Figure 3—figure supplement 2.

Figure 3—figure supplement 2.

(A) Embryos are mounted in a canyon mount made with 1% agarose for confocal imaging of left and right vesicles with a 20X objective. Drugs or dyes are injected into the cardiac chamber and get distributed rapidly throughout the embryo including the perilymph surrounding the ear. (B) Schematic showing the route of lumenal fluid loss upon puncture and movement of dye from perilymph into the lumen. (C) 2.3 nl of 0.5 mM Alexa Fluor 594 tracer dye injected into the cardiac chamber at 30 hpf. The dye enters the circulation and is found to accumulate in the perilymph regions surrounding the vesicle. Puncturing of otic vesicle causes the dye to leak into the lumenal cavity. (D) Injection of dye five minutes post-puncture restricts dye transport to the perilymph, thus demonstrating the rapid sealing property of the otic tissue. (E) 2D confocal micrographs showing ear pairs in embryos with unilateral puncture of the right ear and staged at 25, 30, and 35 hpf. Cell shape change in unpunctured and punctured ears are highlighted using blue and red arrows respectively. Progressively through time, puncturing causes smaller transitions in cell shapes between ear pairs.