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First published online 2 October 2008
doi: 10.1242/dev.028076

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An essential role for frizzled 5 in mammalian ocular development

¹ Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
³ Department of Ophthalmology, and the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁴ Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

View larger version (74K): [in this window] [in a new window]   Fig. 1. Expression of Fz5 in the embryonic eye field and adult retina. (A,B) Whole-mount Fz5CKO-AP/+;Sox2-Cre mouse embryos stained with NBT/BCIP and viewed from the front. At E8.5, Fz5 is expressed in the anterior neural plate (NP). At E9.5, Fz5 expression is predominantly localized to the optic vesicles (OV). In B, the open cephalic neural tube can be seen above the zone of hybridization. (C,D) X-Gal-stained Fz5lacZ/+ embryo at E12.5 showing Fz5 expression in the ventral telencephalon (TE), eye and optic nerve (ON). D shows a vibratome section, the location of which is indicated by the line in C. (E,F) NBT/BCIP-stained flat-mount of a Fz5CKO-AP/+;R26-CreER adult retina. Sparse labeling was achieved by low-dose 4-hydroxytamoxifen injection (see Materials and methods). The boxed region in E is shown enlarged in F. Fz5 expression is restricted to Müller glia (left arrowhead) and to a subset of amacrine cells (right arrowhead). (G) A vertical section through the retina in E showing an AP-labeled Müller glial cell (left) and an amacrine cell (right). Here, and in all other retinal cross-sections, the outer nuclear layer is at the top of the image and the ganglion cell layer is at the bottom.

View larger version (43K): [in this window] [in a new window]   Fig. 2. The major cell types develop normally in the Fz5-/- retina at P30. (A) Amacrine cells, stained for calretinin, residing in the ganglion cell layer and inner nuclear layer show a characteristic trilamination of processes in the inner plexiform layer. (B) Nuclear localization of Islet1 in a subset of amacrine cells and retinal ganglion cells, and the distinctive inner plexiform layer stratification of processes of amacrine cells expressing glutamic acid decarboxylase (GAD). (C) Dopaminergic amacrine cells expressing tyrosine hydroxylase (TH) have processes that are confined to the outermost stratum of the inner plexiform layer. (D) Synaptophysin marks presynaptic terminals in the inner plexiform layer and outer plexiform layer, and glial fibrillary acidic protein (Gfap) marks astrocytes in the ganglion cell layer. (E) Glutamine synthetase (GS) in Müller glia and Gfap in astrocytes. (F) Similar extent of activation of Gfap expression in Müller glia of WT (Fz5+/-) and Fz5-/- mouse retinas following exposure to bright light. Note that the intense Gfap immunoreactivity in the innermost region of the Fz5-/- retina is likely to reflect an excess of astrocytes (see Fig. 9). DAPI staining (blue) in A-E shows the three nuclear layers.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Late degeneration of the Fz5-/- retina. (A,B) Semi-thin, plastic-embedded sections of (A) WT and (B) Fz5-/- mouse retinas at 6 months of age stained with Toluidine Blue. In the Fz5-/- retina, there is a nearly complete loss of photoreceptor outer segments (OS), swelling of inner segments (IS), and cell and neuropil loss in the two plexiform and three nuclear layers (ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer). (C) Fz5-/- retina and choroid at 6 months of age showing extensive retinal folding and detachment. (D,F) High-magnification views from C, showing detachment and folding (D) and rosette formation (F). (E) High-magnification view of the upper right corner of D showing that the subretinal space is populated by macrophages (arrowheads).

View larger version (93K): [in this window] [in a new window]   Fig. 4. Excess mesenchymal cells in the prenatal vitreous cavity and persistent fetal vasculature in the postnatal Fz5-/- eye. (A,B) DAPI-stained (A) WT and (B) Fz5-/- mouse embryonic eyes at E14.5. Arrowhead in B indicates the excess cells between the lens and retina. (C,D) Anti-smooth muscle actin (SMA) immunostaining of whole-mount retinas at P5. The hyaloid vessels and developing retinal vasculature emerge from the optic disk (green arrowheads). The cell mass that is attached to the optic disc in the Fz5-/- retina contains smooth muscle actin (D, red arrowhead). (E-H) Intact retinas viewed from the vitreal face (E,F) and Hematoxylin and Eosin-stained sections through the retina and optic nerve (G,H) at P56. The ectopic tissue (red arrowhead in F) is attached at the optic disc of the Fz5-/- retina (green arrowhead in F) and contains numerous pigment cells. (I-L) Semi-thin, plastic-embedded sections through the central retina showing the vascular structure and connectivity of the intra-vitreal tissue at P56. The serial sections at low magnification (I,J) show ectopic tissue attached to the optic disc (bottom) and the posterior face of the lens (top; green arrowheads). The large blood vessels in I and J are contiguous (red arrowheads). (K) High-magnification view of the boxed region in I, showing an intermingling of pigmented and unpigmented cells surrounding a large blood vessel (asterisk). (L) At the edge of the optic disc, pigmented cells associated with the initial segment of the intravitreal vasculature (asterisk) are contiguous with the pigmented cells of the choroid (arrowheads).

