Zarei S , Zarei K , Fritzsch B , Elliott KL .

R03 DC015333 NIDCD NIH HHS , T35 HL007485 NHLBI NIH HHS

             dendrite morphogenesis
             inner ear development

Xla Wt + Vismodegib (Figure 1 A^2 A^3)
Xla Wt + Vismodegib (Figure 1 A^3)
Xla Wt + Vismodegib (Figure 1 B^1" B^3)
Xla Wt + Vismodegib (Figure 1 B^1" B^3)
Xla Wt + Vismodegib (Figure 1 C" C"^1)
Xla Wt + Vismodegib (Figure 1 C" C"^1)
Xla Wt + Vismodegib (Figure 1 D" D"^1)
Xla Wt + Vismodegib (Figure 2 A B C)
Xla Wt + Vismodegib (Figure 3)
Xla Wt + Vismodegib (Figure 4 A)
Xla Wt + Vismodegib (Figure 4 C)
Xla Wt + Vismodegib (Figure 5 D)
Xla Wt + Vismodegib (Figure 5 E)
Xla Wt + Vismodegib (Figure 5 F)
Xla Wt + Vismodegib (Figure 6)
Xla Wt + Vismodegib (Figure 6 C D)

	Figure 1. Varied concentrations of Vismodegib result in a dose-dependent effect on phenotypic changes. A, Control tadpoles display two distinct olfactory groves (A′). However, increasing Vismodegib concentration results in an increasing tendency toward complete fusion of the two groves (A′-A′′). Animals treated with 6.25 µM Vismodegib exhibited partially fused olfactory epithelia (A′). By 18.75 µM of Vismodegib, all animals exhibited complete fusion of the olfactory epithelia (A′′). B, Eye pigmentation was also affected following treatment with Vismodegib. Compared with control animals, which exhibited full eye pigmentation (B′), animals treated with 6.25 µM Vismodegib began to lose eye pigmentation in the ventral part of the eye (B′). This loss of ventral eye pigmentation was more profound in animals treated with 12.5 µM Vismodegib. About half of animals treated with 18.75 µM had pigment loss in half of the ear [sic- eye] (B′′). In a couple animals treated with 18.75 µM and about one third of animals treated with 21.875 µM Vismodegib had very little eye pigment remaining (B′′). C, Tail development was variably affected as a result of reduced Shh signaling. Animals treated with 12.5, 18.75, and 21.875 Vismodegib exhibited a kink at the caudal portion of their tails (C′) compared with the straight tail of control animals (C′). At 6.25 µM Vismodegib, fewer animals exhibited kinked tails. D, Aberrations in otoconia development were also evident with Vismodegib treatment. Control tadpoles had two distinct otoconia (D′). Some animals treated with 6.25 µM Vismodegib, most animals treated with 12.5 µM Vismodegib, and all animals treated with 18.75 µM Vismodegib had a single otoconia mass (D′). A couple animals treated with 21.875 µM lacked otoconia formation altogether (D′′), whereas the rest had one otoconia mass (D′). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 2.Reduction in ear length along both the anteroposterior and mediolateral axes in Vismodegib treated tadpoles. A, The reduction in ear length along anteroposterior, mediolateral, and dorsoventral axes followed a linear dose response curve. This decrease in ear size was more profound along the anteroposterior dimension of the ear compared with the mediolateral or dorsoventral dimension of the ear. While ears from animals treated with 12.5 µM and greater doses of Vismodegib were significantly reduced in length along the anteroposterior axis from controls, only 18.75 and 21.875 µM Vismodegib-treated animals were significantly reduced along the mediolateral axis from controls. The reduction in length along the anteroposterior axis at the highest dose of Vismodegib was ∼2-fold, whereas along the mediolateral axis, it was only ∼1.3-fold. B, Illustration of lengths measured along the anteroposterior axis and mediolateral axis in the ear of a stage 46 control tadpole. C, Illustration of lengths measured along the anteroposterior axis and mediolateral axis in the ear of a stage 46 tadpole treated with 21.875 µM Vismodegib. Scale bars represent 100 μm. D, The reduction in approximate volume as calculated from the anteroposterior, mediolateral, and dorsoventral lengths from each ear followed a linear dose response curve. The volumes of animals treated with 18.75 µM and 21.875 µM Vismodegib were significantly reduced to ∼1/4th the volume of controls. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 (ANOVA). