Efferent projections to transplanted ears.

<p><b>(A)</b> 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>(B)</b> Immunohistochemistry for tubulin of the ear in A shows all innervation. <b>(C–E)</b> 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. <b>(C′)</b> 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. <b>(F)</b> 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. <b>(G)</b> Immunohistochemistry for tubulin of the ear in G shows all innervation. <b>(H)</b> 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. <b>(I)</b> 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. <b>(J)</b> Immunohistochemistry for tubulin of the ear in I shows all innervation. <b>(K)</b> Immunohistochemistry for VAChT (red) shows the absence of motor terminals on hair cells (HC) of boxed area in I. Scale bar is 100 µm in A, B, F, G, I, J; 25 µm in C, C′, D, E,H, K.</p

Afferent projections to transplanted ears.

<p><b>(A)</b> Embryo showing implantation of lipophilic dyes into the midbrain (blue) and hindbrain (red). The transplanted ear is noted by the arrow. <b>(B)</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. <b>(C)</b> 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. <b>(D)</b> 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. <b>(E)</b> Embryo demonstrating implantations of lipophilic dyes into the native ear (blue) and transplanted ear (red, arrow). <b>(F–I)</b> 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. <b>(I′)</b> 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 µm in B and C, 100 µm in D, F, G, H, I, and I′.</p

Success of tissue transplantation.

<p>Success of transplantation is defined as the detection of transplanted tissue.</p

Transplanted muscle tissue.

<p><b>(A)</b> 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>(B–C)</b> Single z series showing innervation to the eye muscle but not the transplanted somite-derived muscle (GFP, green). <b>(D)</b> 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. <b>(E)</b> 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. <b>(F)</b> 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 µm.</p

Transplanted tissue lacking nicotinic acetylcholine receptors such as heart and liver.

<p><b>(A)</b> 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>(B)</b> Example of axons from the oculomotor nerve (III) projecting to a transplanted heart in addition to eye muscle. <b>(C)</b> 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. <b>(D)</b> Implantations into the midbrain (green) and hindbrain (red) revealed no axons projecting to the liver. <b>(E)</b> 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 µm in A, C; 100 µm in B, D, E.</p

<i>axin1</i> but not <i>axin1(Q162A)</i> rescues anterior defects in Axin1-depleted embryos.

<p>(<b>A–D′</b>) Representative phenotypes of control and Axin1-depleted zebrafish embryos. (<b>A</b>) control uninjected embryo, (<b>B</b>) Axin1-depleted embryo (4 ng <i>axin1</i>-MO), (<b>C</b>) MO injected embryo coinjected with 25 pg <i>axin1</i> mRNA, (<b>D</b>) MO injected embryo coinjected with 25 pg <i>axin1(Q162A)</i> mRNA. (<b>E</b>) Summary table of morphological defects.</p

Both <i>axin1</i> and <i>axin1(Q162A)</i> rescue hyperdorsalization in maternal <i>axin1</i>-depleted embryos.

<p>(<b>A–D</b>) Representative phenotypes of control and <i>axin1</i>-depleted <i>Xenopus</i> embryos obtained following host transfer. Chart showing distribution of phenotypes is inset in each panel, percentages are indicated; black = normal, grey = dorsalized. (<b>A</b>) control uninjected stage 37 embryo, (n = 40) (<b>B</b>) <i>axin1</i>-depleted embryo (4 ng oligo; n = 44), (<b>C</b>) <i>axin1</i>-depleted embryo injected with 60 pg <i>axin1</i> mRNA (n = 32), (<b>D</b>) <i>axin1</i>-depleted embryo injected with 60 pg <i>axin1(Q162A)</i> mRNA (n = 21). (<b>E</b>) Representative realtime RT-PCR of <i>sia1</i>, <i>nr3.1</i> and <i>chd</i> expression in control whole embryos (un), <i>axin1-</i>depleted embryos (<i>axin-</i>) and <i>axin-</i> embryos injected with <i>axin</i> constructs.</p

CAAX-tagged Axin1 is sufficient to inhibit axis formation and to promote Axin1 protein turnover.

<p>(<b>A–D</b>) Localization of CAAX-tagged and untagged Axin1 proteins in <i>Xenopus</i> animal caps. (<b>E–G</b>) Uninjected control embryos (<b>E</b>) and embryos expressing Axin1-CAAX (<b>F</b>) and Axin1(Q162A)-CAAX (<b>G</b>) at the tailbud stage. (<b>H</b>) Immunoblots of stage 9 embryo lysates injected with <i>FLAG-axin1</i> (100 pg, 300 pg) and <i>FLAG-axin1-caax</i> (100 pg, 300 pg). The top panel shows anti-FLAG blotting, the bottom panel shows a non-specific band (n.s.) detected by the anti-FLAG antibody to confirm equivalent loading. (<b>I</b>) Representative realtime RT-PCR of <i>sia1</i>, <i>nr3.1</i> and <i>sizzled (szl)</i> expression in control embryos (un) and in embryos injected with <i>FLAG-axin1</i> (100 pg, 300 pg) or <i>FLAG-axin1-caax</i> (100 pg, 300 pg).</p

Structure-function analysis of the Axin1 RGS domain.

<p>(<b>A</b>) Alignment of RGS domains from human (hAXIN1), mouse (mAxin1), <i>Xenopus</i> (xAxin1) and zebrafish Axin1 (zAxin1) with human RGS4. Blue bars = APC binding interface; orange bars = Gna binding interface. * = residues required for GAP activity in RGS4. Arrow = residue required for RGS4 GAP activity, mutated in this study. (<b>B</b>) Immunoblots showing equivalent protein expression in 24 hpf zebrafish embryos injected with <i>axin1-myc</i> and <i>axin1(Q162A)-myc</i>. (<b>C</b>) Immunoprecipitation of FLAG-tagged Axin1 constructs with HA-tagged Gnao, showing reduced binding of FLAG-Axin1(Q162A) to overexpressed Gnao. (<b>D</b>) FLAG-Axin1 and FLAG-Axin1(Q162A) immunoprecipitate endogenous APC equivalently. (<b>E</b>) FLAG-Axin1 and FLAG-Axin1(Q162A) immunoprecipitate HA-tagged APC-SAMP3 equivalently.</p

Axin1^Q162A does not interact with Gna at the plasma membrane.

<p>(<b>A–D</b>) Immunostaining against Myc showing localization of Axin1-Myc and Axin1(Q162A)-myc in zebrafish embryos, without (A, B) or with coexpression of Gnao (C, D). The arrowhead in C indicates membrane localization of Axin1 upon Gnao expression. Embryos were counterstained with TOPRO3 to show nuclei (purple).</p