Animals
Rana pirica tadpoles were collected in Hokkaido, Japan, from June to September, kept in filtered water, and staged according to [22, 23]. We further subdivided the four- to five-digit formation stages (stages 36 to 37) into five stages (see Fig. S1 for details) to provide a more detailed view on limb development. African clawed frog (Xenopus laevis) tadpoles were kept in 0.1% sea–salt solution at 16 °C and staged according to a developmental table [24]. Post–metamorphic froglets of R. pirica and X. laevis were photographed with a single–lens reflex camera (D5300, Nikon) (Fig. S2).
Lung morphology
For observation of lungs, R. pirica tadpoles at stages 31–37 were anesthetized with 0.003% tricaine methanesulfonate in water, and lungs were dissected immediately under a dissecting microscope to observe the presence of air bubbles as well as vascularization. For anatomical studies, lungs dissected from early stage 37 tadpoles were stained with hematoxylin and eosin as described [25] with slight modifications. Briefly, dissected lungs were fixed in 4% paraformaldehyde (PFA) overnight, and then dehydrated in a graded ethanol series. Ethanol was then replaced with acetone, and the specimens were embedded in Technovit 8100 resin (Heraues–Kulzer). Sections were cut at a thickness of 12 μm, stained with hematoxylin, washed in tap water, stained with eosin, washed in tap water, treated with ethanol, xylene and covered in Mount Quick (Daido Sangyo).
Frequency of air–breathing
The frequency of air–breathing behavior of X. laevis and R. pirica tadpoles (Fig. S3a; Movies 1 and 2) was compared at stages 54–56 and 36–37, the stages in which all five digits of hindlimbs are formed, respectively. The same water source and conditions were used for both species. The number of times the mouth of each tadpole reached the water surface was determined during a total of 10 minutes, excluding times when the tadpole was resting for more than 1 minute. For each species, three individuals were used per experiment, and the experiments were conducted three times each. The air–breathing behavior of R. pirica tadpoles was photographed with a single–lens reflex camera (D5300, Nikon).
Detection of cell death and ROS
Cell death was detected using LysoTracker, Nile Blue, or TUNEL, as previously described [17, 26, 27]. LysoTracker labels the increased lysosomal activity detected in dying cells and around phagocytosed cell debris [28, 29] and has been used to identify the cell death in limb buds of various tetrapods, including X. laevis and coqui frogs [17, 30]. Briefly, tadpoles at stages 32–37 were incubated with 0.5 μM LysoTracker Red (Thermo Fisher Scientific, Waltham, MA) in PBS+ (PBS pH 7.4, 9 mM CaCl2, 3.3 mM MgCl2) for 2 hours, washed and photographed with na LSM780 confocal microscope (Zeiss). Some of the LysoTracker Red-stained tadpoles were then stained with 0.01% Nile blue in filtered water for 20 minutes, washed, and photographed. For TUNEL staining, hindlimb buds from stage 36 tadpoles were cryosectioned at 8–12 μm as described [17], and stained using TUNEL Mix (In situ Cell Death Kit, Roche) according to the manufacturer’s protocol. For detection of cell death and reactive oxygen species, tadpoles at stage 36–37 were incubated with 0.5 μM LysoTracker Green and with 2 μM CellROX Deep Red (Invitrogen) in PBS+ for 2 hours, washed, and photographed with an LSM780 confocal microscope (Zeiss).
Oxygen incubation
Oxygen incubation of tadpoles were carried out as described [17]. Briefly, incubations under high (60%) oxygen atmosphere were made using a multigas incubator (APM–30D, Astec). Normoxic (21%) controls were incubated at atmospheric oxygen levels using a regular incubator (FMU–053, Fukushima Industries). Oxygen levels in the water were measured using a dissolved oxygen meter (DO–5509, Satotech). Tadpoles were immersed in 1.8 cm depth filtered water at 25 °C individually in plastic plates for 3 hours. After oxygen incubation, tadpoles were stained with LysoTracker and photographed, and the number of LysoTracker-positive cells was counted.
Cell counting
We counted the number of the LysoTracker-positive cells as described [31], with modifications. The interdigital region was defined as the area between the outlines of digit primordia recognized by the confocal image obtained through the T–PMT channel. Then, the confocal image was processed using Fiji, ImageJ (https://fiji.sc). Images were converted to 8–bit images, adjusted for contrast, nonlinearly transformed with the gamma function, and binarized with the threshold. We considered the particle size from 5 to 8 to 200 pixels as cells, which were counted using the Analyze Particles function.
Visualization of blood vessels
Blood vessels were visualized as previously described [17, 31]. Briefly, yellow highlighter ink diluted 1:1 in PBS (PUSR80.2 cartridge, Mitsubishi Pencil) was injected using pulled glass capillaries (Narishige) into the blood vessels of anesthetized tadpoles at stages 36 and 37. After 10 minutes, the samples were fixed with 4% PFA and photographed.
Manipulation of air breathing behavior
For short term incubation, R. pirica tadpoles at early stage 36 were reared overnight in a 500 ml beaker without a plastic net (control), or with a plastic net to prevent the tadpoles from rising to the water surface (Fig. 3a). Tap water was filtered and poured into each beaker. For long-term incubation, R. pirica tadpoles at early stage 36 were reared freely (control) or in a 15 ml plastic tube covered with a plastic net for 3 days in a 1.5 L tank (Fig. 3b). Water in the 1.5 L tank was changed daily. The use of a plastic net stopped air breathing behavior, but did not interfere with the amount of dissolved oxygen in the beaker. After incubation in a beaker or a tank, tadpoles were stained with LysoTracker, photographed, and the number of LysoTracker-positive cells was counted.
Statistical analysis
Statistical analyses were performed with Prism 8.0.2 (GraphPad). The data are presented as the mean ± SEM. Significance was determined using two–tailed unpaired t–tests for comparisons between different limbs. Significance was defined as P ≤ 0.05 (∗p < 0.05).
Images
Images were captured using an LSM780 confocal microscope (Zeiss), stereomicroscope (MF16F, Leica; BX61WI, Olympus) with microscope digital camera (DP74, Olympus), or single–lens reflex camera (D5300, Nikon). Pseudocolor images were generated using Zen Black software (Zeiss). The following panels are maximum intensity projections of confocal image stacks: Figs. 1c, 2ab, S5a. The following panels were flipped horizontally: Fig. 1c (stage 32, stage 34, mid stage 36, and late stage 36), Fig. S5b.