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Numerous tissue transplantations have demonstrated that otocysts can develop into normal ears in any location in all vertebrates tested thus far, though the pattern of innervation of these transplanted ears has largely been understudied. Here, expanding on previous findings that transplanted ears demonstrate capability of local brainstem innervation and can also be innervated themselves by efferents, we show that inner ear afferents grow toward the spinal cord mostly along existing afferent and efferent fibers and preferentially enter the dorsal spinal cord. Once in the dorsal funiculus of the spinal cord, they can grow toward the hindbrain and can diverge into vestibular nuclei. Inner ear afferents can also project along lateral line afferents. Likewise, lateral line afferents can navigate along inner ear afferents to reach hair cells in the ear. In addition, transplanted ears near the heart show growth of inner ear afferents along epibranchial placode-derived vagus afferents. Our data indicate that inner ear afferents can navigate in foreign locations, likely devoid of any local ear-specific guidance cues, along existing nerves, possibly using the nerve-associated Schwann cells as substrate to grow along. However, within the spinal cord and hindbrain, inner ear afferents can navigate to vestibular targets, likely using gradients of diffusible factors that define the dorso-ventral axis to guide them. Finally, afferents of transplanted ears functionally connect to native hindbrain vestibular circuitry, indicated by eliciting a startle behavior response, and providing excitatory input to specific sets of extraocular motoneurons.
Figure 1. Evaluation of ear transplantations (AâD) Stage 46 X. laevis embryos showing positions of native ears and transplanted ears (circled). (A) Control animal. (B) Embryo with a third ear transplanted adjacent to the spinal cord. (C) Ventral view of embryo with a third ear transplanted next to the heart. (D) Schematic diagram representing a lateral view of stage 46 X. laevis demonstrating the positions of the native ear (red, A) and the two different transplantations (green, B,C). (E) Control ear and (F) a transplanted ear labeled with antibodies against MyoVI (red) and tubulin (green) demonstrating the presence of hair cells in six distinct epithelia along with Hoechst nuclei counterstain (blue) (Utricle, U; Saccule, S; Lagena, L; Anterior canal, Ac; Horizontal canal, Hc; Posterior canal, Pc) and neurons, respectively. Endolymphatic duct is labeled Ed. Scale bars in AâC are 0.5 mm and 100 µm in EâF Rostral, R; Dorsal, D.
Figure 2. Ear afferent innervation of the spinal cord (A) 3D reconstruction of an ear transplanted adjacent to the spinal cord labeled with antibodies against Tubulin (green) and MyoVI (red) displaying neurons and hair cells, respectively. (BâC) Single optical sections of an X. laevis brain and spinal cord (blue, autofluorescence) in the dorsal (B) and ventral (C) plane following injection of dye (green) into an adjacently transplanted ear shows afferents entering the spinal cord dorsally and ventrally, respectively. White arrowhead indicates the entry point of inner ear afferent projections. (D) Lateral view schematic diagram showing the position of the transplanted ear and the defined midline position (blue dotted line) along the dorsalâventral axis of the spinal cord used to assign entry and projection planes of labeled afferents in BâC. (Dâ²) Analysis of entry point and plane of projection for animals with ears transplanted adjacent to the spinal cord. Serial optical sections were analyzed for entry point of labeled fibers (dorsal, midline, ventral) and for plane of projection (dorsal, ventral). n = 20. (E) Backfilling of inner ear ganglion cells in an ear adjacent to the spinal cord from dye injection into the spinal cord rostral to the transplanted ear. (F) Backfilling (red) of inner ear ganglia and peripheral afferent processes on hair cells in an ear adjacent to the spinal cord from dye injection into the spinal cord. Hoechst nuclei counterstained in blue. Spinal Cord, SC; Hindbrain, HB; Dorsal, D; Ventral, V; Rostral, R; Ganglion cells, G; Utricle, U; Saccule, S; Lagena, L; Anterior canal, Ac; Horizontal canal, Hc; Posterior canal, Pc. Scale bars are 100 µm.
Figure 3, Afferent innervation of the hindbrain by ears transplanted adjacent to the spinal cord (A) Schematic of dye placement for control animals. (Aâ²) Control hemisection of the brain and spinal cord showing ascending spinal fibers (green) enter the hindbrain and fill the descending tract of trigeminal nucleus (V, unlabeled). Native ear projections (red) into the vestibular nucleus in the hindbrain are labeled. Note the lack of overlap between the trigeminal nucleus and vestibular nucleus at higher magnification of Aâ² (B) and of B, shown as a single optical section (Bâ²). (Bâ³) 3D reconstruction of entire stack in B. (C) Schematic of dye placement for animals with ears transplanted adjacent to the spinal cord. (Câ²) Hemisection showing ascending spinal tracts and spinal cord transplanted ear afferent fibers projecting into the hindbrain (green) along the descending tract of V (unlabeled). (D) Higher magnification of Câ² showing inner ear afferents projecting into the vestibular nucleus from the trigeminal nucleus (arrowhead). (Dâ²) Higher magnification of box in D showing projections into the vestibular nucleus (arrowhead) in a single optical section. (Dâ³) 3D reconstruction of entire stack in D. 8 experimental animals were analyzed. Arrows denote the hindbrain/spinal cord boundary. Scale bars are 100 µm in Aâ², B, Bâ³, Câ², Câ³, D and 50 µm in Bâ², Dâ². Vest Ne vestibular nerve, Vest Nu vestibular nucleus, D dorsal, R rostral.
