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. 2022 May 31;11:e76408. doi: 10.7554/eLife.76408

Figure 2. The inner nuclear layer (INL) is a densely packed tissue not dominated by extracellular matrix (ECM).

(A–B) The INL is not dominated by ECM: (A) Tg(lhx-1:eGFP) labels horizontal cells (HCs) (green). Laminin α1 antibody marks laminin (magenta). (B) Tg(ptf1a:dsRed) marks HCs and amacrine cells (ACs) (green). Tg(HA:GFP) labels hyaluronic acid (HA) (magenta). Higher magnification insets show enrichment of anti-Laminin α1 and HA in the basement membrane (BM) (red arrowheads). Scale bars: 50 μm; insets 10 μm. (C–C’) Scheme of structural organization of the retina during development: (C) During HC migration at 48 hours post fertilization (hpf); (C’) after HC layer formation at 70 hpf. HCs (green); photoreceptors (PRs) and retinal ganglion cells (RGCs) (magenta). (D–E”) Brillouin shift maps (right) and their corresponding confocal images (left) of double-transgenic zebrafish of (D–D’’) retina 1 and (E-E’’) retina 2. Top: 48 hpf, and bottom: 70 hpf. Tg(lhx-1:eGFP) labels HCs and ACs (green), Tg(atoh7:RFP) is expressed in RGCs and PRs (magenta). Confocal images were obtained directly after the Brillouin shift measurements. The corresponding Brillouin shift maps of the nuclear layers (outer nuclear layer [ONL], inner nuclear layer [INL], ganglion cell layer [GCL]) show a higher Brillouin shift than the plexiform layers (outer plexiform layer [OPL], inner plexiform layer [IPL]) at 70 hpf. Red dashed boxes in D–E: imaged regions. Scale bar: 10 µm. See also Figure 2—figure supplement 1.

Figure 2—source data 1. Figure 2—figure supplement 1C''.

Figure 2.

Figure 2—figure supplement 1. No collagen is found in the densely packed INL.

Figure 2—figure supplement 1.

(A) Immunofluorescence image of a 48 hours post fertilization (hpf) retina.Tg(h2a:mCh) labels all retinal nuclei (green), and collagen antibody marks collagen type IV (magenta). Bottom panel: Higher magnification inset of the retina show anti-collagen IV (gray). Red arrowhead: basement membrane. Scale bar: 50 μm. (B–B’) Stills from time-lapse of a retina at 50 hpf (B) and 60 hpf (B’). Tg(lhx-1:eGFP) labels HCs (green), Tg(βactin:maKate2-ras) marks cell membranes of all retinal cells (magenta). Scale bar: 50 µm. Right panels: Higher magnification insets of the outlined regions show cell membranes (gray). Dashed line: forming inner plexiform layer (IPL). Scale bar: 10 μm. (C) Confocal sections of Tg(βactin:lap2b-eGFP) labeling nuclear envelopes of all retinal cells (magenta), and Tg(βactin:maKate2-ras) labeling membranes of all retinal cells (green) at 65 hpf. Scale bar: 20 µm. (C’) Higher magnification insets of the outlined region showing nuclear envelopes (left panel) and cell membranes (right panel). Blue dashed line: region for intensity profile measurement in C”. Scale bar: 10 μm. (C’’) Line scan average fluorescent intensity profiles of βactin:lap2b-eGFP (magenta) and βactin:maKate2-ras (green) along the blue dashed line. See Figure 2—source data 1.