*2.3. Tissue Ultrastructure*

TEM was used in this study to properly observe the collagen microstructure and orientation, shown in Figure 5. Images at 3400× magnification reinforce the observations from histology and show unique patterns in the *EC* gular tissue compared to *XL* and *XM*. Higher magnification images (13,500×) were obtained from the areas highlighted with the red box in the first row of Figure 5A–D and shown in the second row (Figure 5E–H). At higher magnification, the intricate orientation and design of the collagen layers can be identified. Figure 5E,F show a tri-dimensional collagen arrangemen<sup>t</sup> defined by the waviness, transitions from circular to rectangular cross-section within a layer, and contrast differences showing axial twisting. Although the layering pattern is present in the *Xenopus* tissues, other patterns are not observed. In contrast, there are discontinuities within the collagen layers of the *Xenopus* tissues shown in Figure 5C,D.

**Figure 5.** TEM images of gular tissue for (**A**) male EC, 3400×. Scale bar 2 μm; (**B**) female EC, 3400×. Scale bar 2 μm; (**C**) XL, 1000×. Scale bar 10 μm; (**D**) XM, 4200×. Scale bar 10 μm; (**E**) male EC, 13,500<sup>×</sup>. Scale bar 500 nm. Green lines represent the crimp angle measurements (θ1 = 80◦, θ2 = 70◦). (**F**) Female EC, 13,500<sup>×</sup>. Scale bar 500 nm. Green lines represent the crimp angle measurements (θ = 77◦). (**G**) XL, 3400×. Scale bar 2 μm. (**H**) XM, 13,500<sup>×</sup>. Scale bar 500 nm. Red boxes indicate the area magnified.

Furthermore, Figure 5E,F can be used to identify key physical characteristics of the crimped collagen structure. Firstly, there is a clear "layering" structure within the bulk tissue, which indicates an alternating collagen bundle orientation across the tissue. In the male *EC*, a collagen layer (Figure 5E) is approximately 500 nm thick, whereas the female *EC* is 667 nm thick. The two figures also show very clearly that the male *EC* gular tissue has more layers within the same field of view. Secondly, calculations were conducted to identify the crimp angle of the collagen bundles. As shown in Figure 5, the angles between the two folded layers of the male *EC* tissues are 80◦ and 70◦, while there is a single fold of 77◦ for the female EC tissues. Those of the *Xenopus* genus were virtually 0◦, meaning that they were nearly straight throughout the tissue.

Some similar features were observed in the rat bladder and are shown in Figure 6. Rat bladder and *EC* gular skin had several features in common, such as out-of-plane orientation and crimping (Figure 6A,B). As shown in Figure 6D, collagen strands change from longitudinal to circular cross-sectional orientation, similar to those seen in Figure 5E,F. However, the rat bladder lacks the layered structure found in *EC* tissues. Rat bladder had a thick collagen bundle (2.5 μm), shifting its orientation all at once, whereas *EC* tissues had smaller bundles stacked on top of each other and alternating their orientation. Figure 6B,E resemble a crimped collagen bundle. This was a short bundle and was seldom identified across the tissue. As expected, a helical structure was observed in the rat bladder (Figure 6C,F). The twisted collagen strands identified in Figure 6F show very clearly the formation of spring-like structures, reminiscent of those found in the human bladder. These observations were frequently found throughout the tissue cross-section.

**Figure 6.** TEM images of the rat bladder. (**A**) Alternating collagen orientation. 17,500<sup>×</sup>. Scale bar 500 nm. (**B**) Crimp structure. 13,500<sup>×</sup>. Scale bar 500 nm. (**C**) Helical structure. 9700×. Scale bar 500 nm. (**D**) Magnified alternating collagen orientation. 33,000<sup>×</sup>. Scale bar 500 nm. (**E**) Magnified crimp structure. 24,500<sup>×</sup>. Scale bar 500 nm. (**F**) Magnified helical structure. 17,500<sup>×</sup>. Scale bar 500 nm.
