To understand why inactivation of
Fzd3 decreases the ERG response, we used immunohistochemistry (IHC) to identify retinal cells and structures that are affected by
Fzd3 inactivation. We first looked at Fzd3 expression. As there is no antibody against Fzd3 that works well in IHC, we took advantage of the LacZ reporter inserted in the KO-first
Fzd3kof allele. At E13.5,
β-gal histochemistry was widely distributed in retinal cells, particularly RGCs and their axons (
Fig. 2A). At P0,
β-gal IHC showed strong expression in the GCL and some amacrine cells in the neuroblast layer (NBL;
Fig. 2B). At P7, in addition to ganglion cells,
β-gal was detected in the INL (
Fig. 2C), consistent with a previous study,
23 suggesting that
Fzd3 is expressed in multiple retinal interneurons. At P14 and P18, this staining pattern was defined more clearly (
Figs. 2D,
2E) and no
β-gal immunoreactivity was detected in the ONL, suggesting that
Fzd3 is expressed in most retinal cells, but not in photoreceptors. As negative controls, wild-type retinas with
β-gal IHC staining showed no specific staining at P0–P14 (
Supplementary Fig. S1) and P18 (
Fig. 2F). To further identify the cell types that express
Fzd3, we double-labeled P18 sections with antibodies to
β-gal and specific cell markers. All PKC
α-positive RBCs (
Fig. 3), all calbindin-positive horizontal cells (
Fig. 2G), and some Brn3a-positive RGCs (
Fig. 2H) expressed
β-gal. Among amacrine cells, ChAT-positive ON-starburst amacrine cells were
β-gal positive (
Fig. 2I, arrow), whereas ChAT-positive OFF-starburst amacrine cells and TH-positive dopaminergic amacrine cells did not coexpress
β-gal (
Figs. 2I,
2J). Triple IHC staining for
β-gal, Isl1 and PKC
α showed that all RBCs expressed
Fzd3 (
β-gal) and Isl1 (
Fig. 3), suggesting that expression of
Fzd3 in RBCs should be abrogated in
Isl1-Cre;Fzd3f/− mutant retina.