(A) A ‘canyon’ template made of plexiglass with each canyon being 1.5 mm deep and 0.4 mm wide. (B) 1% agarose molds are cast using the template. Rows of zebrafish embryos from 16 to 45 hpf stages can be mounted in orientations that position the inner ear closest to the cover-slip for imaging. (C) Embryos are mounted in a dorsal orientation for imaging both ears simultaneously using a 20X, 1.0 NA objective. (D) Embryos are mounted in a dorsolateral orientation for obtaining higher-quality images (otic tissue depth along the path of light is minimized) using a 40X, 1.2 NA objective. (E) Transgenic zebrafish embryos expressing bright nuclear and membrane-specific fluorescent reporters are imaged using a confocal microscope in a time-lapse mode. Embryos are (Tg(actb2:Hsa.H2B-tdTomato)^hm25; Tg(actb2:mem-citrine)^hm26). (F) The acquired images typically cover a field-of-view of 200×200×80 µm. With a sampling of 0.2 × 0.2 × 1.0 µm, image dimensions are 1024 × 1024 × 80. In timelapse experiments, images are acquired every 2 min covering a developmental time-window ranging 12–45 hpf. (G–H) Diagram illustrating the standard (G) dorsal and (H) transverse views of the otic vesicle organ as indicated by the embryonic coordinate axis. The otic vesicle (orange) is situated in between the hindbrain and ectoderm tissues. The locations of the medial, lateral, and pole cell regions is as shown. (I–L) In toto images can be automatically processed using membrane and nuclei segmentation algorithms for estimating cell numbers, cell shapes, and protein expression at a single-cell resolution. Using GoFigure2, an open-source software for visualization, editing, and integrative analysis of in toto data, the segmentation meshes and tracks can be visualized along orthogonal image planes as shown in (I) XY, (J) YZ, (K) XZ, and as (L) triplanar 3D view. (M) Organ size and shape can be semi-automatically or manually processed by placing 2D contours along image planes in the GoFigure2 software platform. The software reconstructs surfaces from contours and computes volumes and surface areas. (N–O) Principal diameters of the lumenal cavity (N, 2⁢Rl) and otic vesicle (O, 2⁢Ro) quantified during growth show the vesicle is elongated along the anteroposterior axis while being approximately symmetric about left-right and dorsoventral axes. The principal diameters monotonically increase during growth and remain correlated. n = 10 embryos per data point. Error bars are SD.

(A) A ‘canyon’ template made of plexiglass with each canyon being 1.5 mm deep and 0.4 mm wide. (B) 1% agarose molds are cast using the template. Rows of zebrafish embryos from 16 to 45 hpf stages can be mounted in orientations that position the inner ear closest to the cover-slip for imaging. (C) Embryos are mounted in a dorsal orientation for imaging both ears simultaneously using a 20X, 1.0 NA objective. (D) Embryos are mounted in a dorsolateral orientation for obtaining higher-quality images (otic tissue depth along the path of light is minimized) using a 40X, 1.2 NA objective. (E) Transgenic zebrafish embryos expressing bright nuclear and membrane-specific fluorescent reporters are imaged using a confocal microscope in a time-lapse mode. Embryos are (Tg(actb2:Hsa.H2B-tdTomato)^hm25; Tg(actb2:mem-citrine)^hm26). (F) The acquired images typically cover a field-of-view of 200×200×80 µm. With a sampling of 0.2 × 0.2 × 1.0 µm, image dimensions are 1024 × 1024 × 80. In timelapse experiments, images are acquired every 2 min covering a developmental time-window ranging 12–45 hpf. (G–H) Diagram illustrating the standard (G) dorsal and (H) transverse views of the otic vesicle organ as indicated by the embryonic coordinate axis. The otic vesicle (orange) is situated in between the hindbrain and ectoderm tissues. The locations of the medial, lateral, and pole cell regions is as shown. (I–L) In toto images can be automatically processed using membrane and nuclei segmentation algorithms for estimating cell numbers, cell shapes, and protein expression at a single-cell resolution. Using GoFigure2, an open-source software for visualization, editing, and integrative analysis of in toto data, the segmentation meshes and tracks can be visualized along orthogonal image planes as shown in (I) XY, (J) YZ, (K) XZ, and as (L) triplanar 3D view. (M) Organ size and shape can be semi-automatically or manually processed by placing 2D contours along image planes in the GoFigure2 software platform. The software reconstructs surfaces from contours and computes volumes and surface areas. (N–O) Principal diameters of the lumenal cavity (N, 2⁢Rl) and otic vesicle (O, 2⁢Ro) quantified during growth show the vesicle is elongated along the anteroposterior axis while being approximately symmetric about left-right and dorsoventral axes. The principal diameters monotonically increase during growth and remain correlated. n = 10 embryos per data point. Error bars are SD.

