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Fig. 1. Subpopulations of ACs express GFP in Pax6-DF4:mGFP transgenic zebrafish lines. (A) Schematic of inner retinal organization in mature vertebrates. Connections between ganglion cells (GCs), amacrine cells (ACs) and bipolar cells (BCs) are localized to the inner plexiform layer (IPL). The IPL is further organized into ON and OFF sublaminae that occupy approximately the inner and outer halves of the IPL, respectively. (B) The construct used to derive Pax6-DF4:mGFP transgenic lines. Expression of the Gap-43GFP fusion protein was driven by a hexamer of the 58 bp pax6 DF4 element, located downstream of the SV40 poly-A sequence, and by an upstream EF1 promoter. The sequence of the highly conserved DF4 element is shown. (C) Cross-sectional view of GFP⁺ ACs and their neurites in the IPL of line 220 7 dpf retina. GFP fluorescence is superimposed on a Nomarski image of a section through the eye. Two bright sublaminae are evident in the IPL (see higher magnification image, inset). (D) Digital rotation of a confocal image stack, providing an orthogonal view of a region of inner retina of a line 220 retinal wholemount at 9 dpf. S1 is an optical slice of this field of view showing sparsely distributed GFP⁺ neurites close to the AC cell bodies. S2-5 are image planes within the boxed region in S1. The two major GFP⁺ sublaminae are S2 and S4; note also sparse innervation of sublaminae S1 and S5 by GFP⁺ neurites. (E) Cross-sectional view of the eye of an 7 dpf line 243 fish. (F) Morphology of individual GFP⁺ cells in line 243. Maximum projection of a confocal image stack (47 µm total) through the inner retina of a retinal whole mount at 35 dpf. (G) Arbors of a few isolated ACs (1, 2 in F) at an IPL depth closer to the GCL. (H) Digital rotation of the complete image stack providing orthogonal views of cells 1 and 2. Cell 1 has a diffuse asymmetric arbor spanning the thickness of the IPL. By contrast, cell 2 has a radially symmetric arbor stratifying in the ON sublamina. (I-K) Cross-sections of adult line 220 retina immunolabeled for ChAT. The two bright GFP⁺ laminae in line 220 coincide with the two major ChAT-immunopositive bands in sublaminae S2 and S4. Some, but not all, GFP⁺ cells are also immunoreactive for ChAT (arrows). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NV, optic nerve; dpf, days post-fertilization; L, lens.

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