Iimura A et al. (2020), Role of TrkA signaling during tadpole tail rege...

XB-ART-56520

Genes Cells 2020 Feb 01;252:86-99. doi: 10.1111/gtc.12740.

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Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis.

Iimura A , Nishida E , Kusakabe M .

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Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

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Species referenced: Xenopus laevis
Genes referenced: bdnf btg3 lrr1 ngf ntrk1 ntrk2 ntrk3 sox2
GO keywords: neurotrophin TRKA receptor binding [+]

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	Figure 1. Xenopus laevis NGF family genes are up-regulated in the regenerating tails of tadpoles. The expression levels of ngf.L/S (a), bdnf.L/S (b), ntf3. L/S (c) and ntf4. L/S (d) were measured by qRT-PCR. Ten tail tips were collected by cutting at the level of 0.5 mm anterior to the original amputation plane at 0, 1, 3 or 6 days post amputation (dpa). The results of two independent experiments are shown. The expression level of each gene was normalized to that of odc, and the normalized gene expression level at 0 dpa was defined as 1.0 in each experiment.
	Figure 2. Expression patterns of Xenopus laevis NGF family genes during tadpole tail regeneration. WISH for ngf (a-f), bdnf (g-l), ntf3 (m-r) and ntf4 (s-x) was performed on intact (N=7), 0, 1, 2, 3, or 6 dpa (N=9 for each time-point) tadpoles from two independent experiments. Magnified views of the boxed areas in e, k, q and w are shown in e', k', q', and w', respectively. Arrowheads and arrows indicate the original amputation planes and the regenerating tadpole tips, respectively. The asterisk (m) indicates the staple holding the tadpole during photographing, bf, border between the outer and inner fins; me, mesenchyme. Scale bar, 1mm, except for e', k', q', and w'.
	Figure 3. Expression patterns of Xenopus laevis Trk family genes during tadpole tail regeneration. WISH for TrkA (a-f), TrkB (g-l) and TrkC (m-r) was performed on intact (N=7), 0, 1, 2, 3, or 6 dpa (N=9 for each time=point) tadpoles from two independent experiments. Magnified views of the boxed areas in e, k, and q are shown in e', k', and q', respectively. Arrowheads and arrows indicate the original amputation planes and the regenerating tail tips, respectively. Broken lines (e', k', and q') indicate the outlines of the regenerating tails. Asterisks (f, k, and r) indicate the staples holding the tadpoles during photographing. nc, notochord; sg, spinal ganglia. Scale bar, 1 mm, except for e', k', and q'.
	Figure 4. The pan-Trk inhibitor K252a and the TrkA inhibitor GW-441756 suppress tail regeneration in Xenopus laevis tadpoles. (a-c) After tail amputation, tadpoles were treated with DMSO or 150 nM K252a for 5 days (DMSO, N=19; K252a, N=20; from two independent experiments). All tadpoles at 5 dpa were analyzed in b and c, and several of them were additionally analyzed in a. Bright-field (upper) and fluorescence (middle and lower) images of tadpoles immunostained with anti-acetylated-alpha-tubulin to detect axons (DMSO, 100% axonal innervation, N=3; K252a, 100% axonal innervation, N=3; from one experiment) (a). The length of the regenerated tails was measured (b). Dorsal fin regeneration and ventral fin regeneration in each tadpole (DMSO, 38 fins from 19 tadpoles; K252a, 40 fins from 20 tadpole) were separately evaluated and classified into three groups: Marked (more than half of the expected size of fully regenerated fins), Mild (less than half of the expected size of fully regenerated fins) and None (no fin regeneration) (c). (d) Tadpoles were treated with DMSO or 150 nM K252a after tail amputation. WISH for sox2 was performed on 1, 2, 3, or 5 dpa tadpoles to detect the spinal cord (N=8 in each condition from one experiment; all embryos in DMSO- treated and K252a- treated groups show similar sox2 expression patterns from 1 dpa to 5 dpa). (e-g) After tail amputation, the tadpoles were treated with DMSO or 1 microM GW-441756 for 5 days (DMSO, N=30; GW-441756, N=30; from three independent experiments). Bright-field observations indicate that the spinal cord is formed in the tails of DMSO- treated and GW-441756- treated tadpoles (N=29/30 and N=27/30, respectively) (e). Each point represents the length of each regenerated tail (f). Dorsal and ventral fin regeneration was separately classified into three groups (DMSO, 60 fins from 30 tadpoles; GW-441756, 60 fins from 30 tadpoles) (g). (h-j) After tail amputation, tadpoles were treated with DMSO or 10 microM ANA-12-treated tadpoles (N=19/20 and N=20/20, respectively) (h). Each point represents the length of each regenerated tail (i). Dorsal and ventral fin regeneration was separately classified into three groups (DMSO, 40 fins from 20 tadpoles; ANA-12, 40 fins from 20 tadpoles) (j). Arrowheads and arrows indicate the amputation planes and the regenerating tail tips, respectively (a, d, e, and h). Magnified views of the boxed areas are shown in bottom panels (a, e, and h). Data points from independent experiments are shown in different colors (b, f, and i). Bars represent the average +/- SD (b, f, and i). **, P<0.01; n.s, not significant; Welch's t-test (b, f, and i) or Mann-Whitney U-test (c, g, and j). sc, spinal cord; nc, notochord (e and h). Scale bars, 1mm, except for the magnified views in a, e, and h.
