Sun J , Yoon J , Lee M , Hwang YS , Daar IO .

ZIA BC010006. 24 National Cancer Institute,USA, ZIA BC010006 Intramural NIH HHS

Xla Wt + efnb1 MO (Fig. 2 A r1 c3)
Xla Wt + raf1 (Fig. 5 r1c4 r2c4)
Xla Wt + spry2 (Fig 1 B)
Xla Wt + spry2 (Fig. 1 B)
Xla Wt + spry2{dn} (Fig. 1. E., F)
Xla Wt + spry2 MO (Fig. 1 D r1 c2)
Xla Wt + spry2 MO (Fig. 1. E r1 c3)
Xla Wt + spry2 MO (Fig. 1. E r1 c3)
Xla Wt + spry2 MO (Fig. 1. E r1 c3)
Xla Wt + spry2 MO (Fig. 2 A r1 c2)
Xla Wt + spry2 MO (Fig 3 A r1 c2, C r1 c2)
Xla Wt + spry2 MO (Fig. 3 B)
Xla Wt + spry2 MO (Fig. 4 A r1 c2, B)
Xla Wt + spry2 MO (Fig. 5 r1c2, r2c2)
Xla Wt + spry2 MO

	Figure 1. Sprouty2 is required for eye development through regulating the population of retina. (a) Whole-mount in situ hybridization reveals that sprouty2 is expressed in the optic vesicle at late neurula stage. Black arrowhead indicates optic vesicle on stage 18 embryo. The eye field was outlined with white dotted line. (b) Embryos were injected with wild type Sprouty2 mRNA (WT) or morpholino-resistant (MOR) form of Sprouty2 alone, or plus Sprouty2 morpholino at one cell stage, and analyzed by Western blot at late gastrula stage. Sprouty2 MO blocks the expression of Sprouty2 WT but not the Sprouty2 MOR. (c) Embryos were injected with Sprouty2 WT RNA or sprouty2 MO or a combination of Sprouty2 MO and Sprouty2 MOR RNA at one cell stage. Phosphorylation of ERK was analyzed by Western blot using embryo lysate from late gastrula stage. (d) D1.1.1 blastomere was injected with Sprouty2 MO with or without Sprouty2 MOR RNA, and the eye size was analyzed at stage 36. Dorsal view of the eyes in Sprouty2 morphant shows a significant reduction in diameter which could be well rescued by Sprouty2 MOR RNA co-injection. Quantification of eye diameter with one-way ANOVA (Dunnett’s multiple comparison), ****P < 0.0001, ***P < 0.001, Error bars indicate ± SD. (e) The D1.1.1 blastomere was injected with GFP mRNA plus Sprouty2 WT or Sprouty2Y55F RNA alone or Sprouty2 MO with or without Sprouty2 MOR RNA. Embryos were sectioned and immunostained with GFP antibody (Green) on the eye region at stage 33. The retina was outlined with an oval dotted line. (f) The Histograms indicate the percentage of embryos with D1.1.1 progeny in the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), ****P < 0.0001, Error bars indicate ± SD.
	Figure 2. Blocking of Ras/Raf/MAPK pathway rescues Sprouty2 MO-induced block to retina population. (a) Indicated morpholinos or RNAs were injected with GFP RNA into D1.1.1 blastomere at 32 cell stage. Embryos were then sectioned and immunostained with GFP antibody (Green) at stage 33. Images were taken of the eye region. The retina was outlined with an oval dotted line. (b) Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signal) within the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001, Error bars indicate ± SD.
	Figure 3. Over-activity of MAPK inhibits positioning of retinal progenitors within retina. (a) The indicated morpholinos or RNAs were injected with GFP mRNA into D1.1.1 blastomere at 32 cell stage. Embryos were then sectioned and immunostained with GFP antibody (Green) at stage 33. Images were taken of the eye region. The retina was outlined with an oval dotted line. Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signals) within the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001, Error bars indicate ± SD. (b) Embryos were injected with RNAs or Morpholinos along with ERK-V5 RNA into D1.1.1 blastomere at 32 cell stage and lysed at late neurula stage for immunoprecipitation with v5 agarose beads. Precipitates were then immunoblotted with phospho-ERK and total ERK antibodies. Three independent repeats were performed. (c) D1.1.1 blastomere was injected with control MO or Sprouty2 MO along with GFP mRNA at 32 cell stage. The injected embryos were either treated with DMSO or U0126 from late gastrula to late neurula stage. Embryos were then sectioned and immunostained with GFP antibody (Green) at stage 33. Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signals) within the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001, Error bars indicate ± SD.
	Figure 4. Sprouty2 regulates retinal progenitor movement into optic vesicle through repressing MAPK activity. (a) D1.1.1 blastomere was injected with GFP RNA plus RNAs and morpholinos as indicated. Embryos were then harvested and analyzed at late neurula stage. The cement gland was outlined with white dotted line and the optic vesicle was outlined with cyan dotted line. Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signals) within the optic vesicle from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001, Error bars indicate ± SD. (b) Embryos were injected with indicated RNAs or morpholinos along with ERK-V5 mRNA into D1.1.1 blastomere at 32 cell stage and lysed at neurula stage for immunoprecipitation with V5 agarose beads. Precipitates were then immunoblotted with phospho-ERK and total ERK antibodies. Three independent repeats were performed.