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 3. Effect of Vismodegib on inner ear sensory epithelia development. A, Animals treated with Vismodegib exhibited a dose-dependent reduction in sensory epithelia development. B, Immunohistochemistry using antibodies against Tubulin (green) and MyoVI (red) to label neurons and hair cells, respectively. Shown here is a control ear. C, 3D reconstruction of hair cells shows that in control animals, there are six distinct sensory epithelia. D,D′, 3D reconstruction of hair cells from an animal treated with 6.25 µM Vismodegib showing four distinct sensory epithelia. E, 3D reconstruction of hair cells from an animal treated with 12.5 µM Vismodegib showing four distinct sensory epithelia. F, 3D reconstruction of hair cells from an animal treated with 18.75 µM Vismodegib showing four distinct sensory epithelia. Sensory epithelial patches were sometimes connected by a few hair cells in a “bridge” (arrow). G-G′, 3D reconstruction of hair cells from an animal treated with 21.875 µM Vismodegib showing three distinct epithelia. In D′ and G′ Hoechst staining was used to 3D reconstruct the inner boundary of the ears, shown in blue. Note the reduction in distinct epithelia (compare outlines in D′,G′) exceeds the reduction in hair cells. Ac, anterior canal crista; Hc, horizontal canal crista; Pc, posterior canal crista; U, utricle; S, saccule; L, lagena. Scale bars represent 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 4. Rostral expansion of hypaxial muscle fibers following Vismodegib treatment. A, The distance from the ear midline from the rostral boundary of the somites was calculated from animals immunostained with antibodies against tubulin (green), MyoVI (red) and counterstained with Hoechsts to label neurons, hair cells and muscle tissue, and nuclei, respectively. A line was drawn through the midline of the ear (dotted line), calculated from the anteroposterior diameter of the ear. From that line, the distance to the rostral boundary of the somites was determined (T-shaped line). Positive values were assigned for distances rostral to the midline and negative values for distances caudal to the midline. B, Means plus or minus standard errors of the mean for both controls and animals treated with 6.25 µM Vismodegib calculated using the method from (A). ****p < 0.0001 C, Coronal section showing that hypaxial muscle fibers (outlined and arrows) expanded between the brain and the ear in animals treated with 6.25 µM Vismodegib. Scale bars represent 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5. Effect of Vismodegib treatment on Mauthner Cell Development. A, 3A10 antibody labeling of a pair of Mauthner cells from a control Xenopus. B, 3A10 antibody labeling of a pair of Mauthner cells from an animal treated with 6.25 µM Vismodegib. Arrowheads for A and B indicate axon crossing at the midline. C, 3D reconstruction of a control Mauthner cell from dextran amine labeling. D, 3D reconstruction of a Mauthner cell from an animal treated with 6.25 µM Vismodegib. E, Shh inhibition results in a significant reduction in the degree of branching of the Mauthner cell in 6.25 µM Vismodegib-treated animals (n = 4) compared with control animals (n = 4). F, Shh inhibition results in an approximately two-fold reduction in axonal diameter of the Mauthner cell in 6.25 µM Vismodegib-treated animals (n = 3) compared with control animals (n = 5). Scale bars represent 100 µm.* p < 0.05, ****p < 0.0001. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6. Impact of Vismodegib treatement on tadpole swimming behavior. A, Schematic of imaging apparatus utilized for recording tadpole swimming behavior and the C-start response following a controlled perturbation, as described by Zarei et al. (2017). B and C, Images of the first 25 frames captured following initiation of the stimulus for both control animals (B) and animals treated with 6.25 µM Vismodegib (C). Control animals on average took 9 frames to elicit the C-start response (purple circle), whereas animals treated with 6.25 µM Vismodegib took 11 frames (green circle), based on the criteria described in Zarei et al. (2017). D, The time to elicit a C-start response was significantly longer in animals treated with 6.25 µM Vismodegib than in control animals. * p < 0.05. E, Although not significant, the maximal flexion in animals treated with 6.25 µM Vismodegib was, on average, tighter than that of control animals, resulting in an atypical U-shaped configuration instead of the normal C-shaped configuration. [Color figure can be viewed at wileyonlinelibrary.com]
	Xenopus inner ear sensory epithelia. B (left): Immunohistochemistry using antibodies against Tubulin (green) label neurons and MyoVI (red) label auditory hair cells. Ac, anterior canal crista; Hc, horizontal [=lateral] canal crista; Pc, posterior canal crista; U, utricle; S, sacculus; L, lagena. In C (right), a 3D reconstruction of hair cells shows that there are six distinct sensory epithelia zones. Scale bars represent 100 μm.
	Neuronal Ab4 (aka 3A10) labeling of a pair of Mauthner cells ( M, arrow head) from the inner ear of Xenopus.

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