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. 2020 Dec 30;9:e60432. doi: 10.7554/eLife.60432

Figure 6. Emx2 affects apical protrusion formation in nascent hair cells (HCs).

(A–H) Time-lapse images of myo6b:YFP-tubulin (wild-type [WT]) transgenic line showing nascent HCs undergoing Rock and Roll. After precursor cell divides (A), sibling HCs form an apical protrusion within 30 min of the Rock phase (B–D, yellow arrowhead, n = 6). When they exchange positions at the Roll phase, they appear to be pivoted at the apex (E–H, yellow arrowheads). Animation of images is shown in respective panels (Rock phase: b–d, Roll phase: e–h). (I–Q) Maximum intensity projection images generated by selected slices along z-axis in WT (I–K, same sample of A–H, n = 3), emx2 loss of function [LOF] (L–N, n = 3) and emx2 gain of function (GOF) (O–Q, n = 3). (i–q) Three-dimensional surface rendering of selected time points of live-imaging of HC pairs (red) in WT (i–k, n = 3), emx2 LOF (l–n, n = 3), and emx2 GOF (o–q, n = 3) neuromasts. Mature HCs are labeled in green. (R) Increases of YFP-tubulin signal intensity at the apical region of nascent HCs of WT (n = 5), emx2 LOF (n = 5), and emx2 GOF (n = 6) during the Rock phase (90 min). The shaded area represents the standard error of the mean (SEM). (S) YFP-tubulin signal intensities of the apical protrusion at the end of Rock phase were significantly different among WT (n = 5), emx2 LOF (n = 5), and emx2 GOF HCs (n = 6, one-way ANOVA, F = 26.97, p<0.0001); post-hoc Dunnett’s multiple comparisons test for WT vs. LOF, p=0.0073, WT vs. GOF, p=0.0052. (T) Increases over time in the length of apical protrusion in WT (n = 5), emx2 LOF (n = 5), and emx2 GOF (n = 5) during the Rock phase (90 min). The shaded area represents the SEM. (U) The length of apical protrusion at the end of the Rock phase in WT (n = 5), emx2 LOF (n = 5), and emx2 GOF (n = 5) were compared. Significance was assessed by using one-way ANOVA, F = 52.15, p<0.0001. Post-hoc Dunnett’s multiple comparisons test for WT vs. LOF is not significant (p=0.148) but significant for WT vs. GOF (p<0.0001). The following figure supplement, source data, videos are available for Figure 6—figure supplement 1. Measurements of signal intensity and length of the apical protrusion using Image J Fiji.

Figure 6—source data 1. Quantification of changes in signal intensity of apical protrusion of hair cells (HCs) during the Rock Phase.
Figure 6—source data 2. Quantification of signal intensity of apical protrusion at the end of Rock phase among wild-type (WT), emx2 loss of function (LOF), and emx2 gain of function (GOF) hair cells (HCs).
Figure 6—source data 3. Quantification of changes in the length of apical protrusion during the Rock Phase.
Figure 6—source data 4. Quantification of the length of apical protrusion at the end of Rock phase among wild-type (WT), emx2 loss of function (LOF), and emx2 gain of function (GOF) hair cells (HCs).

Figure 6.

Figure 6—figure supplement 1. Measurements of signal intensity and length of the apical protrusion using ImageJ Fiji.

Figure 6—figure supplement 1.

(A) A selected maximum intensity projection image was converted into an 8-bit gray scale image. (B) The gray scale image in (A) was converted to Rainbow RBG color mode to visualize the bright green color of the accumulated YFP-tubulin within the apical protrusion. (C) Based on the signal intensity, the region of interest (ROI, yellow outline) was automatically defined by using the wand selection tool of ImageJ Fiji and the intensity measured. (D) The ROI was converted into a mask image (white area). A blue line was drawn across the top of the two hair cell (HC) nuclei, and a pink line joining the tip of the apical protrusion to the blue line at right angle indicates the length of the apical protrusion.
Figure 6—video 1. Time-lapse video of the myo6b:yfp-tubulin hair cell (HC) pair in Figure 6A–H.
Download video file (481.3KB, mp4)
The nascent HC pair (pink and blue dots) extends a protrusion to the apex and the tip of the protrusion is pivoted at the apical surface of the neuromast. The yellow dot indicates the HC precursor.