	Figure 5. Activation of MAPK signal affects eye specific marker gene expression. D1.1.1 blastomere was injected with RNA or morpholino or a combination of both as indicated. Embryos were then collected at stage 18 and subjected to whole-mount in situ hybridization for eye marker genes pax6 and rx1. Asterisk indicates the injected side. Histograms represent the percentage of embryos with normal or reduced expression of rx1 or pax6 from three biological repeats. Quantification with unpaired t test, P < 0.001, Error bars indicate ± SD.
	Supplementary Figure 1. Sprouty2 knockdown does not affect retinal progenitors movement into eye field at early neurula stage. D1.1.1 blastomere was injected with GFP RNA plus control MO or Sprouty2 MO as indicated. Embryos were harvested at stage 15. Both control embryo and Sprouty2 morphant showed normal dispersion of D1.1.1 clones in the eye field that was outlined with purple dotted line. The white dotted line indicates the embryo midline. Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signals) within the eye field from three biological repeats. Quantification with unpaired t test, P > 0.05, Error bars indicate ± SD. ns: no statistical differences between groups.
	Supplementary Figure 2. Knockdown of Sprouty2 does not induce apoptosis in D1.1.1 progeny at late neurula stage. Indicated morpholinos or RNAs were injected with GFP RNA into D1.1.1 blastomere at 32 cell stage. Embryos were sectioned and immunostained with cleaved-caspase 3 antibody at stage 19. Scatterplots represent cleaved-caspase 3+ cells expressing GFP on total GFP+ cells from three biological experiments. Quantification with one-way ANOVA (Dunnett’s multiple comparison), P < 0.0001. Error bars indicate ± SD. ****P < 0.0001, ns: no statistical differences between groups.
	Supplementary Figure 3. Knockdown of Sprouty2 slightly increases proliferation in D1.1.1 progeny at late neurula stage. Indicated morpholinos or RNAs were injected with GFP RNA into D1.1.1 blastomere at 32 cell stage. Embryos were sectioned and immunostained with phospho-Histone 3 (PH3) antibody at stage 19. Scatterplots represent PH3+ cells expressing GFP on total GFP+ cells from three biological experiments. Quantification with one-way ANOVA (Dunnett’s multiple comparison), P < 0.0001. Error bars indicate ± SD. **P < 0.01; ****P < 0.0001; ns: no statistical differences between groups.
	Supplementary Figure 4. Depletion of Sprouty2 prevents D1.1.1 progeny from populating retina and induces cell apoptosis at tadpole stage. Indicated morpholinos or RNAs were injected with GFP RNA into D1.1.1 blastomere at 32 cell stage. Embryos were sectioned and immunostained with cleaved-caspase 3 antibody at stage 33. The retina was outline with oval dotted line. Scatterplots represent cleaved-caspase 3+ cells expressing GFP on total GFP+ cells from three biological experiments. Quantification with one-way ANOVA (Dunnett’s multiple comparison), P < 0.0001. Error bars indicate ± SD. ****P < 0.0001, ns: no statistical differences between groups.
	Supplementary Figure 5. PKCδ is not involved in the regulation of Sprouty2-mediated retina population. (a) Indicated morpholinos or RNAs were injected with GFP mRNA into D1.1.1 blastomere at 32 cell stage. Embryos then were sectioned and immunostained with GFP antibody (Green) at stage 33. Images were taken on the eye region. The retina was outline with oval dotted line. Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signals) within the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001. (b) Embryos were injected with RNAs or Morpholinos as indicated at one cell stage. Injected embryos were lysed at gastrula stage and then immunoblotted with phospho-ERK antibody and total ERK antibody. Three independent repeats were performed.
	Supplementary Figure 6. Sprouty2 is not involved in the regulation of ephrinB1- mediated retina population. (a) Indicated morpholinos or RNAs were injected with GFP mRNA into V1.1.1 blastomere at 32 cell stage. Embryos then were sectioned and immunostained with GFP antibody (Green) at stage 33. Images were taken on the eye region. The retina was outline with oval dotted line. Histograms represent the percentage of embryos with V1.1.1 progeny (GFP positive signals) within the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001. (b) Indicated morpholinos or RNAs were injected with GFP mRNA into D1.1.1 blastomere at 32 cell stage. Histograms represent the percentage of embryos with D1.1.1 progeny (GFP positive signals) within the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), P < 0.0001.

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