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Figure 1 Analysis of chick optic fissure closure. (a) Chicken embryos at HH.St25 and HH.St30 illustrated the optic fissure (OF; arrows) as a non-pigmented region in the ventral aspect of the developing eye. (b) Left: Schematic showing orientation of the developing chick optic fissure with respect to the whole embryonic eye. Dorsal-ventral and proximal-distal axes are indicated. This study focused on the medial optic fissure (marked by white hatching) distal to the developing pecten and optic nerve. Right: brightfield and fluorescent confocal microscopy using memGFP cryosections illustrated the open (arrow) and fused seam (arrowhead) regions in chick OFM. The location and planes of the cut sections along the D-P axis are indicated in the accompanying schematic. (c) Brightfield and fluorescent confocal microscopy of memGFP OFM sections unambiguously defined the location of fusion plates (arrowheads, top and middle panels) at all stages throughout OFC, combined with flat-mounted memGFPs. Bottom panel: representative single plane confocal z-stack projection image clearly indicated FP2. (d) Brightfield microscopy of flat-mounted ventral eyes revealed the tissue dynamics during closure and coinciding with location of fusion plates (FPs). At HH.St29 the medial OFM had narrowed markedly along the P-D axis between the iris and the proximal region, with FP1 and FP2 (arrowheads) closely positioned in the distal OF. At HH.St31 the medial OFM had become fully pigmented in the fused seam, and the distance between FP1 and FP2 (arrowheads) had lengthened in the P-D axis. An opening remained in the OFM at the iris region (asterisk). (e) Histogram to illustrate fused seam length at each HH stage (error bars = s.d.). Quantitative data of OFM progression obtained from flat mounts and cryosections are provided in Table 1. (f) Schematic representation of chick OFC progression in the distal and medial retina. 1. Pre-fusion: A fully open OFM is evident in the ventral retina at stages HH.St25-27; 2. Initiation: At HH.St27-28 the first fused region is observed in the distal-medial OFM; 3. Active fusion: fusion extends briefly in the distal direction but then stops in the presumptive iris to leave an open region throughout development. Fusion proceeds markedly proximally with FP2 extending towards the pecten. 4. Complete fusion: Fusion stops proximally when FP2 meets the fused pecten region. The fusion seam is complete with a complete continuum of both NR and RPE layers in the ventral eye. Abbreviations: L, lens; OC, optic cup, OF, optic fissure; ON, optic nerve; FP, fusion plate; HH, Hamburger Hamilton staging; RPE, retinal pigmented epithelia; NR, neural retina; POM, periocular mesenchyme.