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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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PMP treatments cause stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos treated with DMSO (mock) or 300 to 600 μM PMP from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 or Tbx3. In mock-treated embryos, wild-type expression patterns of these genes are detectable: Pax2 and Vax1b staining is restricted to the OS, Vax2-positive region covers the OS and VR, Tbx3 expression identifies the DR. PMP treatments affect gene expression domains to various degrees depending on the stage of delivery (st. 8 to 10, blastula-early gastrula; st. 12.5 to 14, late gastrula-early neurula; st. 19 to 22, late neurula-early optic vesicle; st. 26, mid optic vesicle). For Pax2 and Vax1b, embryos were grouped into 0 to 3 scores (numbers at the bottom right corner of each image) as explained in the ‘Results’ section and representative eyes for each score group are shown. See text for details. The broken yellow circles highlight the eye region. Scale bar, 200 μm. (B) Quantification of the percentages of embryos stained for Pax2 or Vax1b with 0 to 3 scores in each treatment condition. Embryos stained for Vax2 or Tbx3 were grouped according to the DV extent of Vax2/Tbx3 expression domain (more or less than 90% of the eye for Vax2; more or less than 10% of the eye for Tbx3). The percentages of embryos with strong eye reductions are also indicated (S, small eyes). The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. At least 20 eyes/10 embryos were analysed for each experiment. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B), and hybridized with the indicated probes, confirming stage dependent alterations in the expression domains of Pax2, Vax1b, Vax2 and Tbx3 as detected in whole mount views. Scale bar, 100 μm.
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Fig1: PMP treatments cause stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos treated with DMSO (mock) or 300 to 600 μM PMP from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 or Tbx3. In mock-treated embryos, wild-type expression patterns of these genes are detectable: Pax2 and Vax1b staining is restricted to the OS, Vax2-positive region covers the OS and VR, Tbx3 expression identifies the DR. PMP treatments affect gene expression domains to various degrees depending on the stage of delivery (st. 8 to 10, blastula-early gastrula; st. 12.5 to 14, late gastrula-early neurula; st. 19 to 22, late neurula-early optic vesicle; st. 26, mid optic vesicle). For Pax2 and Vax1b, embryos were grouped into 0 to 3 scores (numbers at the bottom right corner of each image) as explained in the ‘Results’ section and representative eyes for each score group are shown. See text for details. The broken yellow circles highlight the eye region. Scale bar, 200 μm. (B) Quantification of the percentages of embryos stained for Pax2 or Vax1b with 0 to 3 scores in each treatment condition. Embryos stained for Vax2 or Tbx3 were grouped according to the DV extent of Vax2/Tbx3 expression domain (more or less than 90% of the eye for Vax2; more or less than 10% of the eye for Tbx3). The percentages of embryos with strong eye reductions are also indicated (S, small eyes). The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. At least 20 eyes/10 embryos were analysed for each experiment. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B), and hybridized with the indicated probes, confirming stage dependent alterations in the expression domains of Pax2, Vax1b, Vax2 and Tbx3 as detected in whole mount views. Scale bar, 100 μm.

Mentions: PMP treatments started from late blastula/early gastrula stages (st. 8/10.5, Figure 1A,B,C) reproduced the effects of Hh ligand mRNA injections in early embryos [12], as they could cause upregulation of Pax2, Vax1b and Vax2 throughout the eye and downregulation of Tbx3. In these conditions, the majority of treated embryos upregulated Vax2 through most of the eye (73%) and a similar situation was found also for Pax2 (score 3, 92%). Significant numbers of embryos also expressed Vax1b in a broad DV domain (score 3, 47%) or showed severely reduced expression of Tbx3 (22%). The percentage of embryos with strong downregulation of Tbx3 increased when PMP treatments were started from early cleavage stages (st. 4, Additional file 1: Figure S1). When PMP was delivered from early neurula stages (st. 12.5 to 14, Figure 1A,B,C), considerable numbers of embryos with strong expansion of the Pax2 domain were still detectable (score 3, 59%), but the fractions of embryos with a broad spread of Vax2 (24%) or Vax1b (score 3, 14%) were reduced and those with severe Tbx3 reduction were nearly absent (3%). These conditions also resulted in significant numbers of embryos with partial (score 2) dorsal expansion of Pax2 and Vax1b, so that nearly all of the Pax2-stained embryos (97%) and roughly half of the Vax1b-stained embryos (45%) were scored as 2 or 3. No embryos with Vax2 expression covering most of the eye or strongly reduced Tbx3 domain were found following treatments with PMP from late neurula/early optic vesicle stages (st. 18 to 22, Figure 1A,B,C), and a substantial DR domain was specified in these conditions. Pax2, however, was still markedly upregulated by these later treatments. Although Pax2 expression seldom spread through most of the eye (score 3, 6%), almost all the embryos showed a partial dorsal expansion (score 1 or 2, 89%). Vax1b expression domain was also expanded, albeit only within the ventral half of the eye. Finally, no evident effects on Vax2, Vax1b or Tbx3 were observed when PMP was applied from mid optic vesicle stages (st. 25 to 27, Figure 1A,B,C). In contrast, most of the embryos stained with Pax2 probes showed restricted ectopic transcription at the level of the dorsal marginal zone (score 1, 90%). Double staining with Tbx3 probes and an anti-Pax2 antibody confirmed that within the most dorsal retina, PMP treatments started during optic vesicle stages caused localized Pax2 protein expression in the dorsal marginal zone, which partially overlapped with Tbx3 expression (Additional file 2: Figure S2).Figure 1


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)

