Elliott KL et al. (2013), Transplantation of Xenopus laevis tissues to de...

XB-ART-46637

PLoS One 2013 Jan 01;82:e55541. doi: 10.1371/journal.pone.0055541.

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Transplantation of Xenopus laevis tissues to determine the ability of motor neurons to acquire a novel target.

Elliott KL , Houston DW , Fritzsch B .

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The evolutionary origin of novelties is a central problem in biology. At a cellular level this requires, for example, molecularly resolving how brainstem motor neurons change their innervation target from muscle fibers (branchial motor neurons) to neural crest-derived ganglia (visceral motor neurons) or ear-derived hair cells (inner ear and lateral line efferent neurons). Transplantation of various tissues into the path of motor neuron axons could determine the ability of any motor neuron to innervate a novel target. Several tissues that receive direct, indirect, or no motor innervation were transplanted into the path of different motor neuron populations in Xenopus laevis embryos. Ears, somites, hearts, and lungs were transplanted to the orbit, replacing the eye. Jaw and eye muscle were transplanted to the trunk, replacing a somite. Applications of lipophilic dyes and immunohistochemistry to reveal motor neuron axon terminals were used. The ear, but not somite-derived muscle, heart, or liver, received motor neuron axons via the oculomotor or trochlear nerves. Somite-derived muscle tissue was innervated, likely by the hypoglossal nerve, when replacing the ear. In contrast to our previous report on ear innervation by spinal motor neurons, none of the tissues (eye or jaw muscle) was innervated when transplanted to the trunk. Taken together, these results suggest that there is some plasticity inherent to motor innervation, but not every motor neuron can become an efferent to any target that normally receives motor input. The only tissue among our samples that can be innervated by all motor neurons tested is the ear. We suggest some possible, testable molecular suggestions for this apparent uniqueness.

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Species referenced: Xenopus laevis
Genes referenced: slc18a3

