Mapping Central and Peripheral Eye Domains.

<p>Schematic summary of optic vesicle fate mapping results. <b>A:</b> Three distinct optic cup domains can be defined in the dorsal optic vesicle at HH9/10; OCL at the distal OV (green), central RPE situated more proximally (yellow), and central NR at the posterior OV (red). <b>B:</b> Distribution of dye from OV labeling in a forming optic cup at E3. Distinct central NR (red) and RPE (yellow) zones are constrained to the optic cup dorsal to the optic stalk. Distal OV/OCL label (green) distributes equally to inner and outer layers at initial OC stages. <b>C</b>: Representation of labeling results correlated with fate domains of NR, RPE, and anterior inner and outer eye fates at E4.5. Central RPE and NR zones remain restricted to the central eye. An anterior bias is evident in the extent of OCL derived OC tissue, which traverses the boundary between NR and presumptive ciliary body/iris. A-anterior, P-posterior, D-dorsal, V-ventral, Cen-central, Per-peripheral, L-lens, RPE-retinal pigmented epithelium, NR-neural retina, OV-optic vesicle, OCL-optic cup lip, OC-optic cup, se-surface ectoderm, os-optic stalk.</p

The Expression of Eye Field Transcription Factors subdivides the Optic Vesicle.

<p><b>A–H</b>: Coronal sections through HH10 optic vesicles immuno-labeled as indicated on panels. <b>A:</b> DAPI stained coronal section, to highlight the relevant tissues. Asterisk indicates the paraxial mesenchyme in contact with the posterior optic vesicle. Green arrow indicates the targeted site of injection for labeling the central neural retina in previous figures. <b>B:</b> Graphic representation of sections with axes labeled for orientation. <b>C–F:</b> Single immunostaining as indicated. <b>G, H:</b> Double immunostaining as indicated. Scale Bar in A applies to panels C–H. Scale bars = 100 µm.</p

Central and Peripheral Optic Vesicle Domains are Traced to Differentiated Tissues of the Optic Cup.

<p><b>A</b>: Dorsal view of HH10 embryo. GFP-expressing retrovirus, mixed with Cell-Tracker CMFDA, targeted to distal tip of optic vesicle (green fluorescein signal). <b>B:</b> Embryonic day 4; GFP+ cells visible in the eye (green arrowheads). Arrowed line indicates plane of sections. <b>C:</b> Section analysis of eye in B. Labeled cells organized in a spoke pattern extending from the optic cup lip (Box E) into the Hu+ neural retina (asterisk). <b>Inset panel:</b> Adjacent section of area at white asterisk, showing GFP-expressing neurons, including axon-extending ganglion cells. <b>D, E:</b> Adjacent sections of boxed zones in C; regions are slightly more nasal (N) or temporal (T) than boxed areas, indicated in µm. <b>D</b>: Pairs of labeled cells found at the edge of the Hu+ maturation zone. <b>E:</b> Labeled cells found in inner and outer layer at optic cup lip (yellow asterisk). <b>F:</b> Virus/CMFDA solution applied to the posterior optic vesicle. <b>G:</b> Day 4; indistinct GFP+ area visible through eye tissue (green arrowhead). Deep position of GFP within the inner layer accounts for weak signal. <b>H:</b> Section analysis of eye in G. Labeled cells found only in central neural retina. <b>I:</b> Magnification of boxed area in H; GFP+ neurons in retinal columns and extending axons towards optic nerve (yellow arrows). <b>J:</b> Virus/CMFDA applied to dorsal surface of optic vesicle, proximal to distal edge. <b>K:</b> Day 4; GFP+ cells visible in eye (asterisk). <b>L:</b> Sections reveal that visible GFP+ cells are in ectoderm over the optic tissue (green asterisk). Within eye, GFP+ cells found in central RPE (boxed area). <b>M</b>: Magnified view of boxed area on adjacent section, stained for MitF (RPE nuclei), showing GFP-expressing RPE cells (yellow arrow). Labeled cells found only in central RPE. 12 embryos were processed for virus evaluation. Antibodies as indicated, blue is DAPI-stained nuclei. Blue x’s indicate auto-fluorescent blood cells, white dashes on D,I indicate RPE layer. Scale bars = 100 µm D/T-dorsotemporal, V/N-ventronasal, D-dorsal, V-ventral, N-nasal, os-optic stalk.</p

The Peripheral not Central Optic Cup Originates in the Distal Optic Vesicle.

