaced amacrine cells. Dual immunofluorescence staining reveals LMO4 cells in red, Pax6 cells in green and dual labeled cells appear yellow. Amacrine cells are indicated by arrowheads and displaced amacrine cells in the retinal ganglion cell layer by arrows. Scale bar, 50 mm. doi:10.1371/journal.pone.0013232.g003 PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13232