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Dorsoventral patterning of the Xenopus eye involves differential temporal changes in the response of optic stalk and retinal progenitors to Hh signalling.

Wang X, Lupo G, He R, Barsacchi G, Harris WA, Liu Y - Neural Dev (2015)

Bottom Line: In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the ventral retina and the optic stalk, while the effects of Hh inhibition during optic vesicle stages were limited to the reduction of optic stalk size.Our results suggest the existence of two competence windows during which the Hh pathway differentially controls patterning of the eye region.We speculate that this temporal regulation is important to coordinate dorsoventral patterning with morphogenesis and differentiation processes during eye development.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101, China. 13522764597@163.com.

ABSTRACT

Background: Hedgehog (Hh) signals are instrumental to the dorsoventral patterning of the vertebrate eye, promoting optic stalk and ventral retinal fates and repressing dorsal retinal identity. There has been limited analysis, however, of the critical window during which Hh molecules control eye polarity and of the temporal changes in the responsiveness of eye cells to these signals.

Results: In this study, we used pharmacological and molecular tools to perform stage-specific manipulations of Hh signalling in the developing Xenopus eye. In gain-of-function experiments, most of the eye was sensitive to ventralization when the Hh pathway was activated starting from gastrula/neurula stages. During optic vesicle stages, the dorsal eye became resistant to Hh-dependent ventralization, but this pathway could partially upregulate optic stalk markers within the retina. In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the ventral retina and the optic stalk, while the effects of Hh inhibition during optic vesicle stages were limited to the reduction of optic stalk size.

Conclusions: Our results suggest the existence of two competence windows during which the Hh pathway differentially controls patterning of the eye region. In the first window, between the neural plate and the optic vesicle stages, Hh signalling exerts a global influence on eye dorsoventral polarity, contributing to the specification of optic stalk, ventral retina and dorsal retinal domains. In the second window, between optic vesicle and optic cup stages, this pathway plays a more limited role in the maintenance of the optic stalk domain. We speculate that this temporal regulation is important to coordinate dorsoventral patterning with morphogenesis and differentiation processes during eye development.

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Grafts of ShhC25II-soaked beads reproduce the stage-dependent effects of PMP treatments on eye DV patterning. (A) Lateral views of heads of st. 33 embryos that received a graft of control or ShhC25II-soaked beads next to the optic vesicle at the indicated stages and hybridized with probes for Pax2, Vax1b or Vax2. Compared to controls, embryos grafted with ShhC25II beads show stage-dependent increase in the expression domains of ventral eye genes. Scale bar, 100 μm. (B) Quantification of the percentages of embryos with different effects on gene expression domains or eye reductions (S) in each treatment condition. The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B) and hybridized with the indicated probes, confirming stage dependent effects on DV eye patterning as detected in whole mount views. Triangles point to ectopic Pax2 expression in the dorsal marginal zone and arrows to expanded ventral expression domains of Pax2, Vax1b and Vax2, in embryos grafted ShhC25II beads. Stars indicate the position of the beads. Scale bar, 100 μm.
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Fig3: Grafts of ShhC25II-soaked beads reproduce the stage-dependent effects of PMP treatments on eye DV patterning. (A) Lateral views of heads of st. 33 embryos that received a graft of control or ShhC25II-soaked beads next to the optic vesicle at the indicated stages and hybridized with probes for Pax2, Vax1b or Vax2. Compared to controls, embryos grafted with ShhC25II beads show stage-dependent increase in the expression domains of ventral eye genes. Scale bar, 100 μm. (B) Quantification of the percentages of embryos with different effects on gene expression domains or eye reductions (S) in each treatment condition. The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B) and hybridized with the indicated probes, confirming stage dependent effects on DV eye patterning as detected in whole mount views. Triangles point to ectopic Pax2 expression in the dorsal marginal zone and arrows to expanded ventral expression domains of Pax2, Vax1b and Vax2, in embryos grafted ShhC25II beads. Stars indicate the position of the beads. Scale bar, 100 μm.

Mentions: This temporal analysis was validated by upregulating Hh signalling in the eye region by means of grafts of beads soaked in sonic hedgehog (C25II) N-terminus (ShhC25II), a bioactive form of the sonic hedgehog (Shh) protein [22]. ShhC25II beads grafted during early (st. 20 to 24) or mid (st. 25 to 27) optic vesicle stages reproduced the effects of PMP treatments during corresponding developmental windows (Figure 3A,B,C). In either case, a significant part of the dorsal eye remained devoid of expression of the VR marker Vax2 in most of the embryos. Following early optic vesicle grafts, however, partial dorsal upregulation of OS markers was clearly detectable, especially along the nasal region (Pax2, score 2 to 3, 70%; Vax1b, score 2, 25%). Compared to PMP, ShhC25II beads resulted in some embryos expressing Pax2 within a broad DV domain (score 3, 23%) and Vax1b was also more strongly upregulated by ShhC25II at these stages of treatment (compare Pax2 and Vax1b charts in Figures 1B and 2B), suggesting that PMP treatments cause somewhat slower upregulation of the Hh pathway compared to grafts of ShhC25II beads. Grafts performed at mid optic vesicle stages (st. 25 to 27) yielded similar results to PMP treatments, as Vax2 and Vax1b expression domains remained confined to the ventral eye region, while Pax2 was ectopically expressed in the dorsal marginal zone.Figure 3


Dorsoventral patterning of the Xenopus eye involves differential temporal changes in the response of optic stalk and retinal progenitors to Hh signalling.