figure 4. Câstart startle response by transplanted ears. (A) Percentage of animals that displayed a Câstart startle response following stimulation in control animals with no ears and in animals in which the only ear was the transplanted ear adjacent to the spinal cord. (Aâ²) Direction in each trial with a positive response observed in A from animals in which the only ear was the transplanted ear adjacent to the spinal cord. *P < 0.05, ChiâSquare analysis. (B) Schematic of dye placement. (C) Wholeâmount of a hindbrain from an animal that had a response in all four trials, three were in the direction away from the transplanted ear and one in the direction toward the transplanted ear. Arrow designates entry point of transplanted inner ear afferents. M, Mauthner cell. (D) Lateral view of ipsilateral hemisected hindbrain showing projections of transplanted ear afferents (green) in between anterior lateral line (aLL) and trigeminal (V) afferent central projections (red). (E) Lateral view of contralateral hemisected hindbrain showing projections of a transplanted ear afferents (green) in between the region where the anterior lateral line (aLL) and trigeminal (V) nuclei are located. Autofluorescence is in blue. Scale bars are 100 µm.
Figure 5. Multiâ€unit inferior rectus (IR) nerve discharge during activation of native bilateral semicircular canals and a transplanted third ear on the spinal cord. (A) Schematic of a semiâ€intact Xenopus preparation depicting the recording of the right IR nerve during roll motion (black curved double arrow), galvanic vestibular stimulation (GVS) of the contralateral posterior (cPC) and iAC semicircular canal epithelia (iAC; red electrodes) and of the transplanted third ear (green electrodes). (B) Episode of spontaneous IR nerve discharge (black trace) with an average resting rate of ~20 spikes/s (blue trace) in an isolated preparation obtained from a stage 55 tadpole. (C, E, G) Modulated multiâ€unit discharge (black traces) and mean firing rate (lower colored traces) of the same IR nerve during roll motion in the cPCâ€iAC plane (C), during GVS of the cPCâ€iAC (E) and during GVS of the third ear (G); sinusoids indicate the waveform (1 Hz) for natural and galvanic stimulation. (D, F, H) Modulated mean IR nerve firing rate over a single cycle (black traces) ± SEM (colorâ€shaded areas) of the responses shown in C,E,G; the averages were obtained from 20â€120 single cycles, respectively; the colored dotted sinusoids indicate the motion stimulus (D) and GVS of the cPC (F) and third ear (H), and the blue dashed line and horizontal band the mean ± SEM resting rate of the IR nerve. Note that the IR nerve increases firing during motion in direction of the cPC (D), galvanic depolarization of the cPCepithelium (F) and galvanic depolarization of the third ear (*in G, H). Tpos, position signal of the turntable.
Figure 6. Inner Ear Afferent Fasciculation with Lateral Line. (A) Schematic of dye placement for the different transplants. (B) Lateral view of afferents of the pLL projecting over and into the ear (green, dye injection B in panel A). The ear was transplanted adjacent to the spinal cord at stage 32â36. No ganglia were labeled. (C) Lateral view of pLL and inner ear afferents from a caudal dye injection into caudal portion of the pLL nerve (cyan, dye injection C in panel A). G, ganglia. (D) Lateral view of inner ear afferents from a spinal cord injection rostral to the transplanted ear (red, dye injection D in panel A) G, ganglia. (E) Merge of BâD. Cyan of panel C has been replaced by blue. Panels BâE are counterstained for nuclei marker Hoechst (gray). Arrowheads indicate areas of innervation of the inner ear. Scale bars are 100 µm Endolymphatic duct, Ed.
Figure 7. Inner ear afferent fasciculation with the vagus nerve. (A) Schematic of ear transplants showing dye placement for the ears transplanted adjacent to the heart. (B) Ventral view of an animal with an ear transplanted into the heart region showing ear afferents projecting with the vagus nerve (green, dye injection B in panel A; Vagus Ne, arrow). Heart is outlined with a dotted line as determined from autofluorescence background (blue). (C) Ventral view of an animal with an ear transplanted next to the heart. Labeling (green) from dye injection into the vagus nerve (dye injection C in panel A) showing innervation of the ear and labeling of inner ear ganglia. Autofluorescense background (blue). Scale bars are 100 µm Rostral, R; Lateral, L.
Head of a X. laevis embryo, at NF stage 46 , showing position of eyes and otic vesicles (i.e, auditory apparatus or ears), dorsal veiw, anterior up. Scale bar = 0.5cm
Inner ear of X. laevis, NF stage 46 tadpole, labeled with antibodies against MyoVI (red) and tubulin (green) labeling nerves, illustrating the presence of hair cells in six distinct epithelia, along with Hoechst counterstain (blue) labeling nuclei; dorsal left, anterior up.
Key: utricle, U; sacculus, S; lagena, L; anterior canal, Ac; Horizontal [lateral] semicircular canal, Hc; Posterior canal, Pc, Endolymphatic duct Ed. Scale bar = 100 µm.
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