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FIGURE 1
Identification of optimal conditions for microCT analysis of Xenopus tadpoles. (a) Schematic of the microCT analysis method. To analyze the telencephalon regeneration, the telencephalon (orange) of the stage 49 X. laevis tadpole was amputated at the middle (dotted line and scissors), as shown in the left side. Light green represents the diencephalon, and light blue the midbrain. A simplified staining flow is shown on the right side; PTA-stained samples were scanned in 70% or 100% ethanol. No obvious differences in the obtained CT images related to ethanol concentration existed. (b–d) Reconstructed tomograms of a PTA-stained stage 58 X. tropicalis tadpole. (e–g) Fixed with MEMFA and stained with IKI. (h–j) Fixed with 4F1G and stained with IKI. (b, e, h) Frontal sections, (c, f, i) transverse sections, and (d, g, j) transverse sections of the proximal telencephalon. Brightness and contrast for each image were separately adjusted to maximize anatomic visibility. (k, l, m) Schematics of (b–j). ac, abdominal cavity; cp, choroid plexus; gl; gill lobe; ht, heart; itc, inner layer of telencephalon; otc, outer layer of telencephalon; ov, otic vesicle; ptc, proximal telencephalon; tm, trunk muscle. Scale bars: 1 mm in (b, c, e, f, h, i) and 0.5 mm in (d, g, j).
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FIGURE 2
Three-dimensional structure of a PTA-stained Xenopus tadpole revealed by volume rendering. (a, b) Head region of a stage 48 X. laevis tadpole. Transparency for each image was separately optimized to maximize anatomic visibility of the body surface (a) and internal structures (b). (c) Dorsal view of the brain. (d) Magnified dorsal view of the head region. Dorsal structures, including the brain, were virtually removed. (e) Ventral view of the heart. (f) Anterolateral view of the right pronephros and forelimb bud. ao, aortic lateral line; at, atrium; dc, diencephalon; fb, forelimb bud; gh, geniohyoideus; gl, gill lobe; gt, glottis; hb, hindbrain; hv, heart ventricle; ih, interhyoideus; io, infra-orbital lateral line; lm, levator mandibulae; mb, midbrain; ob, olfactory bulb; oh, orbitohyoideus; on, olfactory nerve; ot, outflow tract; pn, pronephros; qha, quadratohyoangularis; sl, supra-orbital lateral line; so, subarcualis obliquus; tc, telencephalon; tm, trunk muscle; vp, velar plate.
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FIGURE 3
Three-dimensional models of the early stages of telencephalon regeneration. (a–f) Three-dimensional models of intact brain (a), brain immediately after amputation (0 dpa), and regenerating brain at 1 (c), 3 (d), 6 (e), and 9 dpa (f). Magenta, green, and cyan colors indicate telencephalon, diencephalon, and midbrain regions, respectively. Asterisks indicate extra regions of interest between the left and right lateral ventricles, which were created due to technical problems during segmentation. Black arrowheads indicate the amputation plane. White arrowheads indicate the anterior ends of the lateral ventricle exposed by amputation. Red arrowheads indicate the narrowed cerebral aqueducts. ca, cerebral aqueduct; dc, diencephalon; iiiv, third ventricle; lv, lateral ventricle; mb, midbrain; mv; midbrain ventricle; tc, telencephalon. Scale bars: 500 μm. (g–j) Plots of the telencephalon length (g), and ventricular-brain volume ratio in the telencephalon (h), diencephalon (i), and midbrain (j) before (intact) and after amputation (0–9 dpa). Black horizontal bars indicate mean values. p-Values were calculated using one-way ANOVA with Tukey HSD post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001). If not stated, then there is no significant difference.
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FIGURE 4
Reconstructed sectional views of the cerebral aqueduct and lateral ventricles. (a) Reconstructed coronal sections of cerebral aqueducts before (intact) and after amputation (0–9 dpa). The coronal planes where the width of the cerebral aqueduct is the largest are shown. Cyan and magenta arrowheads indicate the opened and closed cerebral aqueducts, respectively. dc, diencephalon; iiiv, third ventricle; mb, midbrain; mv; midbrain ventricle; tc, telencephalon. Scale bars: 500 μm. (b) A plot of the width of cerebral aqueduct before (intact) and after amputation (0–9 dpa). Black horizontal bars indicate mean values. p-Values were calculated using one-way ANOVA with Tukey HSD post-hoc test (*p < 0.05; **p < 0.01). If not stated, then there is no significant difference. (c) Reconstructed sagittal sections of lateral ventricles before (intact) and after amputation (0–9 dpa). The sagittal planes where the height of the lateral ventricle is the largest are shown. Green and yellow arrowheads indicate the opened and closed lateral ventricles, respectively.
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FIGURE 5
The transient shrinkage and expansion of the ventricles identified by live-imaging analyses. (a) EGFP expression pattern in the regenerating brain of Xla.Tg(pax6:eGFP)Ogino. Dorsal views. (b) The width of the EGFP expression domain at the position indicated by the arrowheads in (a). Each color represents values measured from the same individual (n = 8). p-Values were calculated using one-way repeated measures ANOVA with pairwise paired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). (c) Fluorescence patterns of RDEX injected into the brain ventricle. Dorsal views. Arrowheads indicate the narrowest region between the diencephalon and midbrain. Scale bars: 500 μm.
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Figure S1. Reconstructed transverse sections of tadpoles stained using three different methods. (a–d) A PTA-stained stage 58 X. tropicalis tadpole. (e–h) Fixed with MEMFA and stained with IKI. (i–l) Fixed with 4F1G and stained with IKI. (a, e, i) Eyes, (b, f, j) gill lobes, (c, g, k) hearts, and (d, g, j) trunk muscles. The insets in (a, d, e, h, i, l) are higher magnification views of the boxed regions. The brightness and contrast for each image were separately adjusted to maximize anatomic visibility. at, atrium; ihv, inner layer of ventricle; ohv, outer layer of ventricle. Scale bars: 500 μm.
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Figure S2. Multi-planar reconstruction of a PTA-stained stage 48 X. laevis tadpole. The blue, green, and orange lines indicate the positions of the frontal, transverse, and sagittal sections, respectively. Scale bars: 1 mm.
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Figure S3. Volume-rendered images of tadpoles stained using three different methods. (Top) A PTA-stained stage 58 X. tropicalis tadpole. (Middle) Fixed with MEMFA and stained with IKI. (Bottom) Fixed with 4F1G and stained with IKI.
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Figure S4. Bright-field images showing shape changes of the amputated brain. The dotted lines show the outlines of the intact brain and amputation planes. Arrowheads indicate the regenerating telencephalon. dc, diencephalon; mb, midbrain; tc, telencephalon.
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Figure S5. Temporal changes in the brain and ventricle volumes during telencephalon regeneration. Plots of the volume of the telencephalon (a), diencephalon (b), midbrain (c), lateral ventricle (d), third ventricle (e), and midbrain ventricle (f) before (intact) and after amputation (0–9 dpa). The black horizontal bars indicate mean values.
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