(A) Diagram illustrating inhibition of mitotic rounding just prior to cytokinesis from lumenal pressure and reactionary support from hindbrain tissue (hb, grey). (B) Diagram illustrating the deformation of the adjacent hindbrain tissue (hb, grey) as the otic vesicle grows from internal pressure. (C–F) 2D confocal micrographs of the otic vesicle at (C) 16 hpf, (D) 24 hpf, (E) 28 hpf, and (F) 32 hpf highlighting the progressive deformation of adjacent hindbrain and ectoderm tissues relative to the dashed-green line. The red and blue arrow heads highlight the progressive deformation in the shape of mitotic cells at contact and non-contact regions, respectively. (G) Quantification of mitotic cell aspect ratios at contact regions (hindbrain-vesicle or ectoderm-vesicle interface, blue markers) and other non-contact regions (anterioposterior poles, red markers, n = 54 mitotic cells total, 5–10 embryos per timepoint, each embryo provided 0–2 mitotic events such that each datapoint represent 4–5 mitotic events, *p<1.0e-4 at 22 hpf and *p<1.0e-5 at 27 hpf, as determined by student t-test (unpaired)). Aspect ratio is measured as the ratio of apico-basal to lateral cell radii. (H) Distribution of division plane orientation relative to the lumenal surface-normal at contact and non-contact cell populations. (I) Distribution of division plane orientation for all cells across three stages 16–25, 25–35, and 35–45 hpf respectively. (J) Quantification of hindbrain deformation measured as the peak indentation depth (relative to the dashed green line segment in C-F). n = 10 embryos per data point. Error bars are SD.

(A) The fabricated pressure probe was calibrated with hydrostatic pressure by reducing the submersion depth stepwise (inset, 1mmH₂O = 9.8 Pa), which demonstrated good linearity. (B) The pressure probes were calibrated against combinations of the capillary inner diameter and the ionic composition of the bath medium. The probes were displaced stepwise (1.27mmH₂O = 12.45 Pa, dotted line) and the histogram of pressure changes is shown. No dependence on the configurations is observed. (C–D) Calculations of membrane deformation on the piezoresistive sensing element. Colors represent different membrane thickness. (C) The numerical results are compared against analytical solutions for the deformation at the membrane center. (Inset) A selected solution is shown for a 5 µm membrane under 250 Pa pressure. The corresponding volume changes are shown in (D) and the volume of a 200 µm diameter sphere is plotted in dotted line as a reference. (E) Calculations of diffusive mixing between endolymph and capillary filling after puncturing. Their initial ionic concentrations are C₀ and 0, respectively. The mean ionic concentration inside the vesicle C decreases over time t at a rate depending on the capillary inner diameter d. (Inset) Selected solutions are shown for d = 5, 15 µm in the first 5 min. (F) Stages in an otic vesicle pressure measurement. Upper: zooming into the first 1.5 min. I: the tip was placed near the vesicle. The hydrostatic pressure was used as the baseline. II: after puncturing, the pressure built up gradually. III: after reaching a plateau, the pressure fluctuated around a mean value. This value was taken as the measurement result. IV: Upon withdrawing the tip from the vesicle, the pressure dropped to the base line, proving that the probe had been sensing the hydrostatic pressure in the enclosed domain.

(A) The fabricated pressure probe was calibrated with hydrostatic pressure by reducing the submersion depth stepwise (inset, 1mmH₂O = 9.8 Pa), which demonstrated good linearity. (B) The pressure probes were calibrated against combinations of the capillary inner diameter and the ionic composition of the bath medium. The probes were displaced stepwise (1.27mmH₂O = 12.45 Pa, dotted line) and the histogram of pressure changes is shown. No dependence on the configurations is observed. (C–D) Calculations of membrane deformation on the piezoresistive sensing element. Colors represent different membrane thickness. (C) The numerical results are compared against analytical solutions for the deformation at the membrane center. (Inset) A selected solution is shown for a 5 µm membrane under 250 Pa pressure. The corresponding volume changes are shown in (D) and the volume of a 200 µm diameter sphere is plotted in dotted line as a reference. (E) Calculations of diffusive mixing between endolymph and capillary filling after puncturing. Their initial ionic concentrations are C₀ and 0, respectively. The mean ionic concentration inside the vesicle C decreases over time t at a rate depending on the capillary inner diameter d. (Inset) Selected solutions are shown for d = 5, 15 µm in the first 5 min. (F) Stages in an otic vesicle pressure measurement. Upper: zooming into the first 1.5 min. I: the tip was placed near the vesicle. The hydrostatic pressure was used as the baseline. II: after puncturing, the pressure built up gradually. III: after reaching a plateau, the pressure fluctuated around a mean value. This value was taken as the measurement result. IV: Upon withdrawing the tip from the vesicle, the pressure dropped to the base line, proving that the probe had been sensing the hydrostatic pressure in the enclosed domain.

