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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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Overexpression ofVP16-Gli1-GRcauses stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos that were unilaterally injected with 250 pg of VP16-Gli1-GR mRNA at the eight-cell stage, treated with dex from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 and Tbx3. Compared to the control side (uninj.), stage-dependent alterations in gene expression domains are detectable on the injected side. Light-blue β-gal staining identifies the injected side. Scale bar, 200 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 gene expression changes as detected in whole mount views. Scale bar, 100 μm.
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Fig4: Overexpression ofVP16-Gli1-GRcauses stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos that were unilaterally injected with 250 pg of VP16-Gli1-GR mRNA at the eight-cell stage, treated with dex from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 and Tbx3. Compared to the control side (uninj.), stage-dependent alterations in gene expression domains are detectable on the injected side. Light-blue β-gal staining identifies the injected side. Scale bar, 200 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 gene expression changes as detected in whole mount views. Scale bar, 100 μm.

Mentions: While both PMP treatments and grafts of ShhC25II-soaked beads caused effective activation of the Hh pathway in the eye, these reagents are likely to affect eye gene expression with some delay after the start of treatment, due to the time needed to build up sufficient amounts of ligands in the ocular extra-cellular space and to activate signal transduction within eye cells. Therefore, to dissect more precisely the time-dependent functions of Hh signalling in eye DV patterning, we employed a previously described Gli1 chimeric construct, encoding for a fusion protein of the Gli1 DNA binding domain with the strong transcriptional activator domain of VP16 and the dexamethasone (dex)-inducible glucocorticoid receptor domain (VP16-Gli1-GR) [23]. In this case, VP16-Gli1-GR mRNA microinjections in early embryos allow accumulation of the fusion protein in eye cells, which can be promptly activated by exposing embryos to dex, leading to rapid transcription of target genes [23,24]. VP16-Gli1-GR mRNA was unilaterally injected into one dorsal animal blastomere at the eight-cell stage, and injected embryos were raised to the desired stage for dex treatment. No effects on eye development or molecular marker expression were seen in injected embryos in the absence of dex, while dex delivery during gastrula or early neurula stages caused strong eye reduction phenotypes (data not shown). Dex treatments from late neurula/early optic vesicle stages (st. 19 to 25, Figure 4A,B,C) did not decrease eye size, but caused partial ventralization of the dorsal eye (Figure 4A,B,C), as shown by significant fractions of embryos where most of the eye expressed Vax2 (30%) or OS markers (Pax2, score 3, 44%; Vax1b, score 3, 33%) and by moderate reduction of Tbx3 expression domain. Double staining for Pax2 protein and Tbx3 mRNA expression showed a partial overlap between Pax2 and Tbx3 staining, indicating that the ectopic Pax2-positive domain extended within the remaining Tbx3-expressing region (Additional file 3: Figure S3). When dex was added starting from mid optic vesicle stages (st. 26 to 27, Figure 4A,B,C), few embryos were found where most of the eye was positive for Vax2 (2%) or for OS markers (Pax2, score 3, 12%; Vax1b, score 3, 20%), while the expression domain of Tbx3 was largely unaffected, thus indicating that DR specification was mostly refractory to ventralization by Gli-mediated signalling by these stages. OS markers, however, were still significantly upregulated by these later treatments, and their expression domains were expanded in the ventral and central retina and, more dorsally, along the nasal part of the retina and/or in small groups of cells scattered in the dorsal half of the eye (Pax2, score 1 to 2, 63%; Vax1b, score 1 to 2, 45%). Finally, when dex treatments were started at the late optic vesicle/early optic cup stage (st. 30 to 32, Figure 4A,B,C), activation of Gli-mediated signalling could still promote ectopic expression of Pax2 and Vax1b in scattered groups of cells within the central/dorsal retina (Pax2, score 1 to 2, 40%; Vax1b, score 1 to 2, 31%). Overall, the effects of VP16-Gli1-GR overexpression were similar to those of PMP treatments and ShhC25II bead grafts, but eye cells remained sensitive to ventralization by VP16-Gli1-GR for longer time windows compared to PMP or ShhC25II treatments. In particular, VP16-Gli1-GR overexpression from late neurula/early optic vesicle stages caused similar effects to PMP treatments from early neurula stages. Likewise, the effects of VP16-Gli1-GR overexpression from mid or late optic vesicle stages were similar to those of PMP/ShhC25II treatments from early or mid optic vesicle stages, respectively.Figure 4


