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Mech Dev
2010 Jan 01;1271-2:62-72. doi: 10.1016/j.mod.2009.10.006.
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Sonic hedgehog is involved in formation of the ventraloptic cup by limiting Bmp4 expression to the dorsal domain.
Zhao L
,
Saitsu H
,
Sun X
,
Shiota K
,
Ishibashi M
.
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Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal-distal and dorsal-ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventraloptic cup morphology.
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19854269
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Fig. 1. Expression analysis of nCre protein and Smo mRNA. Coronal sections of eyes were immunostained with anti-Cre antibody at the 26-somite stage (A–C) and the 36-somite stage (D–F). Control 2 (B) and Smo-CKOs (C) showed nCre immunoreactivity (brown) in the distal to ventral walls of the optic cup. nCre expression (brown) was observed in the neural retina in Control 2 (E) and Smo-CKOs (F). In situ hybridization of Smo mRNA (G–I) at the 20-somite stage (E9.5): Smo mRNA expression was completely abolished in the optic vesicle and optic stalk of Smo-CKOs (I). In situ hybridization of Gli1 mRNA (J–L) at the 26-somite stage (E9.75): Gli1 mRNA expression was completely abolished on optic cup of Smo-CKOs (L). ov: optic vesicle, op: optic cup, os: optic stalk.
Fig. 2. Craniofacial morphology and eye histology. Craniofacial morphology: A–I. (A–C) The optic vesicle was not obviously different at the 26-somite stage (E9.75). (D–F) At the 32-somite stage (E10), Smo-CKOs showed the ventral defect of the optic cup (F, black arrow: vop) and the diencephalon was hypotrophic (F, white arrow). (G–I) P0: No eyes were observed in Smo-CKOs (I, white arrowhead) and the forebrain part was small (I, white arrow), while Contol 1 (G) and Control 2 (H) did not show any abnormality. Eye histology: J–R. (J–L) At the 26-somite stage (E9.75), the optic cup of Smo-CKOs was not obviously different from that of Controls. (M–O) At the 35-somite stage (E10.5), Smo-CKOs displayed the ventral half defect of the optic cup and the hypotrophic lens was detected (O, green arrow). (P–R) At the 40-somite stage (E11), the ventral half of the optic cup and the lens were missing in Smo-CKOs (R). Control 1 (P) and Control 2 (Q) did not show any abnormality. vop: ventral optic cup.
Fig. 3. Cell proliferation and cell death defected in Smo-CKOs. BrdU incorporation analysis was performed (see Section 4). (A–C) At the 21-somite stage (E9.75), Controls and the mutants showed similar incorporation rates at the 21-somite stage (A–C). (D–F) At the 30-somite stage (E10.5), in Control 1 (D, n = 3, 64.66 ± 0.98%) the ventral optic cup showed a comparable rate with Control 2 (E, n = 3, 66.97 ± 0.34%) while Smo-CKOs (F, n = 3, 47.27 ± 0.48%) showed significantly decreased rates of BrdU incorporation (G). (D′–F′) Higher power-views of the ventral optic cups in D–F. (H–J) Caspase-3 was immunostained. In the optic vesicle of Control 1 (H) and Control 2 (I), there were few Caspase-3-positive cells at the 21-somite stage while Smo-CKOs exhibited increased Caspase-3-positive cells in the optic vesicle (J, black arrows). vop: ventral optic cup. P < 0.01.
