Figure 1.
LMO4 null 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 null (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.
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 α-Cre conditionally ablated retina (LMO4 cko). Scale bar, 100 µm.
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 µm.
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 µm.
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 µm.
Figure 6.
Quantitation of Bhlhb5+ amacrine and bipolar cells in P15 retinas.
Immunopositive cells were counted at the peripheral retinas, within 500 µm 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.
Figure 7.
Loss of Bhlhb5+ cells in P0 LMO4 cko retinas.
(A) MO4 is colocalized with Bhlhb5 in amacrine cells (arrows). LMO4 mRNA in the cytoplasm was revealed by in situ hybridization with an antisense probe labeled with biotin (green). Bhlhb5 in the nuclei was revealed by an antibody to Bhlhb5 (red). Inset is enlarged in the lower panels. Scale bars = 100 and 25 µm, top and bottom panels, respectively. (B) Bhlhb5 immunostained cells were significantly fewer in the peripheral retina of LMO4 cko mice compared to littermate controls. Inset is enlarged in the lower panels. Scale bar = 100 µm.
Figure 8.
Electroretinography reveals a deficit in the b-wave in LMO4 cko mice.
ERGs were recorded from 8 week old littermate control (diamonds and solid line) and LMO4 cko (squares and dashed line) mice. (A) b-wave average amplitude was significantly reduced at all light intensities. (B) a-wave amplitude tended to be lower in LMO4 cko mice, but was not significantly different. (C) a-wave latency was not different between control and LMO4 cko mice. All measures are means ± SEM for n = 13 control (WT) and n = 12 LMO4 cko mice.