Perron M et al. (2003), A novel function for Hedgehog signalling in ret...

XB-ART-5643

Development 2003 Apr 01;1308:1565-77. doi: 10.1242/dev.00391.

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A novel function for Hedgehog signalling in retinal pigment epithelium differentiation.

Perron M , Boy S , Amato MA , Viczian A , Koebernick K , Pieler T , Harris WA .

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Sonic hedgehog is involved in eye field separation along the proximodistal axis. We show that Hh signalling continues to be important in defining aspects of the proximodistal axis as the optic vesicle and optic cup mature. We show that two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. We show that this zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, we activated the pathway by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium.

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Species referenced: Xenopus
Genes referenced: acvr2b crx dhh gli1 gli2 gli3 ihh pax2 pax6 pou4f1 ptch1 ptch2 rpe shh smo vax2
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Phenotypes: Xla Wt + Cyclopamine (Fig 7 B)

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	Fig. 1. Expression of Hh pathway components in whole-mount Xenopus embryos. Whole-mount in situ hybridisation on stage 41 embryos using probes as indicated. For each probe a lateral view and a dorsal view are shown. A Gli3 sense probe has been used in A,B. Expression of X-bhh and X-chh in the retina is restricted to the RPE (black arrowheads in C,E). X-chh is also expressed in the hindbrain (white arrowhead in E). In addition to a strong expression in the brain, some expression in the retina can be detected for X-shh, X-Smo, Gli2 and Gli3 (arrowheads in H,N,R,T). Although a strong expression in the brain can be detected, expression of X-Ptc-1, X-Ptc-2 and Gli1 in the retina at this stage is very weak (I-L,O-P). Anterior is towards the right. Scale bar: 300 μm.
	Fig. 2. Expression of X-bhh, X-chh and X-shh on retinal sections. These panels show retinal vibratome sections after whole-mount in situ hybridisation. The probes are indicated for each panel. (A,C,E) Retinas of stages 34-36 embryos. (B,D,F) Retinas of stage 42 embryos. (A) X-shh is expressed in the RPE (arrow) and in the ganglion cell layer (arrowheads). (B) X-shh is strongly expressed in ganglion cells (arrowheads) and a faint staining is visible in a cluster of cells in the outer layer and the RPE (arrow). (C,E) X-bhh and X-chh are expressed in the RPE (arrows). (D,F) X-bhh and X-chh are still strongly expressed in the RPE (arrows), excluding the peripheral pigmented epithelium (bars). le, lens. Dorsal is on the top. Scale bar: 40 μm.
	Fig. 3. Expression of X-Ptc-1, X-Ptc-2, X-Smo, Gli1, Gli2 and Gli3 on retinal sections. These panels show retina vibratome sections after whole-mount in situ hybridisation. The probes are indicated on the left of each series of panels. (A,D,G,J,N,S) Stages 28-30 embryos. (B,E,H,K,O,T) Stage 34-36 embryos. (C,F,I,L,P,U) Stage 40-41 embryos. (M,V) Dorsal CMZ. (Q,R) Ventral CMZ. (A) X-Ptc-1 expression is detected in the periphery of the optic vesicle (arrows). (B,C) X-Ptc-1 expression is still detected in the periphery of the retina albeit at a low level, including the RPE (arrows). (D,G) X-Ptc-2 and Gli1 are expressed in the presumptive RPE (arrows). (E,H) X-Ptc-2 and Gli1 expression is still clearly detected in the RPE (arrows). (F,I) A faint X-Ptc-2 and Gli1 expression is detected in some cells of the RPE (arrows). (J,N,S) Gli2, Gli3 and X-Smo expression is detected in the periphery of the optic vesicle (arrows). (K,O,T) Gli2, Gli3 and X-Smo expression is restricted to the periphery of the retina (arrows). (L,P,U) Gli2, Gli3 and X-Smo expression is restricted to the periphery of the CMZ where stem cells are present. Magnifications of these regions (M,Q,V) show that these genes are also expressed in the peripheral pigmented epithelium (arrowheads point to stained cells with a longitudinal shape that shows its belonging to the RPE). (R) When the RPE has been poorly bleached, we can detect Gli3-positive pigmented cells (arrowhead). Dorsal is towards the top. le, lens; NR, presumptive neural retina; RPE, presumptive RPE; CMZ, ciliary marginal zone. Scale bar: 60 μm for A-L,N-P,S-U; 20 μm for M,Q,R,V.