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)

Overexpression ofVP16-Gli1-GRcauses stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos that were unilaterally injected with 250 pg of VP16-Gli1-GR mRNA at the eight-cell stage, treated with dex from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 and Tbx3. Compared to the control side (uninj.), stage-dependent alterations in gene expression domains are detectable on the injected side. Light-blue β-gal staining identifies the injected side. Scale bar, 200 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 gene expression changes as detected in whole mount views. Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Overexpression ofVP16-Gli1-GRcauses stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos that were unilaterally injected with 250 pg of VP16-Gli1-GR mRNA at the eight-cell stage, treated with dex from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 and Tbx3. Compared to the control side (uninj.), stage-dependent alterations in gene expression domains are detectable on the injected side. Light-blue β-gal staining identifies the injected side. Scale bar, 200 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 gene expression changes as detected in whole mount views. Scale bar, 100 μm.
Mentions: While both PMP treatments and grafts of ShhC25II-soaked beads caused effective activation of the Hh pathway in the eye, these reagents are likely to affect eye gene expression with some delay after the start of treatment, due to the time needed to build up sufficient amounts of ligands in the ocular extra-cellular space and to activate signal transduction within eye cells. Therefore, to dissect more precisely the time-dependent functions of Hh signalling in eye DV patterning, we employed a previously described Gli1 chimeric construct, encoding for a fusion protein of the Gli1 DNA binding domain with the strong transcriptional activator domain of VP16 and the dexamethasone (dex)-inducible glucocorticoid receptor domain (VP16-Gli1-GR) [23]. In this case, VP16-Gli1-GR mRNA microinjections in early embryos allow accumulation of the fusion protein in eye cells, which can be promptly activated by exposing embryos to dex, leading to rapid transcription of target genes [23,24]. VP16-Gli1-GR mRNA was unilaterally injected into one dorsal animal blastomere at the eight-cell stage, and injected embryos were raised to the desired stage for dex treatment. No effects on eye development or molecular marker expression were seen in injected embryos in the absence of dex, while dex delivery during gastrula or early neurula stages caused strong eye reduction phenotypes (data not shown). Dex treatments from late neurula/early optic vesicle stages (st. 19 to 25, Figure 4A,B,C) did not decrease eye size, but caused partial ventralization of the dorsal eye (Figure 4A,B,C), as shown by significant fractions of embryos where most of the eye expressed Vax2 (30%) or OS markers (Pax2, score 3, 44%; Vax1b, score 3, 33%) and by moderate reduction of Tbx3 expression domain. Double staining for Pax2 protein and Tbx3 mRNA expression showed a partial overlap between Pax2 and Tbx3 staining, indicating that the ectopic Pax2-positive domain extended within the remaining Tbx3-expressing region (Additional file 3: Figure S3). When dex was added starting from mid optic vesicle stages (st. 26 to 27, Figure 4A,B,C), few embryos were found where most of the eye was positive for Vax2 (2%) or for OS markers (Pax2, score 3, 12%; Vax1b, score 3, 20%), while the expression domain of Tbx3 was largely unaffected, thus indicating that DR specification was mostly refractory to ventralization by Gli-mediated signalling by these stages. OS markers, however, were still significantly upregulated by these later treatments, and their expression domains were expanded in the ventral and central retina and, more dorsally, along the nasal part of the retina and/or in small groups of cells scattered in the dorsal half of the eye (Pax2, score 1 to 2, 63%; Vax1b, score 1 to 2, 45%). Finally, when dex treatments were started at the late optic vesicle/early optic cup stage (st. 30 to 32, Figure 4A,B,C), activation of Gli-mediated signalling could still promote ectopic expression of Pax2 and Vax1b in scattered groups of cells within the central/dorsal retina (Pax2, score 1 to 2, 40%; Vax1b, score 1 to 2, 31%). Overall, the effects of VP16-Gli1-GR overexpression were similar to those of PMP treatments and ShhC25II bead grafts, but eye cells remained sensitive to ventralization by VP16-Gli1-GR for longer time windows compared to PMP or ShhC25II treatments. In particular, VP16-Gli1-GR overexpression from late neurula/early optic vesicle stages caused similar effects to PMP treatments from early neurula stages. Likewise, the effects of VP16-Gli1-GR overexpression from mid or late optic vesicle stages were similar to those of PMP/ShhC25II treatments from early or mid optic vesicle stages, respectively.Figure 4

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