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

Figure 4. Quantification of frequency and duration of Rock and Roll among hair cells (HCs) in wild-type (WT) and emx2 mutants.

(A) In WT, a HC precursor (yellow dot) divides to form two daughter HCs (pink and blue dots), which first rock (Rock phase) and then frequently roll to exchange positions with each other (Roll phase). (A’) Definition of the Rock and Roll phase (see Materials and methods). (B and C) Compared to WT, emx2 loss of function (LOF) (B) and gain of function (GOF) (C) HC pairs took a shorter time in Rock and Roll and a longer time in the Roll phase, respectively. (D) Frequencies of Roll, No Roll, and Roll back of nascent HC pairs in WT (n = 67 from seven larvae), Emx2 LOF (n = 41 from six larvae), and GOF (n = 59 from seven larvae). Significance was assessed by using chi-squared test with 3 × 3 contingency table (X2 (df = 4)=30.00, p<0.0001, source data 1). A post-hoc 2 × 3 chi-squared test was performed for multiple comparisons. WT vs. LOF; (X2 (df = 2)=7.95, p=0.018), WT vs. GOF; (X2 (df = 2)=10.39, p=0.0055). Post-hoc chi-squared tests were performed for pairwise comparisons with FDR correction. (E–G) Duration of Rock (E), Roll (F), and Rock and Roll (G) of sibling HCs that underwent Rock and Roll in WT (n = 42), emx2 LOF (n = 35), and GOF (n = 19) larvae. Significance was assessed by using MANOVA (Rock, F(df1 = 2, df2 = 93)=5.349, p=0.0063, Wilks’ λ = 0.897; Roll, F(df1 = 2, df2 = 93)=15.638, p<0.0001, Wilks’ λ = 0.748; Rock and Roll, F(df1 = 2, df2 = 93)=20.10, p<0.0001, Wilks’ λ = 0.698), with post-hoc Tukey’s test for pairwise comparisons. *p<0.05, **p<0.01, n.s., not significant.

Figure 4—source data 1. Multiple comparisons of Roll frequencies among wild-type (WT), emx2 loss of function (LOF), and emx2 gain of function (GOF) hair cells (HCs).
Figure 4—source data 2. Quantification of Rock and Roll movements in wild-type (WT), emx2 loss of function (LOF), and emx2 gain of function (GOF) hair cells (HCs).

Figure 4.

Figure 4—figure supplement 1. Localization of Emx2 expression in wild-type (WT), loss of function (LOF), and gain of function (GOF) neuromasts at 2.0 dpf.

Figure 4—figure supplement 1.

(A–C) Only hair cells (HCs) (B, Gfp) in the anterior neuromast of WT are positive for anti-Emx2 staining (C). Inset in B shows the bidirectional hair bundle orientation pattern. (D–F) No HCs (E) are positive for anti-Emx2 staining (F) in emx2 LOF neuromast and all hair bundles are oriented in P→A direction (E). (G–J) An emx2 GOF neuromast. (G) Merged image of Gfp (H), anti-Emx2 (I), and mCherry (J) expression. Anti-Emx2 staining only detected endogenous Emx2 in the anterior HCs (I), whereas exogenous Emx2 expression as indicated by mCherry expression is present in all HCs (J). All hair bundles are oriented in A→P direction and two immature HCs show the kinocilium located in the center (magenta arrows).
Figure 4—video 1. Time-lapse video of the wild-type (WT) (myo6b:actb1-Gfp) hair cell (HC) pair shown in Figure 4A.
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The HC precursor (yellow dot) divides to form two sibling HCs (pink and blue dots) that first rock and then roll to exchange positions with each other. Zero minute represents the beginning of Rock, immediately after cell division. Rock duration: 0–96 min, Roll duration: 102–186 min.
Figure 4—video 2. Time-lapse video of a hair cell (HC) pair in wild-type (WT) (myo6b:actb1-Gfp) neuromast.
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The HC precursor (yellow dot) divides to form two sibling HCs (pink and blue dots) that do not roll to exchange positions with each other. Zero minute represents the beginning of Rock immediately after cell division.
Figure 4—video 3. Time-lapse video of the emx2 loss of function (LOF);myo6b:actb1-Gfp hair cell (HC) pair shown in Figure 4B.
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The HC precursor (yellow dot) divides to form two sibling HCs (pink and blue dots) that rock briefly and then roll to exchange positions with each other. Zero minute represents the beginning of Rock, immediately after cell division. Rock duration: 0–66 min, Roll duration: 72–120 min.
Figure 4—video 4. Time-lapse video of the emx2 gain of function (GOF); myo6b:actb1-Gfp hair cell (HC) pair shown in Figure 4C.
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The HC precursor (yellow dot) divides to form two sibling HCs (pink and blue dots) that first rock and then roll to exchange positions with each other, which takes longer than the wild-type (WT) in Figure 4—video 1. Zero minute represents the beginning of Rock, immediately after cell division. Rock duration: 0–108 min, Roll duration: 114–222 min.