Forristall CA et al. (2014), Embryological manipulations in the developing X...

XB-ART-48509

Dev Dyn 2014 Oct 01;24310:1262-74. doi: 10.1002/dvdy.24116.

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Embryological manipulations in the developing Xenopus inner ear reveal an intrinsic role for Wnt signaling in dorsal-ventral patterning.

Forristall CA , Stellabotte F , Castillo A , Collazo A .

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The inner ear develops from an ectodermal thickening known as the otic placode into a complex structure that is asymmetrical along both the anterior-posterior (A-P) and dorsal-ventral (D-V) axes. Embryological manipulations in Xenopus allow us to test regenerative potential along specific axes and timing of axis determination. We explore the role of Wnt signaling with gain and loss of function experiments. In contrast to A or P half ablations, D or V half ablations almost never result in mirror duplications or normal ears. Instead there is a loss of structures, especially those associated with the ablated region. Rotation experiments inverting the D-V axis reveal that it is determined by stage 24-26 which is just before expression of the dorsal otic marker Wnt3a. Conditional blocking of canonical Wnt signaling results in reductions in the number of sensory organs and semicircular canals which could be placed in one of three categories, the most common phenotypes being similar to those seen after dorsal ablations. There is less regenerative potential along the D-V axis. Wnt3a protein alone is sufficient to rescue the severe loss of inner ear structures resulting from dorsal but not ventral half ablations.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 pc.1 wnt3a
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	Figure 1. A–K′: Dorsal and ventral half ablations. Dorsal views of inner ears in living tadpoles at approximately stage 48. A–D,F: Five different examples of dorsal half ablations (Dorsal on the figure), ventral half remaining. E: Control, normal ear with sensory organs labeled. G–K: Four different examples of ventral half ablations (Ventral on the figure), dorsal half remaining. F,G: Both ears shown with ablated ear on Left side, Control ear on Right. G: Black dotted lines indicate the edges of the lateral semicircular canal. A–C,F-K: Brightfield images. (A′–C′,D,E,H′–K′) Fluorescent images. A,C,G: Ablations done at stage 26. B,F,I,J: Ablations done at stage 31. D,H,K: Ablations done at stages 29–31. Anterior is to the top of all images except D and H where anterior is to the top left and top right respectively. A′,H′: These images are extended focus composites of at least 2 different focal planes using the extended depth of field plugin (see Methods). Black arrows point to otoliths and in I it also indicates the lumen of a partially formed semicircular canal. Black arrowheads point to incomplete invaginations that would have formed semicircular canals except in H where it points to lumen of partially formed canal. White arrows point to labeled hair cells in unidentified sensory organs. hb, hindbrain; as, anterior semicircular canal (scc); ls, lateral scc; ps, posterior scc; uo, utricular otolith; so, saccular otolith; ac, anterior crista; lc, lateral crista; pc, posterior crista; um, utricular macula; sm, saccular macula. Scale bar = 500 μm.Download figure to PowerPoint
	Figure 2. A–L′: Dorsal third ablations have less severe phenotypes than dorsal half ablations. Dorsal views of inner ears in living tadpoles at approximately stage 47–48. A–L: Twelve examples of dorsal one-third ablations (Dorsal Third on the figure), ventral two-thirds remaining. A–H,L′: Bright field images. A′–D′,I–L: Fluorescent images. I′-K′: Overlay of fluorescence and bright field images. A,A′: Abnormal semicircular canal (black arrowhead) with nearly normal sensory organs, including posterior crista (white arrow). (B,C) Nearly completed semicircular canal formation (black arrowheads) but with abnormal fused otolith (black arrows). B′: Smaller posterior cristae with labeled hair cells in saccular macula (white arrow). C′: Nearly normal pattern of sensory organs although smaller, like utricular macula (white arrow). D,J′: Abnormal semicircular canals (black arrowhead) and single fused otolith (black arrow). D′,J: Sensory organs somewhat patterned, although smaller with ectopic hair cells, white arrow points to anterior crista. A′-D′,I,K,L: White arrowheads point to lateral cristae. (A′,K,L) White arrows point to posterior cristae. E–G: Brightfield images of ears with increasingly severe phenotypes. E: Nearly normal semicircular canals (black arrowhead) and otoliths (black arrow). F: Nearly normal semicircular canals (black arrowhead) with abnormal fused otolith (black arrow). G,H: Abnormal canals (black arrowhead) and otolith (black arrow). I,I′,K,K′: Normal pattern of sensory organs but reduced in size, especially in K, and abnormal otoliths. I: Upper white arrow points to saccular macula, lower white arrow points to posterior crista. (L,L′) Nearly normal sensory organ pattern and otoliths. B,C,A′–C′,E–G: These images are extended focus composites of at least 2 different focal planes using the extended depth of field plugin (see Methods). White arrowheads point to the lateral cristae. Anterior is to the top of all images except D and G where anterior is to the top right. Abbreviations: um, utricular macula; hb, hindbrain. Scale bars = 500 μm with bar in L′ applying to all panels but H.Download figure to PowerPoint

	Figure 4. A–I′: Inhibiting Canonical Wnt signaling results in three main phenotypes. Dorsal views of inner ears in living tadpoles at approximately stage 45–47. A: The uninjected control ear. B,C: Two different examples of the least severe, Type III (Indicated on the figure), phenotype. D–F: Three examples of intermediate, Type II (Indicated on the figure), phenotype. G–J: Four examples of the most severe, Type I (Indicated on the figure), phenotype. A–J: DIC images. A′–F′,H′,I′: Fluorescent images. Black arrows point to otoliths. Black arrowheads point to incomplete invaginations that would have formed semicircular canals. White arrows point to labeled hair cells. All images collected with confocal microscope using a two-photon laser. Fluorescent images are projections of multiple planes of focus using the extended depth of field plugin (see Methods). hb, hindbrain; as, anterior semicircular canal (scc); ls, lateral scc; ps, posterior scc; uo, utricular otolith; so, saccular otolith; ac, anterior crista; lc, lateral crista; pc, posterior crista; um, utricular macula; sm, saccular macula. Scale bar = 200 μm.Download figure to PowerPoint
	Figure 5. A–D: Wnt3a rescue of dorsal half ablation. Dorsal views of intact living tadpoles at stage 48. Fluorescent confocal Images (A′,B,C′,D) and brightfield images overlaid with red fluorescence (A,C) Control unoperated side on left, bead recipient right (stage 48). A,A′,B: Two examples of bead recipients with full rescue. C,C′: PBS soaked bead recipient with no rescue of dorsal ablation on right side. D: Bead recipient with a mirror anterior duplication. Asterisk indicates bead still present at tadpole stage. Fluorescent confocal images are maximum intensity projections of multiple planes of focus. hb, hindbrain; as, anterior semicircular canal (scc); ls, lateral scc; ps, posterior scc; uo, utricular otolith; so, saccular otolith; ac, anterior crista; lc, lateral crista; pc, posterior crista; um, utricular macula; sm, saccular macula. Scale bar = 200 μm.Download figure to PowerPoint
	Figure 6. Expression of fluorescently tagged β-catenin morpholino. A: Lateral view of stage 33/34 tadpole injected on one side at two cell stage showing morpholino containing tissues which include the inner ear (o; outlined by white dots), eye (e), brain (b), epidermal skin (s), muscle cells (m). B: Higher magnification view of same tadpole. Arrow indicates labeling in inner ear, highlighting stronger staining in dorsal to ventral stripe down center of otocyst. Scale bar = 200 μm. Download figure to PowerPoint.