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Dev Growth Differ
2023 Feb 01;652:86-93. doi: 10.1111/dgd.12841.
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Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl.
Matsubara H
,
Kawasumi-Kita A
,
Nara S
,
Yokoyama H
,
Hayashi T
,
Takeuchi T
,
Yokoyama H
.
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Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the "agarose-embedded heat shock (AeHS) method" involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.
Figure 2. Limb-specific gene induction in X. laevis. (a) Application of the agarose-embedded heat shock (AeHS) method to X. laevis froglets. Multiple froglets can be heat-shocked simultaneously. Each froglet on a 60-mm dish was occasionally moistened with distilled and deionized water to prevent drying. (b) A wild-type froglet and (c) an Xla.Tg (Xla.hsp70:EGFP)Izutsu froglet at 1 day after heat shock. GFP was detected in the intact leftforelimb and regenerating rightblastema at 7 days postamputation (dpa) of the Tg froglet. All six Tg froglets and both wild-type froglets used showed consistent results. (d) Magnified view of the rectangle in (c). (e–g) Transverse sections at different levels along the proximal–distal axis, as shown in (d). Note that green fluorescence was directly emitted by GFP without using an anti-GFP antibody. An anti-Myosin heavy chain antibody (MF20) was used to visualize skeletal muscle in the limb stump. A, anterior; P, posterior; D, dorsal; V, ventral. Arrowheads indicate the amputation plane of a limb. Scale bar = 2 mm for (b–d) and 100 μm for (e–g). Abbreviations: GFP, Green Fluorescent Protein; DAPI, 4',6-Diamidino-2-phenylindole
FIGURE S1. Damages caused by the agarose-embedded heat shock (AeHS) method at high temperatures and AeHS in an intact hindlimb of X. laevis. (a) Magnified view on the posterior side of a hindlimb at day one after AeHS at too high temperatures (≥38°C). Burn-like reddish region (arrowhead) and detaching skin (arrow) were seen as damages. (b, b’) Hindlimb-specific gene expression using the AeHS method at one day after the heat-shock. An intact righthindlimb of an Xla.Tg (Xla.hsp70:EGFP)Izutsu froglet was subjected to AeHS at an optimal temperature (33–37°C) for 30 min. (b) Bright field and (b’) fluorescent image of the froglet. (c, c’) Hindlimb-specific gene expression was induced using Bacto agar instead of agarose at one day after heat-shock. An intact righthindlimb of an Xla.Tg (Xla.hsp70:EGFP)Izutsu froglet was subjected to heat-shock using 1.25% Bacto agar (Wako, 018-15811) instead of agarose at an optimal temperature (3337°C) for 30 min. (c) Bright field and (c’) fluorescent image of the froglet. GFP was broadly detected in the distal part (autopod) of the righthindlimb. Scale bar = 2 mm.
FIGURE S2. Agarose-embedded heat shock (AeHS) method at different temperatures and comparison with gene induction using a heated metal probe in X. laevis. (a-f) AeHS at 33–37°C and that at 30–33°C were applied to the right and leftforelimb blastemas of the same Tg froglet, respectively, at 7 dpa (days post amputation). All four froglets used showed consistent results. (g-h) AeHS at 33–37°C and local heat-shock using a temperature stimulator to the posterior side were applied to the right and leftforelimb blastemas of the same Tg froglet, respectively, at 14 dpa. Local heat-shock by a temperature stimulator using a heated metal probe was applied to the most posterior side (around the dorsal-ventral boundary) of the blastema at 37°C for 10 min as previously reported (Kawasumi-Kita et al., 2015). Both of the froglets used showed consistent results. (a, c, e, g, i, k) Bright field and (b, d, f, h, j, l) fluorescent images of the Xla.Tg (Xla.hsp70:EGFP)Izutsu froglets. Right and leftforelimb blastemas of the same froglet were photographed in exactly the same condition on the same day. Scale bar = 2 mm for (a, b, g, h) and 1 mm for (c-f, i-l).
