LMO4 in Retinal Development mouse embryos die before birth, it was not possible to study the requirement for this gene in postnatal retina function in this mouse strain. Thus, we conditionally ablated LMO4 in the developing retina by crossing LMO4flox mice with a-Cre-IRES-GFP transgenic mice where the Cre recombinase and a GFP reporter are driven by the retinal neural progenitor-specific Pax6 enhancer (Pax 6 alpha enhancer) [34]. a-Cre transgene is active in the peripheral retinal progenitors of the embryo and stably expressed in mature amacrine cells [8]. We confirmed expression of the GFP reporter predominantly in the ventral peripheral retinal progenitor cells at E14.5 (data not shown). By P0, all amacrine cells expressed the GFP reporter (and by inference the Cre-recombinase) (Figure S1). Immunostaining showed that LMO4 is ablated in the peripheral retina of the a-Cre/LMO4flox mice at P0; to simplify, we abbreviate these as LMO4 conditional knockout (LMO4 cko) mice (Figure 2C). By immunostaining with cell-type specific markers at P15, we further determined how conditional ablation of LMO4 in retinal progenitors affects development of the ‘‘peripheral’’ retina at the optic nerve plane (Figure 4). Nuclei stained with DAPI showed no obvious anatomical difference in the retina of the LMO4 cko mice compared to littermate controls. The pan-amacrine cell marker Pax6 showed a slight (15%) reduction of amacrine cells in LMO4 cko mice. Using amacrine subtype specific markers, we found a 30% reduction in the number of GABAergic (Bhlhb5+) amacrine cells (Figure 4), but no difference in the number of cholinergic (ChAT+) or dopaminergic (TH+) amacrine cells (Figures 5 & 6A). In addition to amacrine cells localized at the inner region of inner nuclear layer, Bhlhb5 also labels subsets of cone bipolar cells localized at the outer region of the inner nuclear layer [7]. There was also a 30% reduction in the number of Bhlhb5+ cone bipolar cells in LMO4 cko mice compared to littermate controls. In contrast, there was no difference in the number of rod bipolar cells that are immuno-positive for protein kinase C (PKC) (Figures 5 & 6B). Since we observed a 30% reduction in the number of cells labeled with a pan-bipolar cell marker Chx10 (Figures 4 & 6B), this result suggests that in addition to Bhlhb5+ OFF- cone bipolar cells, LMO4 is also required for differentiation of other Bhlhb5-negative cone bipolar cells. Figure 4. Selective loss of Bhlhb5 amacrine cells and cone bipolar cells in the peripheral retina of LMO4 cko mice. Neuron-specific markers were used to phenotype peripheral retinas at P15 to reveal the functional consequences of LMO4 ablation in retinal progenitors. These are representative sections of those used for quantitation in Figure 6. DAPI stained all retinal nuclei of the ganglionic cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL); Pax6 labeled all amacrine cells; Syntaxin labels amacrine cell bodies and the IPL; Bhlhb5 labels GABAergic amacrine cell and OFF-cone bipolar cell nuclei; Chx10 labels bipolar cell nuclei. A significant reduction in Pax6+, Bhlhb5+, Chx10+ cells was observed in LMO4 cko mice. Scale bars, 100 mm. doi:10.1371/journal.pone.0013232.g004 PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13232