Wang X, Lupo G, He R, Barsacchi G, Harris WA, Liu Y - Neural Dev (2015)

Grafts of ShhC25II-soaked beads reproduce the stage-dependent effects of PMP treatments on eye DV patterning. (A) Lateral views of heads of st. 33 embryos that received a graft of control or ShhC25II-soaked beads next to the optic vesicle at the indicated stages and hybridized with probes for Pax2, Vax1b or Vax2. Compared to controls, embryos grafted with ShhC25II beads show stage-dependent increase in the expression domains of ventral eye genes. Scale bar, 100 μm. (B) Quantification of the percentages of embryos with different effects on gene expression domains or eye reductions (S) in each treatment condition. The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B) and hybridized with the indicated probes, confirming stage dependent effects on DV eye patterning as detected in whole mount views. Triangles point to ectopic Pax2 expression in the dorsal marginal zone and arrows to expanded ventral expression domains of Pax2, Vax1b and Vax2, in embryos grafted ShhC25II beads. Stars indicate the position of the beads. Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4373414&req=5

Fig3: Grafts of ShhC25II-soaked beads reproduce the stage-dependent effects of PMP treatments on eye DV patterning. (A) Lateral views of heads of st. 33 embryos that received a graft of control or ShhC25II-soaked beads next to the optic vesicle at the indicated stages and hybridized with probes for Pax2, Vax1b or Vax2. Compared to controls, embryos grafted with ShhC25II beads show stage-dependent increase in the expression domains of ventral eye genes. Scale bar, 100 μm. (B) Quantification of the percentages of embryos with different effects on gene expression domains or eye reductions (S) in each treatment condition. The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B) and hybridized with the indicated probes, confirming stage dependent effects on DV eye patterning as detected in whole mount views. Triangles point to ectopic Pax2 expression in the dorsal marginal zone and arrows to expanded ventral expression domains of Pax2, Vax1b and Vax2, in embryos grafted ShhC25II beads. Stars indicate the position of the beads. Scale bar, 100 μm.
Mentions: This temporal analysis was validated by upregulating Hh signalling in the eye region by means of grafts of beads soaked in sonic hedgehog (C25II) N-terminus (ShhC25II), a bioactive form of the sonic hedgehog (Shh) protein [22]. ShhC25II beads grafted during early (st. 20 to 24) or mid (st. 25 to 27) optic vesicle stages reproduced the effects of PMP treatments during corresponding developmental windows (Figure 3A,B,C). In either case, a significant part of the dorsal eye remained devoid of expression of the VR marker Vax2 in most of the embryos. Following early optic vesicle grafts, however, partial dorsal upregulation of OS markers was clearly detectable, especially along the nasal region (Pax2, score 2 to 3, 70%; Vax1b, score 2, 25%). Compared to PMP, ShhC25II beads resulted in some embryos expressing Pax2 within a broad DV domain (score 3, 23%) and Vax1b was also more strongly upregulated by ShhC25II at these stages of treatment (compare Pax2 and Vax1b charts in Figures 1B and 2B), suggesting that PMP treatments cause somewhat slower upregulation of the Hh pathway compared to grafts of ShhC25II beads. Grafts performed at mid optic vesicle stages (st. 25 to 27) yielded similar results to PMP treatments, as Vax2 and Vax1b expression domains remained confined to the ventral eye region, while Pax2 was ectopically expressed in the dorsal marginal zone.Figure 3

Bottom Line: In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the ventral retina and the optic stalk, while the effects of Hh inhibition during optic vesicle stages were limited to the reduction of optic stalk size.Our results suggest the existence of two competence windows during which the Hh pathway differentially controls patterning of the eye region.We speculate that this temporal regulation is important to coordinate dorsoventral patterning with morphogenesis and differentiation processes during eye development.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101, China. 13522764597@163.com.

ABSTRACT

Background: Hedgehog (Hh) signals are instrumental to the dorsoventral patterning of the vertebrate eye, promoting optic stalk and ventral retinal fates and repressing dorsal retinal identity. There has been limited analysis, however, of the critical window during which Hh molecules control eye polarity and of the temporal changes in the responsiveness of eye cells to these signals.

Results: In this study, we used pharmacological and molecular tools to perform stage-specific manipulations of Hh signalling in the developing Xenopus eye. In gain-of-function experiments, most of the eye was sensitive to ventralization when the Hh pathway was activated starting from gastrula/neurula stages. During optic vesicle stages, the dorsal eye became resistant to Hh-dependent ventralization, but this pathway could partially upregulate optic stalk markers within the retina. In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the ventral retina and the optic stalk, while the effects of Hh inhibition during optic vesicle stages were limited to the reduction of optic stalk size.

Conclusions: Our results suggest the existence of two competence windows during which the Hh pathway differentially controls patterning of the eye region. In the first window, between the neural plate and the optic vesicle stages, Hh signalling exerts a global influence on eye dorsoventral polarity, contributing to the specification of optic stalk, ventral retina and dorsal retinal domains. In the second window, between optic vesicle and optic cup stages, this pathway plays a more limited role in the maintenance of the optic stalk domain. We speculate that this temporal regulation is important to coordinate dorsoventral patterning with morphogenesis and differentiation processes during eye development.

Show MeSH
Related in: MedlinePlus