Figure 2. Otic vesicle expansion and early pillar morphology are mechanically coupled.

A-A’. Representative images of an OV at 54 hpf before and after ablation. B-B’. Magnified insets of the boxed regions in A-A’ showing relaxation of the pillar and decreased aspect ratio. C. Quantification of OV lumen volume before and after ablation at the three developmental stages indicated. P-values as labeled (two-tailed paired t-test). D. Quantification of pillar aspect ratio before and after ablation at the developmental stages indicated. Pillar measured at each staged ablation is indicated along the x-axis. P-values as labeled (two-tailed paired t-test). E-E’. Representative images of an OV before and 4 hours post-treatment (hpt) by soaking in DMSO. F-F’. Magnified insets of the boxed regions in E-E’ showing minimal change in pillar aspect ratio after DMSO treatment. G-G’. Representative images of an OV before and 4 hpt by soaking in Forskolin. H-H’. Magnified insets of the boxed regions in G-G’ showing elongation of the pillar and increase in aspect ratio after Forskolin treatment. I. Quantification of OV lumen volume before and 4 hpt in DMSO or Forskolin at the developmental stages indicated. J. Quantification of pillar aspect ratio before and 4 hpt in DMSO or Forskolin at the developmental stages indicated. Pillar measured at each staged treatment is indicated along the x-axis. K. Schematic of the fluid driven physical model of OV lumen and pillar dynamics. Symbols are defined to the right of the schematic, along with assumptions made in the model, and the equations of water and ion flux across boundaries. L. Computed values of pillar volume (blue, pL) and lumen volume (orange, pL) over time after simulated OV ablation. Scatter points indicate experimentally measured values. M. Computed values of effective pillar aspect ratio over time after simulated OV ablation. N. Computed phase diagram of change in lumen pressure (kPa) over a four-hour period as a function of lumen ion influx and effective epithelial viscosity. O. Computed values of lumen volume (pL) over time for wild type (WT, blue) and Fsk (red) conditions. P. Computed values of effective pillar aspect ratio over time defined as a function of lumen radius divided by pillar radius for WT (blue) and Fsk (red) conditions. Q. Computed phase diagram showing the change in aspect ratio (ΔAR) over a four-hour period as a function of lumen ion influx ($\mid {\tilde{J}}_{s2}\mid$) and epithelial viscosity ($\alpha \eta ∕R_{2,0}$). Here R₂₀ denotes the reference lumen radius used for nondimensionalization. R. Representative images of OVs in the staged time series used for the quantification in S. S. Quantification of pillar aspect ratio and OV lumen volume (pL) for embryos pooled at the following developmental stages: anterior pillars (pink) quantified at 54 hpf, posterior (green) pillars measured at 60 hpf, and ventral (blue) pillars measured at 72 hpf. Linear fit to the data shown as dashed line. T. Quantification of posterior pillar aspect ratio and OV lumen volume (pL) for control embryos from S (grey), before and after soaking with Forskolin (purple and magenta), and before and after OV ablation (green and orange). Linear fit to the control data shown as grey dashed line. Linear fit to all data shown as black dashed line. “n” denotes number of embryos. Scale bar values indicated in each image. P-values as labeled (two-tailed paired t-test).