Figure 1—figure supplement 1.

Figure 1—figure supplement 1.

(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.