	Fig. 4. The retinal pigmented epithelium includes several cell populations. Double in situ hybridisation was performed with X-Smo in blue (arrows in A) and X-bhh in red (weak in bright field, A; strong under fluorescence, arrows in B). (C) Overlapping staining shows that some cells are negative for expression of both genes in between the two expression domains (white and black arrows give the approximate limits of X-bhh and X-Smo expression domains, respectively). (D,E) Immunostaining with the antibody XAR1 on retinal cross-section of stage 36 and 42, respectively. Staining is detected in the RPE except in the peripheral pigmented epithelium (bars in E). (F) In situ hybridisation on stages 38 embryos with a Xotx5 probe. Xotx5 is expressed in the photoreceptor layer (arrowhead) and strongly in the RPE (arrows), excluding the peripheral pigmented epithelium (bars). (G-I) Staining for BrdU uptake in retina cross-sections of stage 42 embryos. (G) The retina in bright field; (H) BrdU immunostaining. (I) An overlap of G and H shows that some BrdU-positive cells are present in the peripheral pigmented epithelium (arrowheads). Arrows indicate some BrdU-positive cells in the CMZ. (J-L) Double staining for BrdU uptake and Gli3 expression. Gli3 staining is shown in J, BrdU immunostaining in K. Double staining in L shows that some BrdU-positive cells in the peripheral pigmented epithelium are also Gli3 positive (arrow). le, lens; CMZ, ciliary marginal zone. Scale bar: 45 μm in A-F; 15 μ m in G-L.
	Fig. 5. Overexpression of dnPKA leads to proximodistal defects. Embryos were injected at the two-cell stage with RNA for dnPKA (B,D), then cultured until stage 39. (A,C) Control embryos. In situ hybridisation on cross-sections was then performed using Pax2 (A,B) or Pax6 (C,D) probes (dark blue). Overexpression of dnPKA leads Pax2 expression to expand in the neural retina compared with the control (arrowheads), whereas it inhibits expression of Pax6 in the ventral retina (arrowhead). le, lens. Dorsal is towards the top. Scale bar: 40 μ m.
	Fig. 6. Cyclopamine treatment blocks Hh signalling in Xenopus. Whole-mount in situ hybridisation on embryos incubated in cyclopamine solution from stage 20 onwards (B,D,F,H,J,L) or control embryos (A,C,E,G,I,K). The probes are indicated for each panel. (C-L) Stage 41 embryos; (A,B) stage 30 embryos. Cyclopamine treatment leads to a strong reduction of Gli1, X-Ptc-1 and X-Ptc-2 expression. Some Gli1 expression remains only in the tailbud (arrowhead in B). Expression of both Gli2 and Gli3 is not affected. Anterior is towards the left. Scale bar: 1.35 mm in A,B; 300 μm in C-L.
	Fig. 7. Cyclopamine treatment leads to proximodistal defects. A brightfield view of a retina of an embryo incubated in cyclopamine from the two-cell stage (B) shows no closure of the ventral RPE (arrowhead) and ectopic pigment in the central retina (arrow) compared with a control retina (A). In situ hybridisation or immunostaining on retinal sections of stage 39 embryos incubated in cyclopamine solution from the two-cell stage (D,F,H,J,L) or control embryos (C,E,G,I,K). The probes or the antibodies are indicated for each panel. All sections are 12 μm cryostat sections except in E,F,I,J, which are 50 μm vibratome sections. Pax2 expression is strongly reduced in the cyclopamine-treated retina. Only a faint expression remains (arrow in D). Pax6 expression invades the optic stalk region (arrow in F). Ectopic photoreceptors are detected in the optic stalk region (arrowhead in H) and in the central retina along the ectopic pigment (arrow in H). Brn3.0 expression is detected in the optic stalk region (arrow in J). Vax2 expression is also moved towards the optic stalk region (arrowhead in L) but is still present and restricted to the ventral part only, as in the control (K). The dorsoventral borders are indicated with arrows in K and L.
	gli1.1 (GLI family zinc finger 1, gene 1 ) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 41, lateral view, head only, anterior right.
	gli3 (GLI family zinc finger 3) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 41, lateral view, head region only, anterior right.
	gli1.1 (GLI family zinc finger 1, gene 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 28, lateral view, anterior right.
	gli2 (GLI family zinc finger 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 41, lateral view, head region only, anterior right.
	ihh (indian hedgehog) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 41, head region only, lateral view, anterior right.
	dhh (desert hedgehog) gene expression in Xenopus laevis embryos, NF stage 41, assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	smo (smoothened, frizzled family receptor) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 41, head region only, M: lateral view, anterior right, dorsal up; N: dorsal view, anterior right.
	ptch1 (patched 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, head region only, lateral view, anterior right, dorsal up.
	ptch1 (patched 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, head region only, dorsal view, anterior right.
	gli2 (GLI family zinc finger 2) gene expression in Xenopus laevis embryo,head region, assayed via in situ hybridization, NF stage 41, lateral view, anterior right, dorsal up.