Figure 3. Otic vesicle expansion canalizes variability in pillar morphology.

A. Representative confocal images of 3D-rendered posterior buds at contact from membrane mNeonGreen-expressing embryos at 54 hpf, demonstrating initial variability in bud contact angle. B. Representative confocal images of 3D-rendered posterior pillars from membrane-mNeonGreen-expressing embryos at the stages indicated, demonstrating straightening of posterior pillars after initial shape variability. C. Schematic defining pillar angle metric used to describe pillar straightness quantitatively. D. Scatter plot of canalization of posterior bud contact or pillar angle (degrees) vs otic vesicle lumen volume (pL) quantified in embryos pooled at the following developmental stages: 54 hpf (green, bud contact), 60 hpf (orange, pillars), 66 hpf (blue, pillars), and 72 hpf (pink, pillars). E. Plot of coefficient of variation calculated from embryos in D binned by lumen volume in 90-pL bins. F. Representative confocal images of 3D-rendered posterior pillars from membrane-mNeonGreen-expressing embryos at the stages indicated, before and 1 hour post treatment (hpt) with DMSO as a control or Forskolin in DMSO. G. Quantification of OV lumen volume (pL) before and 1 hpt in DMSO or Forskolin with DMSO at 60 hpf. P-values as labeled (two-tailed unpaired t-test). H. Quantification of posterior pillar angle (degrees) before and 1 hpt in DMSO or Forskolin with DMSO at 60 hpf. P-values as labeled (two-tailed unpaired t-test). I. Representative confocal images of 3D-rendered OVs from membrane-mNeonGreen-expressing embryos at the stages indicated, before and after 2-photon laser ablation of the OV to reduce lumen volume and pressure. J. Quantification of OV lumen volume (pL) before and after 2-photon laser ablation of the OV at 60 hpf. P-values as labeled (two-tailed paired t-test). K. Quantification of posterior pillar angle (degrees) before and after 2-photon laser ablation of the OV at 60 hpf. P-values as labeled (two-tailed paired t-test). L. Plot of change in anterior pillar angle (degrees, A_n-A₀) scaled to initial potential for pillar straightening at pillar formation (180°-A₀) vs change in OV lumen volume (pL, V_n-V₀) scaled to initial OV lumen volume at pillar formation (V₀); data pooled from all timepoints (n=42) quantified from 5 embryos that were timelapse imaged from pillar formation to pillar straightening (colored by embryo). Spearman coefficient of pooled data as shown (0.758). M. Plot of scaled change in pillar angle (degrees) vs scaled change in OV lumen volume (pL); data pooled from all timepoints quantified from 5 time-lapsed embryos (anterior pillar in wild type, WT, grey) and for perturbed conditions (anterior pillar from 54 hpf ablation, blue, posterior pillar from 60hpf Ablation, pink, posterior pillar from 60 hpf Forskolin, orange). Change quantified from before and after perturbation. Spearman coefficient of pooled data as shown (0.909). Coefficient of variation (cv) indicated in certain graphs. Scale bar values indicated in each image. “n” denotes number of embryos.