PMP treatments cause stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos treated with DMSO (mock) or 300 to 600 μM PMP from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 or Tbx3. In mock-treated embryos, wild-type expression patterns of these genes are detectable: Pax2 and Vax1b staining is restricted to the OS, Vax2-positive region covers the OS and VR, Tbx3 expression identifies the DR. PMP treatments affect gene expression domains to various degrees depending on the stage of delivery (st. 8 to 10, blastula-early gastrula; st. 12.5 to 14, late gastrula-early neurula; st. 19 to 22, late neurula-early optic vesicle; st. 26, mid optic vesicle). For Pax2 and Vax1b, embryos were grouped into 0 to 3 scores (numbers at the bottom right corner of each image) as explained in the ‘Results’ section and representative eyes for each score group are shown. See text for details. The broken yellow circles highlight the eye region. Scale bar, 200 μm. (B) Quantification of the percentages of embryos stained for Pax2 or Vax1b with 0 to 3 scores in each treatment condition. Embryos stained for Vax2 or Tbx3 were grouped according to the DV extent of Vax2/Tbx3 expression domain (more or less than 90% of the eye for Vax2; more or less than 10% of the eye for Tbx3). The percentages of embryos with strong eye reductions are also indicated (S, small eyes). The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. At least 20 eyes/10 embryos were analysed for each experiment. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B), and hybridized with the indicated probes, confirming stage dependent alterations in the expression domains of Pax2, Vax1b, Vax2 and Tbx3 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

Fig1: PMP treatments cause stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos treated with DMSO (mock) or 300 to 600 μM PMP from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 or Tbx3. In mock-treated embryos, wild-type expression patterns of these genes are detectable: Pax2 and Vax1b staining is restricted to the OS, Vax2-positive region covers the OS and VR, Tbx3 expression identifies the DR. PMP treatments affect gene expression domains to various degrees depending on the stage of delivery (st. 8 to 10, blastula-early gastrula; st. 12.5 to 14, late gastrula-early neurula; st. 19 to 22, late neurula-early optic vesicle; st. 26, mid optic vesicle). For Pax2 and Vax1b, embryos were grouped into 0 to 3 scores (numbers at the bottom right corner of each image) as explained in the ‘Results’ section and representative eyes for each score group are shown. See text for details. The broken yellow circles highlight the eye region. Scale bar, 200 μm. (B) Quantification of the percentages of embryos stained for Pax2 or Vax1b with 0 to 3 scores in each treatment condition. Embryos stained for Vax2 or Tbx3 were grouped according to the DV extent of Vax2/Tbx3 expression domain (more or less than 90% of the eye for Vax2; more or less than 10% of the eye for Tbx3). The percentages of embryos with strong eye reductions are also indicated (S, small eyes). The number of experiments performed for each probe and treatment condition is indicated on top of the corresponding histogram bar. At least 20 eyes/10 embryos were analysed for each experiment. (C) Histological sections of eyes of st. 33 embryos treated as in (A) and (B), and hybridized with the indicated probes, confirming stage dependent alterations in the expression domains of Pax2, Vax1b, Vax2 and Tbx3 as detected in whole mount views. Scale bar, 100 μm.
Mentions: PMP treatments started from late blastula/early gastrula stages (st. 8/10.5, Figure 1A,B,C) reproduced the effects of Hh ligand mRNA injections in early embryos [12], as they could cause upregulation of Pax2, Vax1b and Vax2 throughout the eye and downregulation of Tbx3. In these conditions, the majority of treated embryos upregulated Vax2 through most of the eye (73%) and a similar situation was found also for Pax2 (score 3, 92%). Significant numbers of embryos also expressed Vax1b in a broad DV domain (score 3, 47%) or showed severely reduced expression of Tbx3 (22%). The percentage of embryos with strong downregulation of Tbx3 increased when PMP treatments were started from early cleavage stages (st. 4, Additional file 1: Figure S1). When PMP was delivered from early neurula stages (st. 12.5 to 14, Figure 1A,B,C), considerable numbers of embryos with strong expansion of the Pax2 domain were still detectable (score 3, 59%), but the fractions of embryos with a broad spread of Vax2 (24%) or Vax1b (score 3, 14%) were reduced and those with severe Tbx3 reduction were nearly absent (3%). These conditions also resulted in significant numbers of embryos with partial (score 2) dorsal expansion of Pax2 and Vax1b, so that nearly all of the Pax2-stained embryos (97%) and roughly half of the Vax1b-stained embryos (45%) were scored as 2 or 3. No embryos with Vax2 expression covering most of the eye or strongly reduced Tbx3 domain were found following treatments with PMP from late neurula/early optic vesicle stages (st. 18 to 22, Figure 1A,B,C), and a substantial DR domain was specified in these conditions. Pax2, however, was still markedly upregulated by these later treatments. Although Pax2 expression seldom spread through most of the eye (score 3, 6%), almost all the embryos showed a partial dorsal expansion (score 1 or 2, 89%). Vax1b expression domain was also expanded, albeit only within the ventral half of the eye. Finally, no evident effects on Vax2, Vax1b or Tbx3 were observed when PMP was applied from mid optic vesicle stages (st. 25 to 27, Figure 1A,B,C). In contrast, most of the embryos stained with Pax2 probes showed restricted ectopic transcription at the level of the dorsal marginal zone (score 1, 90%). Double staining with Tbx3 probes and an anti-Pax2 antibody confirmed that within the most dorsal retina, PMP treatments started during optic vesicle stages caused localized Pax2 protein expression in the dorsal marginal zone, which partially overlapped with Tbx3 expression (Additional file 2: Figure S2).Figure 1

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