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Fig. 5. Irx1 is required for PPR and otic gene expression (A) Injecting a dominant-negative Irx1 construct (DN-Irx1) results in loss of Six1 PPR expression (red arrows) in every embryo. Black arrows denote normal expression on control (ctrl) side. (B) Injecting DN-Irx1 results in loss of Sox11 PPR expression (red arrows) in every embryo. Black arrows denote normal expression on control side. (C) Injecting DN-Irx1 results in loss of Six1 otic expression (red arrow) in every embryo. Black arrow denotes normal expression on control side. (D) Injecting DN-Irx1 results in loss of Pax2 otic expression (red arrow) in every embryo. Black arrow denotes normal expression on control side. A, B are anterior views; C, D are side views, all with dorsal to the top. np, neural plate; e, eye. |
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Fig. 9. Increased Irx1 alters placode gene expression. (A) A low dose (200 pg) of Irx1 mRNA expanded the Sox11 PPR domain (between the red arrows) compared to control side (between the black arrows); in contrast, a higher dose (400 pg) reduced it. nt, neural tube. (B) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) in Sox11 PPR domain after injection of different doses of Irx1 mRNA. There was no difference between 400 pg and 800 pg (p > 0.05). (C) 800 pg of Irx1 mRNA expanded the Sox11 PPR domain (between the red arrows) in a minority of cases (see B), but in a majority of cases it also caused ectopic Sox11 expression in the lateral ectoderm (outlined by red dashes). (D) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) in Sox11 PPR domain after activation of Irx1-EnR-hGR at st 14 or st 16. Analyses were performed as described in Fig. 4. Each experimental group was significantly different from the no Dex group, but not significantly different from each other (p > 0.05). Control, uninjected embryos treated with Dex did not show an asymmetry in Sox11 expression (Dex st 14: 0%, n = 14; Dex st 16: 4.5%, n = 22). (E) Two examples showing that treatment of Irx1-EnR-hGR injected embryos with Chx 40 min before st 14 Dex treatment resulted in ectopic Sox11 expression throughout the lateral ectoderm (red arrows) compared to discrete PPR expression (between black arrows) and neural plate (np) expression on control side. (F) A low dose (200 pg) of Six1 mRNA expanded the Sox11 PPR domain (between red arrows) compared to control side (between the black arrows), whereas a high dose (800 pg) reduced it. (G) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) in Sox11 PPR domain after injection of different doses of Six1 mRNA. 800 pg showed a significantly different frequency compared to 200 pg (*, p < 0.05), but not 400 pg. (H) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) in Sox11 PPR domain after activation of Six1-hGR at st 14 or st 16. Analyses were performed as described in Fig. 4. Each experimental group was significantly different from the no Dex group, but not significantly different from each other (p > 0.05). Control, uninjected embryos treated with Dex did not show an asymmetry in Sox11 expression (Dex st 14: 0%, n = 14; Dex st 16: 4.5%, n = 22). (I) Irx1 expression in two placodes (black arrows) is distinct on the control side but nearly undetected on the Sox11-MO injected side (red arrows) in nearly half of embryos. (J) Increasing Sox11 prevents Irx1 expression from resolving into distinct placodes, indicated on the control side by black arrows. It remains broad and faint (between red arrows), as is typical of an earlier stage PPR. (K) Increasing Sox11 reduces Irx1 otic expression (red arrow) compared to control side (black arrow) of same embryo. e, eye; nt, neural tube. A, E, F, I, and J are anterior views; C is an anterior-lateral view; K are lateral views; all with dorsal to the top. |