<p><b>A:</b> Scheme of the predicted distribution of dye directed to the OV of an HH10 embryo. The distal-most tip (red) is predicted to generate central neural retina. Central RPE is predicted to arise from the dorsal OV (yellow dots). OCL (green dots) origin is not known <b>B:</b> Dorsal view of a 12-somite embryo after DiI targeted to the distal OV (red arrow). <b>C:</b> Lateral view of the embryo in B following re-incubation. Dye distributed to the dorsal eye, from the OCL towards the central eye (white bar). Asterisk marks dye in the ectoderm. Pink line marks boundary between lens and OCL. <b>D–G:</b> Coronal sections of the embryo in B, C. <b>D:</b> Lower magnification for orientation. <b>E–F:</b> Dye in the dorsal OCL. Dye is present in the OCL and adjacent inner and outer layers (arrows). <b>G:</b> Dye is absent in both central neural retinal and RPE. <b>H:</b> DiI (red) and DiO (green) targeted to anterior and posterior distal OV. <b>I:</b> The embryo in H following re-incubation. DiO is distributed around the temporal OCL (green arrows) and DiI around the nasal OCL (red arrows). <b>J–K:</b> Transverse section through an embryo targeted at the anterior distal OV. Dye is restricted to the OCL and into the peripheral inner and outer layers (arrow). <b>L:</b> Graph showing dye distribution in the optic cup following distal OV labeling. Most embryos showed dye distributed to the OCL and excluded from the central neural retina or RPE. <b>M:</b> Scheme of the measuring strategy to analyze dye distribution. <b>N:</b> Plot of the distribution of distal OV targets against the midpoint of dye distribution around the OCL. Targeting through the posterior to anterior of the distal OV trends from temporal to nasal OCL. NR-neural retina, RPE-retinal pigmented epithelium, OCL-optic cup lip, L-lens, A-anterior, P-posterior, Pr-proximal, Di-distal, D-dorsal, V-ventral, T-temporal, N-nasal, OV-optic vesicle, OCL-optic cup lip. Scale bars = 100 µm.</p

Identification of Central Optic Cup Fields in the Optic Vesicle.

<p><b>A:</b> Schematic dorsal view of an optic vesicle at HH9/10 summarizing non-distal OV zones. <b>B:</b> Dye targeted to the posterior OV (arrow). <b>C–E:</b> Coronal sections of B following re-incubation. <b>C:</b> Low magnification. <b>D–E:</b> Higher magnification showing dye distributed in the central neural retina (red arrows). Dye is excluded from the OCL and posterior RPE. <b>F:</b> Dorsal view with DiI at the distal OV (red arrow) and DiO proximal (green arrows). <b>G–I:</b> Coronal section through the embryo in F following reincubation. <b>G:</b> DiI is distributed to the OCL. The central limit of DiI distribution is indicated (red arrow). <b>H:</b> DiO is distributed through the central RPE. The peripheral limit of DiO distribution is indicated (green arrow). <b>I:</b> Composite of G/H. DiI (red) in the OCL and DiO (green) in the central RPE do not overlap. <b>J:</b> Dorsal view of dye label proximal in the OV (red arrow). <b>K:</b> The same embryo following re-incubation. Dye is constrained towards the back of the eye (arrow). Pink line marks boundary between lens and OCL. <b>L–N:</b> Coronal sections through the embryo in J, K. <b>L:</b> DiI (red arrows) is in the central RPE and brain (red arrows) but absent from other eye domains. <b>M:</b> Higher magnification of the boxed area in L. DiI (red arrows) is distributed in the central RPE and in the brain. <b>N:</b> More temporally positioned section through the optic stalk. DiI is distributed from the central RPE through the optic stalk to the brain (red arrows). <b>O–R:</b> Optic vesicle labeling distributing in non-eye tissues. <b>O, Q:</b> Optic vesicle labeling in HH9 embryos. <b>P, R:</b> Distribution of dye in the CNS (arrows) following the labeling in O and Q respectively. No dye was distributed to the eye. <b>S</b>: Scheme summarizing dye distribution of OV dye labels into the central OC. Central RPE arose from the dorsal OV (yellow) and central NR from the posterior OV, adjacent to paraxial mesoderm (red). Proximal OV label distributed label to the CNS (grey). OV-optic vesicle, CNS-central nervous system, L-lens, NR-neural retina, RPE-retinal pigmented epithelium, A-anterior, P-posterior, Pr-proximal, Di-distal, D-dorsal, V-ventral, b-brain, L-lens. Scale bars = 100 µm.</p