View larger version (36K): [in this window] [in a new window]   Fig. 5. Incomplete closure of the optic fissure in early retinal development produces a coloboma in the neonatal Fz5-/- eye. (A,B) X-Gal-stained whole-mounts of (A) Fz5lacZ/+;Sox2-CreER (Fz5+/-) and (B) Fz5CKO-AP/lacZ;Sox2-CreER (Fz5-/-) mouse eyes at E13.5 as viewed from the back, showing failed optic fissure closure in the Fz5-/- retina (red arrowheads). Green arrowheads indicate the optic nerve. (C,D) Vertical sections through the eyes shown in A,B. The red bracket in D (and H) demarcates the open ventral fissure. (E,F) Front view of X-Gal-stained eyes at E15.5. Note the tear-drop shape to the Fz5-/- iris. (G,H) Vertical sections through the eyes shown in E,F. (I,J) P1 eyes showing the typical Fz5-/- coloboma (arrowheads in J) and microphthalmia. (K,L) Anti-neurofilament (NF) immunostaining of whole-mount P1 retinas to visualize retinal ganglion cell axons. The optic disc is at the upper right (green arrowhead), and the open fissure is to the right in the Fz5-/- retina (red arrowhead). Misdirected bundles of retinal ganglion cell axons are seen in the Fz5-/- retina (white arrowheads). In all panels, dorsal is upward, as indicated in A (D, dorsal; V, ventral).

View larger version (52K): [in this window] [in a new window]   Fig. 6. Decreased expression of markers for ventral patterning during early Fz5-/- eye development. (A-F) The intact eye and surrounding tissue viewed from the cornea following whole-mount in situ hybridization. Dorsal is upward, as indicated in A. In the Fz5-/- mouse retina, ventral expression of Raldh3 at E10.5 and of Vax2 at E12.5 is decreased (arrowheads) whereas dorsal expression of Tbx5 (bracket) is minimally affected. (G,H) At E12.5, immunolocalization of Pax2 in the Fz5-/- retina extends beyond the immediate vicinity of the optic disc (arrowheads). (I,J) The pan-retinal distribution of Sox2 immunostaining is similar in WT and Fz5-/- retinas at E12.5; note the smaller size of the Fz5-/- eye.

View larger version (60K): [in this window] [in a new window]   Fig. 7. Increased apoptosis in the Fz5-/- retina at E10.5. (A,B) Whole-mount mouse embryos immunostained for cleaved caspase 3 showing increased apoptosis in the ventral Fz5-/- optic cup (arrowhead). The intact optic cup is seen from the cornea. Dorsal is upward, as indicated in A. (C-F) Horizontal sections from littermate embryos harvested 1 hour after injection with BrdU. Sections were stained with DAPI (C,D) or immunostained for BrdU and activated caspase 3 (E,F). The boxed regions in C,D show the locations of E,F. In the Fz5-/- optic cup, many more cells and cell fragments in both the inner (future retina) and outer (future retinal pigment epithelium) layers contain activated caspase 3. Proliferation appears to be grossly normal in the Fz5-/- optic cup.

View larger version (104K): [in this window] [in a new window]   Fig. 8. Comparison of Pax2 immunolocalization during and after optic fissure closure in WT versus Fz5-/- retinas. In A-L, pairs of nearby sections are shown in order to better illustrate the staining and visualize the three-dimensional anatomy. (A-D) Anti-Pax2 immunostaining at E13.5 showing the posterior expansion of the domain of Pax2 expression in the Fz5-/- optic fissure. Arrowheads indicate the posterior border of the optic fissure and the zone of Pax2 expression that tracks the optic fissure. (E-H) At E14.5, when Pax2 is no longer detected in the ventral retina in the WT mouse, it continues to mark the ventral fissure in the Fz5-/- retina. A-H are sectioned in the horizontal plane; nasal is upward, as indicated in A (N, nasal; T, temporal). (I-L) At E14.5, Pax2 abundance in the optic disc and optic nerve is similar in WT and Fz5-/- retinas. (M,N) Whole-mount immunostaining at E16.5 showing Pax2 in the ventral fissure region of the Fz5-/- retina. Insets show a plane of focus within the optic disc, showing a ring of Pax2-positive cells around the optic nerve. (O,P) The number and lateral distribution of Pax2-expressing astrocyte precursors at the vitreal face of the retina are increased in the Fz5-/- eye.

View larger version (68K): [in this window] [in a new window]   Fig. 9. An increase in the number of astrocytes in the Fz5-/- retina. (A,B) Flat-mount mouse retinas at P5 immunostained for Gfap to visualize astrocytes. The optic disc is at the lower left in all panels (asterisk). (C-H) Flat-mount retinas at P42 showing astrocytes (Gfap) and the vasculature (lectin IB4) on the inner face of the retina. Merged images from C-F are shown in G,H. At P42, the retinal vasculature appears normal. At both ages (P5 and P42), the Fz5-/- retina has an increased density of astrocytes compared with the WT.

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