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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. 6. Increased Irx1 reduces PPR and otic gene expression. (A) Increasing Irx1 by mRNA injection (right side) reduced the size of the Six1 expression domain in the PPR. Black arrows denote Six1 expression on the control side; red arrow denotes the reduced domain on the injected side. (B) Increased Irx1 reduced the size of the Eya1 expression domain in the PPR. (C) The percentage of embryos that showed the same phenotype as in A and B when injected with Irx1 mRNA. Irx1–800 pg caused the Six1 phenotype significantly more frequently (*, p < 0.05) than Irx1–200 pg. There were no significant differences across Irx1 mRNA doses for Eya1 (p > 0.05). Numbers above the bars indicate sample size. (D) Increased Irx1 reduced the size of the Sox9 and Pax2 domains in the otic vesicle (red arrows) compared to control side of same embryo (black arrows). (E) Embryos were injected with Irx1-EnR-hGR mRNA (400 pg) and treated with Dexamethasone (Dex) at indicated stages. Analyses of Six1 expression were performed as described in Fig. 4. Each experimental group was significantly different from the no Dex group, and significantly different from each other (p < 0.05). Control, uninjected embryos treated with Dex did not show an asymmetry in Six1 expression (Dex st 14: 0%, n = 25; Dex st 16: 0%, n = 15; Dex st 18: 0%, n = 25). A, B are anterior views; D are side views, all with dorsal to the top. |
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Fig. 7. Irx1 may affect PPR gene expression by downregulating Fgf. (A) When Irx1 mRNA (400 pg) was injected into animal blastomeres, an open blastopore phenotype (arrows) was observed at closed neural tube stages. Dorsal view, anterior (a) to the top, posterior (p) to the bottom. (B) The frequency of this phenotype depended upon the cell injected (see Fig. 1 for blastomere nomenclature). (C) Irx1 mRNA (400 pg) injection reduced the size of the Fgf8 expression domain in the PPR. Black arrows denote Fgf8 expression in the PPR on the control side; red arrows denote the reduced domain on the injected side. (D) In the majority of embryos co-injected with Irx1 plus cFgfr1 mRNAs, Six1 expression was restored (76.7%, n = 60). Black arrows denote control side; red arrows denote injected side. C, D are anterior views, dorsal to the top. |