Fig. 4. Pax6 and Pax2 expression patterns in the optic vesicle/cup and stalk. (A–I) Pax6 mRNA expression. (A–C) At the 27-somite stage (E9.75), all embryos showed normal expression patterns of Pax6. (D–F) At the 30-somite stage (E10.25), Smo-CKOs showed disturbed expression of Pax6 in the ventral optic cup (F, vop, red arrowhead). (G–I) At the 35-somite stage (E10.5), In Smo-CKOs (I) the Pax6 mRNA was confined to the dorsal optic cup only while Pax6 mRNA was expressed in both the ventral optic cup and the dorsal optic cup of Control 1 (G) and Control 2 (H). Coronal sections of eye: J–R. (J–L) SS25 (E9.75): The double immunostaining of Pax2 (green) and Pax6 (red). Pax2 and Pax6 were coexpressed in the optic vesicles (yellow). (M–O) At the 27-somite stage (E9.75), Pax2 expression was not obviously different among all genotypes. (P–R) At the 32-somite stage (E10.25), Pax2 expression domain in the mutant optic stalk (R) was similar to those seen in Control 1 (P) and Control 2 (Q). Pax2 expression of the dorsal optic cup was not detected in the mutants (R, black arrowhead). (S–U) At the 40-somite stage (E11), Pax2 expression was reduced in Smo-CKOs (U), compared to Control 1 (S) and Control 2 (T) not only in the optic cup but also in the optic stalk. os: optic stalk. dop: dorsal optic cup. vop: ventral optic cup.
Fig. 5. Vax1 and Vax2 expression patterns in the optic vesicle and cup. (A–R) Side views of whole mount in situ hybridization. (A′–R′) In situ hybridization on coronal sections. (A–C, A′–C′) At the 20-somite stage (E9.5), the Vax1 mRNA expression pattern in Smo-CKOs was not obviously different from that in Controls. (D–F, D′–F′) At the 24-somite stage (E9.75), comparing with Control 1 (D and D′) and Control 2 (E and E′), Vax1 mRNA was not expressed on the dorsal optic cup of Smo-CKOs (F, F′, red arrow). (G–I, G′–I′) At the 30-somite stage (E10), in Smo-CKOs (I and I′), Vax1 mRNA was downregulated in the ventral optic cup (black arrow) while Control 1 (G and G′) and Control 2 (H and H′) showed Vax1 expression in the ventral optic cup (black arrow). (J–R, J′–R′) Vax2 expression patterns were examined. (J–L, J′–L′). At the 22-sfomite stage (9.5), Vax2 expression patterns in the optic cup were not obviously different in all genotypes. (M–O, M′–O′) At the 24-somite stage (E9.75), comparing with Control 1 (M and M′) and Control 2 (N and N′), Vax2 mRNA was not expressed in the dorsal optic cup of Smo-CKOs (O, O′, red arrow). (P–R, P′–R′) At the 38-somite stage (E10), Vax2-positive ventral optic cup disappeared completely in Smo-CKOs (R′, asterisk) while Control 1 (P and P′) and Control 2 (Q and Q′) showed Vax2 expression in the ventral optic cup (black arrow). dop: dorsal optic vesicle/cup. vop: ventral optic vesicle/cup.
Fig. 6. Bmp4 expression is up-regulated in the Smo-CKO eye region. (A–C) At the 18-somite stage (E9.25), the mutant embryos exhibited slightly increased Bmp4 mRNA in the optic vesicle (C, dotted circle), compared to Controls (A and B). (D–F, D′–F′)At the 21-somite stage (E9.5), the Bmp4 mRNA was greatly increased in the optic vesicle (ov) of the Smo-CKOs (F, F′, red arrow). (G–I, G′–I′) At the 27-somite stage (E10), in Control 1(G and G′) and Control 2 (H and H′), Bmp4 mRNA was confined to the dorsal optic cup only, while in Smo-CKOs (I) Bmp4 mRNA was detected in both the ventral optic cup (vop) and the dorsal optic cup (dop).
Fig. 7. Raldh2/Raldh3 expression patterns are not altered in the mutants. (A–C) At the 24-somite stage (E9.75), Raldh2 expression pattern in Smo-CKOs (C) was not obviously different from that in Controls (A and B). (D–F) At the 25-somite stage (E9.75), Raldh3 expression pattern in Smo-CKOs (F) was not obviously different from that in Controls (D and E).
Fig. 8. Schematic representation of the relationship among Shh, Vax1/2, Pax2/6 and Bmp4. Shh signaling directly or indirectly mediates Vax1/2 expression in the eye field. Vax1 and Vax2 are suppressed by Bmp4 in the dorsal optic vesicle. This suppression might be mediated by Tbx and unknown factors. Vax1 and Vax2 directly inhibit Pax6 expression and maintain Pax2 expression. Pax2 and Pax6 transcriptionally repress each other.
