Rose CS and James B (2013), Plasticity of lung development in the amphibian...

XB-ART-47732

Biol Open 2013 Dec 15;212:1324-35. doi: 10.1242/bio.20133772.

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Plasticity of lung development in the amphibian, Xenopus laevis.

Rose CS , James B .

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Contrary to previous studies, we found that Xenopus laevis tadpoles raised in normoxic water without access to air can routinely complete metamorphosis with lungs that are either severely stunted and uninflated or absent altogether. This is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions can be raised to forego this function without adverse effects. This study compared lung development in untreated, air-deprived (AD) and air-restored (AR) tadpoles and frogs using whole mounts, histology, BrdU labeling of cell division and antibody staining of smooth muscle actin. We also examined the relationship of swimming and breathing behaviors to lung recovery in AR animals. Inhibition and recovery of lung development occurred at the stage of lung inflation. Lung recovery in AR tadpoles occurred at a predictable and rapid rate and correlated with changes in swimming and breathing behavior. It thus presents a new experimental model for investigating the role of mechanical forces in lung development. Lung recovery in AR frogs was unpredictable and did not correlate with behavioral changes. Its low frequency of occurrence could be attributed to developmental, physical and behavioral changes, the effects of which increase with size and age. Plasticity of lung inflation at tadpole stages and loss of plasticity at postmetamorphic stages offer new insights into the role of developmental plasticity in amphibian lung loss and life history evolution.

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Species referenced: Xenopus laevis
Genes referenced: acta2 acta4 actc1 actl6a

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	Fig. 1. Lung development in untreated Xenopus.(A–C) Frontal sections (anterior is up) showing lungs (arrows) at (A) NF 46 just before first breath (fb), (B) NF 47 three days after fb showing expansion of lumen, and (C) NF 47 six days after fb showing thinning of walls of trachea (t), bronchi (b) and lungs (lu) except at the posterior tips; brown are BrdU-labeled nuclei. Lungs show similar levels of BrdU labeling as other tissues before and after inflation. (D–G) Whole mounted lungs (anterior is left) with fluorescently stained smooth muscle actin at (D) NF 47 showing closely spaced bands throughout the length, (E) NF 52 showing difference in spacing and branching of actin bands anteriorly (left arrows) and posteriorly (right arrow) and pulmonary artery, (F) NF 52 with an uninflated tip, and (G) NF 52 with full inflation showing closely spaced bands anteriorly (left) and widely spaced bundles posteriorly (right). (H–J) Frontal sections (anterior is left) with fluorescently stained smooth muscle actin (red) and nuclei (white) at (H) NF 54 showing a continuous layer of actin, (I) NF 54 showing thickening of the lung wall and the appearance of epithelial folds with actin bundles (a) in the crest and rudimentary veins (v) in the base, and (J) NF 58 showing a primary alveolar septum (s) comprised of two epithelial sheets and an expanded crest containing an actin bundle (a) and vein (v). (K) Frontal section of more advanced septa at NF 58 showing flattening and extension of the actin layer (a, brown) and well developed veins (v), lung epithelium (e) and peritoneum (p). (L) Frontal section through the peripheral space of the lung (anterior is left) with fluorescently stained smooth muscle actin (red) and nuclei (white) at NF 63 showing primary (ps) and secondary alveolar septa (ss) in the middle regions, epithelial folds (arrows) in the anterior and posterior regions, and an actin-rich band (ab) posteriorly. (M,N) Lateral views (anterior is left) of live albinos at (M) NF 58 showing lateral subdivision of the smooth muscle-bound compartments and (N) NF 66 showing lungs extending the full length of the pleuroperitoneal cavity. (O) Frontal section (anterior is up) through an adult lung showing the undivided central space, partitioned peripheral spaces and cartilages (c, blue), smooth muscle bundles (a), pulmonary artery (pa) and veins (v) lining the border between these spaces. Scale bars are 0.5 mm for A–C and H–L, and 1 mm for D–G, M–O.
	Fig. 2. Lung development in air-deprived (AD) and air-restored (AR) Xenopus.(A,B) Frontal sections of AD tadpoles at (A) NF 46 showing normal lung development and (B) NF 49 showing expanded bronchi (b) and compressed, folded lungs (arrows). (C) Ventral view of a dissected AD NF 57 tadpole showing translucent stunted lungs against a blue background. (D,E) Frontal sections of an AD NF 57 tadpole showing (D) ventrally a compressed, thick walled bronchus with a pulmonary artery (pa) and small lumen anterior of the peritoneum (p) and (E) dorsally a compressed, folded lung abutting the liver (li) and gut tube (gt); arrows indicate incipient epithelial folds and brown is BrdU labeling. (F) Frontal section of a stunted AD NF 57 lung with fluorescently stained smooth muscle actin (red) and nuclei (white) showing little actin adjacent to the epithelium (arrows) and partial filling of the lumen by blood cells (the continuous actin layer is in the wall of the esophagus, e). (G–I) Frontal sections of AD NF 66+ frogs showing (G) ventrally a compressed bronchus, (H) dorsally the absence of a lung and the bronchus extending into the peritoneum-lined space normally occupied by the bronchial diverticulum, and (I) cartilage (c, blue) and BrdU labeling (brown) in the wall of a compressed bronchus. (J,K) Sagittal sections through the wall of a 19-month old AD lung showing a thick connective tissue layer containing cartilage (c), veins, and bands of actin fibers (pink in J and brown in K), and large incipient epithelial folds (arrows) protruding into the lumen (lower side). (L) A tissue-filled lung showing a thin wall, vein (v) and lumen filled with blood cells. (M) A section through the spleen of an adult frog showing the red (rp) and white pulp (wp). (N–P) Dorsal views of whole-mounted actin-stained lungs and esophagus (e) of (N) an AD NF 51 tadpole with weakly defined, closely spaced actin bands, (O) an AR NF 53 tadpole 9 days after air restoration with actin bundles more closely spaced than in untreated NF 52 lungs, and (P) an AR NF 59 tadpole 4 days after with widely spaced actin bundles but not the lateral subdivisions seen in untreated NF 59 lungs. Anterior is up for frontal views A–I and left for sagittal sections J,K. Scale bars are 0.5 mm for A,B; 1 mm for C, N and O; 0.1 mm for D–M; and 2 mm for P.
	Fig. 3. Swimming and breathing behaviors as a function of days after air restoration for experiments 1 and 2 on 8-week old tadpoles and frogs.(A) Swimming behaviors for tadpoles in bowl 3 and controls of experiment 1. (B) Breathing frequencies for tadpoles in bowls 1–3 of experiment 1. (C) Breathing frequencies and relative lung sizes for tadpoles in bowls 1–5 of experiment 2; error bars indicate maximum and minimum lung lengths of specimens sampled on each day. (D) Breathing frequencies and POS values of the water for tadpoles in bowls 3–5 of experiment 2. (E) Floating behavior for frogs in bowls 1–3 of experiment 1; bowl 1 and 2 specimens were sampled or died before day 3; controls (not shown) did not float. (F) Breathing frequencies for frogs in bowls 1–3 and controls of experiment 1.

References [+] :

Barja de Quiroga, Hyperoxia decreases lung size of amphibian tadpoles without changing GSH-peroxidases or tissue peroxidation. 1989, Pubmed