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	Figure 1. Stage 46 Xenopus laevis. (A) Embryo with a transplanted ear containing otoconia completely replacing the eye. Inset shows higher magnification of the transplanted ear. (B) Embryo with a transplanted ear containing otoconia medial to the reformed eye. A residual ear (RE) regrew in the native location. Inset shows higher magnification of the transplanted ear and remaining portion of the eye (circled). (C) Embryo with a transplanted, empty vesicle medial to the eye, which has formed a secondary eye more caudal. A residual ear regrew in the native location. Inset shows higher magnification of the transplanted ear and remaining portion of the eye (circled). (D) Embryo with transplanted GFP-expressing somite-derived muscle tissue medial to the eye. (E) Embryo with transplanted somite-derived muscle tissue to replace the ear. (F) Embryo with a transplanted donor GFP-expressing eye to the trunk, replacing a somite. Inset shows the GFP expression in the transplanted eye and surrounding transplanted eye muscle. (G) Embryo with a transplanted heart completely replacing the eye. (H) Embryo with a transplanted liver completely replacing the eye. Native, unmanipulated ears are labeled arand are circled with a black dotted line. Eyes, native and reformed, are indicated by ye Arrows indicate transplanted tissues; transplanted ears are circled in addition with a white dotted line. Scale bar is 1 mm in A, B, C, F, H; 0.5 mm in D, E. doi:10.1371/journal.pone.0055541.g001
	Figure 2. Afferent projections to transplanted ears. (A) Embryo showing implantation of lipophilic dyes into the midbrain (blue) and hindbrain (red). The transplanted ear is noted by the arrow. (B) Transplanted ear with ganglion cells (GC) projecting to hair cells (HC) in the inner ear and along the trigeminal nerve (V, red) back to the hindbrain. The optic nerve (II) is green. (C) Transplanted ear labeled with GFP reveals delaminated ganglion cells (GC), some of which project back to the brain () along the trigeminal nerve (V) as noted by colocalization with lipohilic dye. Other ganglion cells (*) did not colocalize with lipophilic dyes. (D) Transplanted ear labeled with GFP reveals delaminated ganglion cells (GC) which project back to the brain along the oculomotor nerve (III). Inset is higher magnification of boxed area showing the GFP labeled otic ganglion cells. (E) Embryo demonstrating implantations of lipophilic dyes into the native ear (blue) and transplanted ear (red, arrow). (F) Brains from embryos following lipophilic implantation into the native ear (green) and transplanted ear (red) reveal variation in afferent projections from the transplanted ear. Note: some of the lipophilic dye-labeled projections are from cranial nerves that were labeled transcellularly from the afferents. (I′) Stack of eight z-series confocal images from I showing hindbrain projections from the transplanted ear to the alar plate, probably the vestibular nucleus. Scale bar is 1 mm in A and E, 50 in B and C, 100 in D, F, G, H, I, and I′. doi:10.1371/journal.pone.0055541.g002
	Figure 3. Efferent projections to transplanted ears. (A) Implantations of lipophilic dyes into the midbrain (green) and hindbrain (red) revealed axon projections from the oculomotor nerve (III) to hair cells of the transplanted ear (circled). (B) Immunohistochemistry for tubulin of the ear in A shows all innervation. (C) Immunohistochemistry for VAChT (red) confirms motor terminals on hair cells (HC) of boxed areas in B. Insets show higher magnification of VAChT staining at the base of hair cells. (C′) Single z-series images at the base of the hair cells (lower left) showing VAChT-positive terminals and at the apex (upper right) devoid of VAChT staining. (F) Implantations of lipophilic dyes into the midbrain (green) and hindbrain (red) revealed axon projections from the trochlear nerve (IV) to hair cells in the transplanted ear (circled). Afferent axons projected along the trigeminal nerve to the hindbrain, demonstrated by the colocalization of ganglion cells (GC) with the red lipophilic dye. (G) Immunohistochemistry for tubulin of the ear in G shows all innervation. (H) Immunohistochemistry for VAChT (red) confirms motor terminals on hair cells of boxed area in G. Inset shows higher magnification of VACHT staining at the base of hair cells. (I) Implantations of lipophilic dyes into the midbrain (green) and hindbrain (red) revealed ganglion cells (GC) projecting along the oculomotor nerve (III). For this ear, the oculomotor nerve innervated the eye muscles ventral to the transplanted ear. (J) Immunohistochemistry for tubulin of the ear in I shows all innervation. (K) Immunohistochemistry for VAChT (red) shows the absence of motor terminals on hair cells (HC) of boxed area in I. Scale bar is 100 in A, B, F, G, I, J; 25 in C, C′, D, E,H, K. doi:10.1371/journal.pone.0055541.g003
	Figure 4. Transplanted muscle tissue. (A) Implantations of dye into the midbrain (red) and hindbrain (blue) revealed axon projections from oculomotor nerve to the eye muscles but not to the transplanted somite-derived muscle (GFP, green). (B) Single z series showing innervation to the eye muscle but not the transplanted somite-derived muscle (GFP, green). (D) Implantations of dye into the hindbrain at the level of the trigeminal (red) and the glossopharyngeal, vagus, and hypoglossal (green) revealed axons projecting to transplanted somite-derived muscle tissue, likely from the hypoglossal nerve. (E) Implantations of dye ventral to the spinal cord revealed spinal motor neuron innervation of surrounding somite-derived muscle but not to the GFP-positive eye muscle (green) transplanted with the eye. (F) Implantations of dye ventral to the spinal cord revealed spinal motor neuron innervation of surrounding somite-derived muscle, but not to the jaw muscle transplanted from a dextran-injected embryo. Scale bar is 100 . doi:10.1371/journal.pone.0055541.g004
	Figure 5. Transplanted tissue lacking nicotinic acetylcholine receptors such as heart and liver. (A) Implantations into the midbrain (red) and hindbrain (blue) revealed trigeminal innervation of the transplanted GFP-positive heart. The oculomotor (III) only innervated nearby eye muscle tissue. (B) Example of axons from the oculomotor nerve (III) projecting to a transplanted heart in addition to eye muscle. (C) Immunohistochemistry for tubulin (green) and VAChT (red) demonstrate that axons from the oculomotor nerve project to ganglion cells associating with the heart but not on the heart muscle itself. (D) Implantations into the midbrain (green) and hindbrain (red) revealed no axons projecting to the liver. (E) Implantations into the midbrain (green) and hindbrain (red) showed nerve fibers passing over the liver, but not innervating it. Transplanted tissue is circled. Scale bar is 50 in A, C; 100 in B, D, E. doi:10.1371/journal.pone.0055541.g005

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