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Int J Dev Biol
2010 Jan 01;5410:1443-51. doi: 10.1387/ijdb.103061ke.
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Transplantation of Xenopus laevis ears reveals the ability to form afferent and efferent connections with the spinal cord.
Elliott KL
,
Fritzsch B
.
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Previous comparative and developmental studies have suggested that the cholinergic inner ear efferent system derives from developmentally redirected facial branchial motor neurons that innervate the vertebrate ear hair cells instead of striated muscle fibers. Transplantation of Xenopus laevis ears into the path of spinal motor neuron axons could show whether spinal motor neurons could reroute to innervate the hair cells as efferent fibers. Such transplantations could also reveal whether ear development could occur in a novel location including afferent and efferent connections with the spinal cord. Ears from stage 24-26 embryos were transplanted from the head to the trunk and allowed to mature to stage 46. Of 109 transplanted ears, 73 developed with otoconia. The presence of hair cells was confirmed by specific markers and by general histology of the ear, including TEM. Injections of dyes ventral to the spinal cord revealed motor innervation of hair cells. This was confirmed by immunohistochemistry and by electron microscopy structural analysis, suggesting that some motor neurons rerouted to innervate the ear. Also, injection of dyes into the spinal cord labeled vestibular ganglion cells in transplanted ears indicating that these ganglion cells connected to the spinal cord. These nerves ran together with spinal nerves innervating the muscles, suggesting that fasciculation with existing fibers is necessary. Furthermore, ear removal had little effect on development of cranial and lateral line nerves. These results indicate that the ear can develop normally, in terms of histology, in a new location, complete with efferent and afferent innervations to and from the spinal cord.
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21302254
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Fig. 2. Development of the transplanted ear. (A) MiR-183 in sensory epithelia in the native ear and in surrounding cranial ganglia as well as ganglia next to the ablated ear (i.e. Trigeminal ganglia, V). (B) Lateral view of the transplanted ear with miR-183 positive sensory epithelia and ganglion cells; anterior is to the right. (C) Acety- lated tubulin (green) and MyoVI (red) immunostaining of a transplanted ear showing hair cells. (D) Magnification of region in C show kinocilia (white arrows) on hair cells. (E) Green fluorescent protein (GFP)-labeled transplanted ear reveals GFP-positive delaminating sensory ganglion cells (G), a few of which (G*) are colocalized with lipophilic dye (red). Lipophilic dye injections were ventral to the spinal cord. (F) 2 m thick transverse section through the dorsal and lateral view of transplanted ear show- ing a sensory epithelium with hair cells and an endolymphatic duct. (G) Transmission electron micrograph of two hair cells in the transplanted ear with synaptic ribbons (black arrows). The boxed areas are shown at higher magnification in Fig. 3 K,L. U, utricle; S, saccule; Ac, anterior canal crista; Hc, horizontal canal crista; Pc, posterior canal crista; G, ganglion cell(s); HC, hair cells K, kinocilium; S, stereocilia; ED, endolymphatic duct. Scale bar is 0.5 mm in (A,B); 25 um in (C,D); 50 um in (E), 0.1 mm in (E inset); 10 um in (F) and 5 um in (G)
Fig. 3. Afferent and efferent innervation of transplanted ears. (A) Transplanted ear that had dextran amine (red) injected into the spinal cord. Autofluorescence of the tissue is green. Inset: higher magnification of boxed area. (B) Transplanted ear that had lipophilic dyes injected into the spinal cord rostral (red) and caudal (green) to the ear. (C) Acetylated tubulin immunostaining (red) of boxed area in B showing ganglion cells apposed to hair cells. Autofluorescence of the tissue is green. (D) Cells in the ventral spinal cord positive for dextran amine dyes that had been injected into the transplanted ear. (E) Neuromasts in the skin showing vesicular acetylcholine transporter (VAChT)-positive terminals on the hair cells. Autofluorescence is blue. (F) Hair cells in a control ear immunostained for acetylated tubulin and VAChT. (G) Transplanted ear that had lipophilic dye injected ventral to the spinal cord show nerve fibers extending to the ear. (H) Transplanted ear in G immunostained for acetylated tubulin. (I) Transplanted ear in G immunostained for acetylated tubulin and VAChT. Inset: higher magnification of boxed area. Red arrows indicate VAChT immunostaining. (J) Transplanted ear immunostained for acetylated tubulin and VAChT. Inset: single optical section of VAChT staining (red arrows) at the base of hair cells. (K) Electron micrograph of an afferent terminal on a hair cell. A synaptic ribbon (arrow) is observed on the hair cell above the afferent terminal (L) Electron micrograph of an efferent terminal with vesicles (circled) making a synapse on a hair cell. These TEM images were obtained from ears previously processed for immunohis- tochemistry to confirm the presence of VAChT, hence poor preservation of membranes. GC, ganglion cell; HC, hair cell; MN, motor neuron cell body; NM, neuromasts; Aff, afferent terminal; Eff, efferent terminal; V, vesicles; Mt, mitochondria. Scale bar is 100 um in (A,B,C,D,G,H); 25 um in (E,F,I,J) and 1 um in (K,L).
Fig. 4. Development of cranial and lateral line nerves. Acetylated tubulin immunostaining of cranial nerves in a control embryo (A) and in embryos in which the ear was transplanted (B,C). (D) Acetylated tubulin (green) immunostaining of lateral line nerves in the cranial skin of a control embryo (E) Acetylated tubulin (green) and Hoechst (blue) immunostaining of a later-stage transplanted embryo. Acetylated tubulin (green) immunostaining of lateral line nerves in the cranial skin of a control embryo (F) and an embryo in which the ear was transplanted (G). (H) Higher magnification of boxed area in E. Areas where native ears are present and where ablated ears had occupied are circled with solid and dotted lines, respec- tively. Native ears are labeled ar Eyes (A,B) and skin over the eyes (D,E,F,G) are labeled ye Cranial nerves are labeled with Roman Numerals. LL, lateral line; U, utricle; S, saccule; L, lagena; Ac, anterior canal; Hc, horizontal canal; Pc, posterior canal. Scale bar is 250 m.
slc18a3 ( solute carrier family 18 (vesicular acetylcholine), member 3 ) gene expression (in red) in sections of Xenopus laevis embryos, NF stage 46, as assayed by immunohistochemistry.
Copyright University of the Basque Country Press , 2010
myo6 (myosin VI) gene expression (in red) in sections of Xenopus laevis embryos, NF stage 46, as assayed by immunohistochemistry.
