LMO4 in Retinal Development Figure 5. Ganglion cells, cholinergic and dopaminergic amacrine cells, rod bipolar and horizontal cells appear normal in the peripheral retina of LMO4 cko mice. No significant difference was seen with immunostaining markers: Brn3b, retinal ganglion cells (RGC); vesicular GABA transporter (vGAT), GABAergic neurons; choline acetyltransferase (ChAT), cholinergic amacrine cells; tyrosine hydroxylase (TH), dopaminergic amacrine cells; PKC, rod bipolar cells, calbindin, horizontal cells; prox1, amacrine, bipolar and horizontal cells; rhodopsin, rod photoreceptors; peanut agglutinin (PNA), cone photoreceptors. Scale bars, 100 mm. doi:10.1371/journal.pone.0013232.g005 No difference in the number of retinal ganglion cells (Brn3b+), horizontal (calbindin+), rod (rhodopsin+) or cone (peanut agglutinin+, PA+) cells was observed (Figure 5). In addition, the number of photoreceptor nuclei in the outer nuclear layer was not different (data not shown). Taken together, these results indicate a role for LMO4 in the development of Bhlhb5+ (inhibitory GABAergic) amacrine and cone bipolar cells. Since the LMO4 antibody that worked well in our hands is raised in goat, as is the Bhlhb5 antibody, to compare their expression we performed dual in situ and immunostaining, using biotinylated LMO4 anti-sense mRNA and Bhlhb5 antibody. We found that Bhlhb5 protein was co-expressed with LMO4 mRNA in peripheral developing retinal cells at P0 (Figure 7A). These ‘‘peripherally localized’’ Bhlhb5 amacrine cells were significantly reduced in LMO4 cko mice at P0 (Figure 7B). However, no reduction in Bhlhb5-immunopositive retinal cells was observed at E14.5 in LMO4 cko mice (Figure S2), indicating that loss of LMO4 impairs Bhlhb5+ amacrine cell differentiation between E14.5 and P0. To test the possibility that GABAergic amacrine and OFF-cone bipolar subtypes were initially generated but later died of apoptosis in LMO4 cko retinas, retinal sections were immunostained with anti-activated-caspase-3. No increase in apoptotic cells in LMO4 cko retinas was detected at P0 (Figure S3). Taken together, targeted deletion of LMO4 specifically diminished the generation of selective Bhlhb5+ OFF-cone bipolar cells and GABAergic amacrine cells. To address whether reduced expression of Bhlhb5 was related to loss of LMO4, we cloned a fragment of the mouse Bhlhb5 promoter and tested the effect of LMO4 knock-down on a luciferase reporter by transient transfection of cultured neuronal cells (Figure S4). Bhlhb5 promoter activity was specifically reduced by 50% when LMO4 was knocked down by shRNA, and this mechanism may contribute to reduced numbers of Bhlhb5+ neurons in LMO4 cko retinas. Impaired retinal response in a-Cre/LMO4flox mice To test whether the reduction of Bhlhb5+ GABAergic amacrine cells in LMO4 cko mice affects retinal function, we performed electroretinography (ERG) on dark-adapted adult LMO4 cko or littermate control mice. ERG measures the electrical response of the various components of the retina to light stimulation. The response normally comprises a hyperpolarization ‘‘a-wave’’ and a depolarization ‘‘b-wave’’ that measure photoreceptor and inner layer function, respectively. ERG recording revealed a significant reduction in the amplitude of the b-wave (Figure 8A), consistent with the loss of Bhlhb5 amacrine cells. Both the amplitude and latency of the a-wave (Figure 8B, C) were not affected (Figure 8C) consistent with similar photoreceptor cell numbers in LMO4 cko and littermate controls. In addition, to compare the function of the cone pathways, we also measured the light-adapted, long-flash (i.e., ON-OFF) ERG. This method uses a background photopic light to saturate the rods, effectively isolating cone photoreceptor pathways; and long duration flashes are used to separate responses from downstream ON- and OFF- bipolar cells with ON-bipolar being favored at flash onset and OFF-bipolar cells at offset. Despite a reduction in Bhlhb5+ type 2 Off-cone bipolar cells [7], we did not observe a difference in ON- or OFF-bipolar cell responses of LMO4 cko mice (data not shown). Discussion Germline ablations of essential transcription regulatory factors that cause embryonic lethality preclude the analysis of their function at later developmental stages. This issue is especially problematic for the study of perinatal differentiation. In particular, the development of the retina is not complete until two weeks after birth. Here, we selectively ablated LMO4 in peripheral retinal progenitors and found that LMO4 is required for visual function Figure 6. Quantitation of Bhlhb5+ amacrine and bipolar cells in P15 retinas. Immunopositive cells were counted at the peripheral retinas, within 500 mm from the tip, in the plane of the optic nerve. (A) A reduction in the density of amacrine cells labeled with the pan-amacrine Pax6 antibody and Bhlhb5 antibody that labels GABAergic amacrine cells was measured in LMO4 cko mice compared to littermate controls. No difference in cholinergic (ChAT+) or dopaminergic (TH+) amacrine cell density was observed. (B) A reduction in the density of bipolar cells labeled with the pan- bipolar Chx10 antibody and Bhlhb5+ cone bipolar (CB) cells was observed in LMO4 cko mice. However, PKC+ rod bipolar (RB) cell density was not different. Filled bars are for littermate controls and open bars are for LMO4 cko mice. The bars indicate s.e.m. and the asterisks indicate p,0.05. doi:10.1371/journal.pone.0013232.g006 PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13232