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Figure 1.
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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Figure 3.
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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Figure 4.
Overexpression of
VP16-Gli1-GR
causes 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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Figure 6.
CPM treatments cause stage dependent effects on eye DV polarity. (A) Lateral views of heads of st. 33 embryos treated with ethanol (mock) or 100 μM CPM from the indicated stages and hybridized with probes for Pax2, Vax1b, Vax2 or Tbx3. Compared to controls, CPM-treated embryos show stage-dependent changes in gene expression domains along the anteroposterior (AP; Pax2, Vax1b) or the DV (Vax2, Tbx3) axes of the eye. Scale bar, 200 μm. (B) Quantification of the mean AP (Pax2, Vax1b) or DV (Vax2, Tbx3) width/height of gene expression domains, normalized to total width/height of the eye, in the eyes of embryos treated as in (A). The number of eyes analysed for each probe and treatment condition is indicated within the corresponding histogram bar. Error bars show standard deviations. *P < 0.05; ns, non-significant (P ≥ 0.05) according to two-tailed Student’s t-test. (C) Histological sections of eyes of st. 33 embryos treated with ethanol or CPM from st. 13 and hybridized with the indicated probes. CPM-treated eyes show a reduction of Pax2, Vax1b and Vax2 expression domains along the eye proximodistal axis. Yellow brackets highlight the proximodistal extent of the whole ventral eye region. Scale bar, 100 μm. |
