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doi: 10.1242/10.1242/dev.00391

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A novel function for Hedgehog signalling in retinal pigment epithelium differentiation

¹ Laboratoire d'Embryologie Moléculaire, Bat. 445 Université Paris XI, 91405 Orsay, France
² Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK
³ Institut fuer Biochemie, Humboldtallee 23, 37073 Göttingen, Germany

View larger version (117K): [in a new window]   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.

View larger version (87K): [in a new window]   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.

View larger version (81K): [in a new window]   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.

View larger version (82K): [in a new window]   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 µm in G-L.

View larger version (137K): [in a new window]   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 µm.

View larger version (55K): [in a new window]   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.

View larger version (107K): [in a new window]   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.

View larger version (102K): [in a new window]   Fig. 8. Cyclopamine treatment leads to RPE defects. An embryo incubated in cyclopamine solution (B) displays an abnormal pigmentation of the RPE (arrow) compared with the control embryo (A). Whole-mount in situ hybridisation (K-N) or retinal sections after whole-mount in situ hybridisation (G-J,Q-X), or immunostaining on retinal sections (C-F,O,P) of stage 40 embryos incubated with or without cyclopamine solution, as indicated, from stage 20 onwards. The probes or the antibodies are indicated for each panel. Cyclopamine does not interfere with the staining of ß-tubulin (C,D), R2-12 (E,F), Brn3.0 (G,H; the arrow indicates the expression in the ganglion cell layer in G), Xotx2 (I,J; the arrow in I indicates the expression in the inner nuclear cell layer, while the arrowhead indicates the expression in the CMZ) or Vax2 (K,L; the arrows indicate the ventral part of the retina expressing Vax2). Pax2 expression is strongly reduced (N; the arrow in M indicates Pax2 normal expression in the ventral retina). XAR1 immunostaining is completely abolished in the RPE in cyclopamine treated embryos (arrows in P), compared with control staining (arrows in O). XMitf expression is decreased in the presence of cyclopamine in the central RPE (arrow in R compared with Q) and abolished in the more peripheral region of the RPE (arrowheads in R compared with Q). Xotx5 expression is decreased mainly in the ventral region of the RPE in cyclopamine-treated embryos (arrow in T). X-bhh and X-chh expression in cyclopamine-treated embryos is reduced in the RPE, mainly in the peripheral part of the expression domain (arrows in V and X). le, lens; GCL, ganglion cell layer; INL, inner nuclear layer; PR, photoreceptor layer. Scale bar: 300 µm in A,B,K-N; 30 µm in C,D,G-J,O-X; 20 µm in E,F.

View larger version (33K): [in a new window]   Fig. 9. (A) A molecular gradient in the RPE of Xenopus tadpole embryos. The most peripheral region of the RPE expresses X-Smo, Gli2 and Gli3. BrdU-positive cells have been found in this zone 1. More centrally, in zone 2, these genes are inactivated and Hh genes are not activated. In zone 3, the Hh genes are expressed: X-bhh and X-chh strongly and X-shh weakly. Xotx5 is also expressed in zone 3. The XAR1 antibody also gives a staining in zone 3. (B) Model for the activity of Hh signalling during several steps of retinogenesis. From the midline, Hh signalling is required in eye field separation. This source of Hh signalling is then required in the establishment of the proximodistal axis of the retina. Later during retinogenesis, a source of Hh signalling emanating from the RPE is required for the proper RPE differentiation. The eye development is represented in blue, the mature RPE in black, Hh signalling gradients are in pink and areas affected when Hh signalling is blocked are indicated with yellow stars.