(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.

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 (ΔVlVl). (C–D) Lumenal fluid flux Ω in punctured (blue) and unpunctured (red) vesicles during regeneration showing the increase in Ω in punctured embryos and gradual decay to the unpunctured levels. (E–F) Plot showing Ω as a function of Δ⁢VlVl. The data was pooled across multiple embryos for the punctured (blue) and unpunctured (red) ears throughout their regeneration period until 45 hpf. Ω in punctured ears (blue) is positively correlated with Δ⁢VlVl while unpunctured ears (red) continued growing with an approximately constant flux.

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 (ΔVlVl). (C–D) Lumenal fluid flux Ω in punctured (blue) and unpunctured (red) vesicles during regeneration showing the increase in Ω in punctured embryos and gradual decay to the unpunctured levels. (E–F) Plot showing Ω as a function of Δ⁢VlVl. The data was pooled across multiple embryos for the punctured (blue) and unpunctured (red) ears throughout their regeneration period until 45 hpf. Ω in punctured ears (blue) is positively correlated with Δ⁢VlVl while unpunctured ears (red) continued growing with an approximately constant flux.

2D+time confocal movie showing growth and regeneration inhibition from ouabain treatment. Labelling by (Tg(actb2:Hsa.H2B-tdTomato)^hm25; Tg(actb2:mem-citrine)^hm26) from 30 to 34 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 embryo was soaked in 100μM ouabain to inhibit Na⁺-K⁺-ATPase. 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. Throughout the imaging session, regular growth in the left ear and regeneration of the right ear are completely inhibited.

(A) Pole cells are tracked from 25 hpf (left) to 30 hpf (right) as they treadmill from high-curvature to low-curvature (dashed blue curves) tissue regions. Mean curvature of the lumenal surface is used as a measure of the local curvature. (B) Quantification of the cell aspect ratios and mean curvature shows that cells retain their shapes independent of the location on the lumenal surface (n = 10, **p<5.0e-5). Error bars are SD. (C) For the measurement of single-cell data on shape deformations in Figure 6I, mosaically labeled cells were tracked in Tg(actb2:GFP-Hsa.UTRN) before and after puncture. Dextran TexasRed was injected into a single blastomere of 16 cell stage embryos for tracking positions of cells before and after puncture. Confocal 3D image datasets were collected before and after puncture and co-registered. The confocal micrographs show XY, YZ, XZ, and a 3D triplanar view centered on the otic vesicle of a single embryo staged at 30 hpf before (top) and after puncture (bottom). Cells positive for TexasRed fluorescence (examples shown in red, blue, and white arrows) were matched before and after puncture. For each cell, the deformation and fluorescence localization were manually measured using the GoFigure2 software.

(A) Pole cells are tracked from 25 hpf (left) to 30 hpf (right) as they treadmill from high-curvature to low-curvature (dashed blue curves) tissue regions. Mean curvature of the lumenal surface is used as a measure of the local curvature. (B) Quantification of the cell aspect ratios and mean curvature shows that cells retain their shapes independent of the location on the lumenal surface (n = 10, **p<5.0e-5). Error bars are SD. (C) For the measurement of single-cell data on shape deformations in Figure 6I, mosaically labeled cells were tracked in Tg(actb2:GFP-Hsa.UTRN) before and after puncture. Dextran TexasRed was injected into a single blastomere of 16 cell stage embryos for tracking positions of cells before and after puncture. Confocal 3D image datasets were collected before and after puncture and co-registered. The confocal micrographs show XY, YZ, XZ, and a 3D triplanar view centered on the otic vesicle of a single embryo staged at 30 hpf before (top) and after puncture (bottom). Cells positive for TexasRed fluorescence (examples shown in red, blue, and white arrows) were matched before and after puncture. For each cell, the deformation and fluorescence localization were manually measured using the GoFigure2 software.