Nakajima K et al. (2022), Intravital staining to detect mineralization in...

XB-ART-59300

Dev Growth Differ 2022 Sep 01;647:368-378. doi: 10.1111/dgd.12804.

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Intravital staining to detect mineralization in Xenopus tropicalis during and after metamorphosis.

Nakajima K , Yabumoto S , Tazawa I , Furuno N .

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Observing mineralization is essential for studying skeletal development, maintenance, and regeneration. Calcein and alizarin red have long been used to visualize mineralization in fixed specimens, but this requires the target animals to be sacrificed. However, several intravital bone-staining methods have been developed to visualize mineralized tissues in living animals. These methods have been applied to study fin rays and transparent fishes. Xenopus tropicalis is an excellent experimental animal model for studying bone formation and regeneration because skeletal mineralization begins during the free-living tadpole period, and its regenerative ability changes during metamorphosis. However, intravital bone staining of X. tropicalis has only been reported for tadpoles, and no details on its specificity or appropriate experimental conditions are available. Here, we compared the calcein- and alizarin red S (ARS)-staining methods and optimized these methods for tadpoles and juvenile frogs during and after metamorphosis. Staining with 0.01% ARS yielded acceptable signaling for young tadpoles, whereas calcein either at 0.1 or 0.01% occasionally showed artifactual staining of unmineralized tissues. In addition, 0.1% calcein or 0.1% ARS staining showed a higher signal-to-noise ratio with juvenile frogs compared to staining at 0.01%. We propose the use of 0.01% ARS for tadpoles before stage 61 and 0.1% ARS thereafter for staining mineralized tissues. Using this method, we found that ossification of the neural arches occurred at stage 51 in X. tropicalis. This method enables precise staging and manipulation based on the visualized bone structure.

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Species referenced: Xenopus tropicalis
Genes referenced: ptpn11
GO keywords: bone mineralization [+]

