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. 2019 Oct 1;8:e39596. doi: 10.7554/eLife.39596

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© 2019, Mosaliganti et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 6. — (A) Schematic for experimentally measuring tissue material properties ( $k$ , $μ$ ). The strain of a cell (highlighted in orange) before and after puncturing $(c - a)$ is inversely proportional to the elasticity modulus k. Changes in resting cell-shapes observed over time ( $d - b$ ) is inversely proportional to the viscosity parameter µ. (B–C) Quantification of normalized change in wall thickness ( $(c - a) / a$ , (B) and resting wall thickness ( $(c - d) / c$ , (C) post-puncture near the hindbrain (medial, blue), ectoderm (lateral, red), and anteroposterior regions (poles, green). Puncturing was done at 25, 30, 35, and 40 hpf. n = 5 for each data point in (B–C). (D) Overall lumenal surface area growth rate (blue markers) showing compensatory contributions from proliferation (red) and cell stretching (green). (E–F) Timelapse confocal imaging using Tg(actb2:GFP-Hsa.UTRN) and Tg(actb2:myl12.1-eGFP) embryos report the dramatic apical localization of F-actin (D) and Myosin II (E) respectively prior to lumenization through 12-16hpf. Through early growth between 16–22 hpf, cells at the poles and lateral regions (red arrows) retain their fluorescence while medial cells lose their fluorescence (blue arrows). (G–H) 3D rendering of F-actin (G) and myosin II (H, right) data at 30 hpf show co-localization to apicolateral cell junctions as cells stretch out. (I) Quantification of long-term cell shape deformation ( $\frac{Δ h}{h}$ ) between 16–22 hpf as a function of the rate of change in apical concentration ( $\frac{Δ u}{u}$ ) of F-actin (blue markers) and Myosin II (red). n = 22. (J) Quantification of the short-term puncture-induced deformation in cell shapes ( $\frac{Δ h}{h}$ ) as a function of the normalized apical concentration ( $\frac{u}{< u >}$ ) of F-actin (blue markers) and Myosin II (red). < u > represents the mean apical fluorescent intensity across the vesicle. Error bars are SD, n = 22. (K) Quantification of fluid flux in embryos treated with 2 mM cytochalasin D at different developmental stages (hpf). Before 25 hpf, embryos failed to grow ( $Ω \approx 0$ ) or lose lumenal volume. After 25 hpf, embryos increased their secretion rate by 2-5X over wild-type values (dashed black line, 1 µm/hr, n = 15). (L) Quantification showing the change in apical Myosin II fluorescence ( $\frac{Δ u}{u}$ ) as positively correlated with fluid flux ( $Ω$ , n = 16). (M) Quantification of vesicle shape change show maximal change in dorsoventral radius (green markers) compared to the mediolateral (blue) and anteroposterior radius (red, n = 12). Related to Figure 6—figure supplement 1 and Figure 6—videos 1–4.

Figure 6—figure supplement 1. — (A) Schematic for experimentally measuring tissue material properties ( $k$ , $μ$ ). The strain of a cell (highlighted in orange) before and after puncturing $(c - a)$ is inversely proportional to the elasticity modulus k. Changes in resting cell-shapes observed over time ( $d - b$ ) is inversely proportional to the viscosity parameter µ. (B–C) Quantification of normalized change in wall thickness ( $(c - a) / a$ , (B) and resting wall thickness ( $(c - d) / c$ , (C) post-puncture near the hindbrain (medial, blue), ectoderm (lateral, red), and anteroposterior regions (poles, green). Puncturing was done at 25, 30, 35, and 40 hpf. n = 5 for each data point in (B–C). (D) Overall lumenal surface area growth rate (blue markers) showing compensatory contributions from proliferation (red) and cell stretching (green). (E–F) Timelapse confocal imaging using Tg(actb2:GFP-Hsa.UTRN) and Tg(actb2:myl12.1-eGFP) embryos report the dramatic apical localization of F-actin (D) and Myosin II (E) respectively prior to lumenization through 12-16hpf. Through early growth between 16–22 hpf, cells at the poles and lateral regions (red arrows) retain their fluorescence while medial cells lose their fluorescence (blue arrows). (G–H) 3D rendering of F-actin (G) and myosin II (H, right) data at 30 hpf show co-localization to apicolateral cell junctions as cells stretch out. (I) Quantification of long-term cell shape deformation ( $\frac{Δ h}{h}$ ) between 16–22 hpf as a function of the rate of change in apical concentration ( $\frac{Δ u}{u}$ ) of F-actin (blue markers) and Myosin II (red). n = 22. (J) Quantification of the short-term puncture-induced deformation in cell shapes ( $\frac{Δ h}{h}$ ) as a function of the normalized apical concentration ( $\frac{u}{< u >}$ ) of F-actin (blue markers) and Myosin II (red). < u > represents the mean apical fluorescent intensity across the vesicle. Error bars are SD, n = 22. (K) Quantification of fluid flux in embryos treated with 2 mM cytochalasin D at different developmental stages (hpf). Before 25 hpf, embryos failed to grow ( $Ω \approx 0$ ) or lose lumenal volume. After 25 hpf, embryos increased their secretion rate by 2-5X over wild-type values (dashed black line, 1 µm/hr, n = 15). (L) Quantification showing the change in apical Myosin II fluorescence ( $\frac{Δ u}{u}$ ) as positively correlated with fluid flux ( $Ω$ , n = 16). (M) Quantification of vesicle shape change show maximal change in dorsoventral radius (green markers) compared to the mediolateral (blue) and anteroposterior radius (red, n = 12). Related to Figure 6—figure supplement 1 and Figure 6—videos 1–4.