|
Display additional annotations [+]
|
| |
Fig. 8. Increased Irx1 alters BZ and NC gene expression. (A) Increased Irx1 expands the width of the Pax3 domain (red bar) compared to control side (black bar). (B) Increased Irx1 expands the width of the Zic1 neural crest domain (red bar) compared to control side (black bar). (C) Increased Irx1 expands the width of the Zic2 neural crest domain (red bar) compared to control side (black bar). (D) Increased Irx1 extends the anterior-posterior extent of the Tfap2α neural crest domain (between red arrows) compared to control side (between black arrows). (E) Increased Irx1 either expands (left embryo) or reduces (right embryo) the Foxd3 domain. inj, injected side; ctrl, control side. (F) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) of Foxd3 domains after injection of different doses of Irx1 mRNA. The frequencies of the Foxd3 phenotypes were not significantly different between 200 pg and 400 pg, but 800 pg caused reduction of Foxd3 at a significantly higher frequency (*, p < 0.05). (G) Increased Irx1 reduced the Sox9 neural crest domain (red arrows) compared to control side (black arrows). It also reduced the size of the Sox9-expressing otic placode (outlined in green). (H) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) in Sox9 neural crest (NC) domains after injection of different doses of Irx1 mRNA. These phenotypes were not significantly different across the doses (p > 0.05). (I) Percentages of embryos showing reduced (orange), expanded (blue) or no change (NC, grey) in Sox9 otic placode expression after injection of different doses of Irx1 mRNA. These phenotypes were not significantly different across the doses (p > 0.05). All embryos are anterior views with dorsal to the top. Numbers in brackets above bars are sample sizes. |
|
Display additional annotations [+]
|
| |
Fig. 3. Increasing Six1 levels represses Irx1 expression. (A) Six1 or Six1+Eya1 mRNAs (right sides) reduce the size of the Irx1 expression domain in the PPR or placodes. Black arrows denote Irx1 PPR/placode expression on the control side; red arrows denote the same on the injected side, which also is indicated by the pink βGal lineage tracer. np, neural plate; nt, neural tube. (B) The percentage of embryos showing the same Irx1 phenotypes as in (A). *, indicates a significant increase (p < 0.05) in frequency comparing 800 pg to either 200 pg or 400 pg of Six1 mRNA. Co-expressing Six1 +Eya1 mRNAs does not significantly alter the frequency of the phenotype (p > 0.05). There were no significant differences between Six1 or Six1 +Eya1 mRNAs. Numbers above bars indicate the sample size. (C) The Six1 repressive construct (Six1EnR, 400 pg) also reduces Irx1 PPR expression (red arrow). (D) The Six1 activating construct (Six1VP16, 400 pg) broadens the Irx1 PPR domain (red arrow), as indicated by the red bar (compare to control width, black bar). (E) Six1 and Six1 +Eya1 mRNAs reduce Irx1 expression in the otic vesicle. Black arrows denote otic vesicle on control side, and red arrows denote it on injected side of same embryo. (F) The percentage of embryos that show the same phenotype as in (E) when injected with Six1, Six1 +Eya1 or Six1EnR mRNA. Numbers above bars indicate the sample size. There are no significant differences (p > 0.05) across the groups. (G) Six1 also reduces Sox9 and Pax2 expression in the otic vesicle. Black arrows denote otic vesicle on control side, and red arrows denote it on injected side of same embryo. A, C, D are anterior views, E, G are side views, all with dorsal to the top. |
|
Display additional annotations [+]
|
| |
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. |