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	Figure 1. Calcein staining of the hindlimb during and after metamorphosis. All experiments were done with Xenopus tropicalis. (a, b, f, g) Unstained tadpoles at stages 56 (a, f) and 64 (b, g). (c, d, e, hân, pâs) Tadpoles stained with 0.01% calcein at stages 52 (c, h), 55 (d, i), 56 (e, j), 58 (k, p), 60 (l, q), 62 (m, r), and 64 (n, s) as indicated. (o, t) Juvenile frogs stained with 0.01% calcein. (aâe, kâo) Bright-field images. (fâj, pât) Fluorescence photographs. Scale bar, 1âmm. il, ilium; fe, femur; ti, tibia-fibula; ta, tarsus; me, metatarsus; ph, phalange. Images are presented in lateral view, with the top of each image indicating the dorsal direction and the left of each image indicating the anterior direction
	Figure 2. Calcein staining of the spine during and after metamorphosis. (a, b, f, g) Unstained tadpoles at stages 56 (a, f) and 64 (b, g). (c, d, e, hân, pâs) Tadpoles stained with 0.01% calcein at stages 52 (c, h), 55 (d, i), 56 (e, j), 58 (k, p), 60 (l, q), 62 (m, r), and 64 (n, s) as indicated. (o, t) Juvenile frogs stained with 0.01% calcein. (aâe, kâo) Bright-field images. (fâj, pât) Fluorescence photographs. Scale bar, 1âmm. Images are presented in the dorsal view, with the top of each image indicating the right side of the specimen and the left of each image indicating the anterior direction
	Figure 3. Calcein staining of clavicles and coracoids during and after metamorphosis. (a, b, f, g) Unstained tadpoles at stages 56 (a, f) and 64 (b, g). Tadpoles stained with 0.01% calcein at stages 52 (c, h), 55 (d, i), 56 (e, j), 58 (k, p), 60 (l, q), 62 (m, r), and 64 (n, s) as indicated. (o, t) Juvenile frog were stained with 0.01% calcein. (aâe, kâo) Bright-field images. (fâj, pât) Fluorescence photographs. Scale bar, 1âmm. he, heart; gi, gill; cl, clavicle; co, coracoid. Images are presented in ventral view, with the top of each image indicating the anterior direction and the left of each image indicating the right side of the specimen
	Figure 4. Alizarin red S staining during and after metamorphosis. Tadpoles stained with 0.01% ARS at stages 51 (aâf, s, t), 60 (gâl), and a juvenile frog (mâr). (a, d, g, j, m, p) Hindlimbs (lateral view; top of image, dorsal direction; left side of image, anterior direction). (b, e, h, k, n, q, s, t) Spine (dorsal view; top of image, right side of the specimen; left side of the image, anterior direction). (c, f, i, l, o, r) Clavicles and coracoids (ventral view; top of image, anterior direction; left side of image, right side of specimen). (s, t) Cleared-and-stained whole-mount (CSW) specimens stained with ARS and Alcian blue for calcified and cartilage tissues, respectively. (aâc, gâi, mâo, s) Bright-field photographs. (dâf, jâl, pâr, t) Fluorescence photographs. ta, tarsus; me, metatarsus; ve, vertebrae
	Figure 5. Quantification of fluorescent signals by staining with 0.01% and 0.1% calcein and ARS. (aâl) Fluorescence photographs of juvenile frogs stained with 0.01% (aâc) or 0.1% (dâf) calcein, or 0.01% (gâi) or 0.1% (jâl) ARS. (a, d, g, j) Hindlimbs (lateral view; top of image, dorsal direction; left side of image, anterior direction). (b, e, h, k) Spine (dorsal view; top of image, right side of specimen; left side of image, anterior direction). (c, f, i, l) Clavicles and coracoids (ventral view; top of image, anterior direction; left side of image, right side of specimen). White lines on the hindlimb, spine, and coracoids indicating the lines for which fluorescent intensity were quantified in m, n, and o, respectively. ta, tarsus; ur, urostyle; il ilium; ve, vertebrae; co, coracoid. White arrowheads indicating artificial signals. Scale bar, 1âmm. (mâo) Fluorescent intensity values along the lines in aâl selected for staining with 0.01% calcein (dashed green lines), 0.1% calcein (solid green lines), 0.01% ARS, (dashed red lines) or 0.1% ARS (solid red lines)
	Figure 6. Precise staging based on bone structure. Tadpoles at late stage 55 (a, d, g, j), early stage 56 (b, e, h, k), and late stage 56 (c, c', f, f', i, l) stained with 0.01% ARS. (aâf, c', f'). Lateral views of the left hindlimbs. (gâl) Lateral view of the right hindlimbs. (c', f') After amputation of the phalanges. (aâc, c', gâi) Brightfield photographs. (dâf, f'â², jâl) Fluorescence photographs. Scale bar, 1âmm. il, ilium; fe, femur; ti, tibia-fibula; ta, tarsus; me, metatarsus; ph, phalange
	Figure 7. Intravital staining with ARS and cleared-and-stained whole-mount (CSW) specimens. An albino tadpole at stage 60 stained with 0.01% ARS (aâh) and a juvenile frog stained with 0.1% ARS (iâp). (a, e, i, m) Bright-field images. (b, f, j, n) Fluorescence photographs. (c, d, g, h, k, l, o, p) CSW specimens generated after sacrifice of these individuals. Brightfield (c, g, k, o) and fluorescence (d, h, l, p) photographs of CSW specimens stained with ARS and Alcian blue for calcified and cartilage tissues, respectively. (aâd, iâl) Dorsal views. (eâh, mâp) Ventral view. (q-s) Magnified images of the boxed areas in panels b and c. (t) Left forelimb of a stage-60 tadpole (c, g) from a different angle. Scale bar, 1âmm. Panels aâp and qât show identical scales. pm, premaxilla; ma, maxilla; sm, septomaxilla; na, nasal; ra, radioulna; me, metacarpal; ph, phalange