Figure 4. Pressure negatively regulates fluid flux.

(A) Numerical calculation of fluid flux ($\mathrm{\Omega }$) as a function of time using Equation 6 by fitting quadratic polynomials to volume and surface area data. (B–D) Confocal 2D micrographs with XZ (top) and XY (bottom) planes depicting the regeneration of a punctured right vesicle (blue) relative to the unpunctured vesicle (left) from (A) 30 hpf right after puncture, to (B) 32.5 hpf, and to (C) 35 hpf. (E–F) Quantification of the recovery of volume and wall thickness symmetry. The y-axis plots the difference in lumenal volumes normalized to the unpunctured lumenal volume ($\frac{\mathrm{\Delta }{V}_l}{V_l}$, E) and similarly for wall thickness ($\frac{\mathrm{\Delta }h}{h}$, F). Error bars are SD. (G) Fluid flux $\mathrm{\Omega }$ in the punctured ears (blue) and unpunctured ears (red). Error bars are SD. (H) Scatterplot showing $\mathrm{\Omega }$ as a function of volume asymmetry ($\frac{\mathrm{\Delta }{V}_l}{V_l}$) in punctured (blue) and unpunctured (red) ears. n = 10 for each data point in (E–H) Related to Figure 4—figure supplement 1 and Figure 4—video 1.

Figure 4—figure supplement 1. The otic vesicle regenerates to stage-specific volumes when punctured between 25–45 hpf.

In addition to experiments at 30 hpf reported in Figure 4, puncturing was conducted at 25 hpf (A, C, E) and 35 hpf (B, D, F). (A–B) Volume regeneration and establishment of contralateral symmetry after unilateral puncturing. The y-axis plots the difference in lumenal volumes normalized to the unpunctured volume (${\displaystyle \frac{\mathrm{\Delta }{V}_l}{V_l}}$). (C–D) Lumenal fluid flux $\mathrm{\Omega }$ in punctured (blue) and unpunctured (red) vesicles during regeneration showing the increase in $\mathrm{\Omega }$ in punctured embryos and gradual decay to the unpunctured levels. (E–F) Plot showing $\mathrm{\Omega }$ as a function of $\frac{\mathrm{\Delta }{V}_l}{V_l}$. The data was pooled across multiple embryos for the punctured (blue) and unpunctured (red) ears throughout their regeneration period until 45 hpf. $\mathrm{\Omega }$ in punctured ears (blue) is positively correlated with $\frac{\mathrm{\Delta }{V}_l}{V_l}$ while unpunctured ears (red) continued growing with an approximately constant flux.

Figure 4—video 1. Otic vesicle catch-up growth after puncture.

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DOI: 10.7554/eLife.39596.011

2D+time confocal movie of zebrafish otic vesicle regeneration from puncture. Labelling by (Tg(actb2:Hsa.H2B-tdTomato)^hm25; Tg(actb2:mem-citrine)^hm26) from 27 to 33 hpf. Each frame has a spatial-sampling of 0.4 µm along X-Y axis, 1.0 μm along the Z-axis, and with temporal-sampling of 5 min across frames. The movie shows the transverse (XZ) and saggital (XY) views. The right vesicle of the embryo was punctured prior to imaging. At the start of the imaging session, the right ear is significantly smaller that the left ear. The movie shows the rapid regeneration of the right vesicle to a developmentally appropriate size, as depicted by the left ear (control). At the end of the imaging session, both the ears are similar in size.