	Figure 5. Spatial expression patterns of Xenopus laevis ngf in early development. The expression of ngf at stage 11 (a), 13 (b), 15 (c), 20 (d), 25 (e), 30 (f), 35 (g) and 40 (h) was analyzed via WISH. N=11 in each stage, from two independent experiments. Lateral views with dorsal up (e-h). bf, border between the outer and inner fins; ov, otic vesicle. Scale bar, 1mm.
	Figure 6. Spatial expression patterns of Xenopus laevis TrkAin early development. The expression of TrkA at stage 11(a), 13 (b), 15 (c), 20 (d), 25 (e), 30 (f), 35 (g), and 40 (h) was analyzed via WISH. N=6 in stage 11, from one experiment; N=11 each in other stages, from two independent experiments. D, dorsal; V, ventral (a-c). Sagittal sections of embryos are shown in right panels with dorsal to the right (a and b). A transverse section of the embryo is shown in the right panel with dorsal up (c). Lateral views with dorsal up (e-h). Magnified views of the boxed areas are shown in right bottom (c) or right (e and f) panels. yp, yolk plug; dm, dorsal mesoderm; vm, ventral mesoderm; nc, notochord; sg, spinal ganglion; tn, trigeminal nerve; vn, vestibulocochlear nerve. Scale bar, 1mm, except for the magnified views in c, e, and f.
	Figure 7. TrkA is required for gastrulation and subsequent body axis development during gastrula and neurula stages in Xenopus laevis. (a) Schematic representation of the Xenopus laevis TrkA.S, Xenopus laevis TrkA.L and Xenopus tropicalis TrkA (xtTrkA) proteins. C1 and C2, cysteine clusters; LRR1-3, leucine-rich repeats; Ig-like1 and Ig-like2, immunoglobulin-like domains. (b) Alignment of nucleotide sequences around the start codons of TrkA.S, TrkA.L and xtTrkA. The putative start codons are boxed. The putative untranslated and coding sequences are indicated in lower and upper case, respectively. The target sequences of TrkA.S MO and TrkA.L MO are underlined. (c) Embryos were injected with the indicated MOs (15 ng) into the dorsal marginal zone at the four-cell stage. Vegetal views at stage 12 are shown with dorsal up. Magnified views of the boxed areas are shown in the bottom panels, and blue lines indicate the outlines of the cells. Control MO, 100% normal blastopore, N=12; TrkA.S MO, 100% large blastopore, N=12; from three independent experiments. (d-f) Embryos were coinjected with the indicated MO (15 ng) and GFP mRNA (750 pg) into the dorsal marginal zone at the four-cell stage (control MO+ GFP mRNA, N=39; TrkA.S MO + GFP mRNA, N=38; from two independent experiments). Injected embryos were subjected to analysis at stage 14. Vegetal views are shown in bright-field (upper) and fluorescence (lower) micrographs (d). Blastopore closure phenotypes were classified into two groups: Normal, blastopore closed; Severe, blastopore remained open (e). Body axis phenotypes were classified into two groups: Normal, GFP-labeled dorsal marginal cells converged toward the body axis; Severe, GFP-labeled dorsal marginal cells remained broadly distributed (f). ** P<0.01, Mann-Whitney U-test (e and f). Scale bars, 1 mm, except for the lower panels in c (100 micro m).
	Figure 8. TrkA knockdown leads to defects in body axis elongation during tailbud stages in Xenopus laevis. (a-c) Embryos were injected with the indicated MO (15 ng) and indicated mRNA (750 pg) into the dorsal marginal zone at the four-cell stage. Injected embryos were subjected to analysis at stage 39 (control MO+ GFP mRNA, N=62; TrkA.S MO + GFP mRNA, N=64; TrkA.S MO + TrkA.L mRNA, N=64; from four independent experiments). Lateral views are shown with dorsal up (a). Each point represents the body length of each embryo (b). Eye phenotypes were classified into three groups: Severe, no apparent eyes, Mild, eyes with small size and/or distorted (e.g., noncircular) lans/optic cup; Normal, eyes with normal size and morphology (the left and the right eye in one embryo were separately evaluated; control MO + GFP mRNA, 124 eyes from 62 embryos; TrkA.S MO + GFP mRNA, 128 eyes from 64 embryos; TrkA.S MO + TrkA.L mRNA, 128 eyes from 64 embryos) (c). (d and e) Embryos were injected with the indicated MO (15 ng) into the ventral marginal zone at the four-cell stage and subjected to analysis at stage 39 (control MO, N=28; TrkA.S MO, N=27; from two independent experiments). Lateral views are shown with dorsal up (d). Each point represents the body length of each embryo (e). (f-g) Embryos were injected with the indicated MO (80 ng) into the dorsal marginal zone at the four-cell stage and subjected to analysis at stage 39 (control MO, N=30; TrkA.L MO, N=30 from two independent experiments). Lateral views shown with dorsal up (f). Each point represents the body length of each embryos (g). Data points from independent experiments are shown indifferent colors (b, e, and g). Bars represent the average +/- SD (b, e, and g). **P<0.01, Mann-Whitney U-test (c) or Welch's t-test (b,e, and g). Scale bars, 2mm (a, d, and f).