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. 2024 Jul 10;631(8021):654–662. doi: 10.1038/s41586-024-07623-6

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 12 — a, Whole-mount immunostaining of myosin heavy chain (myosin) in avian hindlimb at stages HH30 and HH32 of embryonic development. Ventral views of the limbs are shown, with limb margins outlined in yellow. Lower panels: Zoomed-in views of the boxes in the upper panels. The foot and shank muscles are labelled. b-e, Longitudinal sections of stage HH33 limbs. b, Co-staining of myosin and TUNEL, showing the muscular ventral death zone (MVDZ) at the zeugopod area. c, Co-staining of TUNEL and 4-HNE at the MVDZ in (b). d, e, Co-immunostaining of myosin and 4-HNE in the foot (d) and shank (e) regions. Degenerating muscles display a rounded and beaded appearance. Data shown (a-e) are representative of three biological repeats. f, g, 4-HNE and TUNEL signals are abundant at the central region relative to the lateral regions of the limb zeugopod. Upper panels: Immunostaining of 4-HNE (f) and TUNEL staining (g) in stage HH32 limbs. Lower panels: The relative mean values of 4-HNE (f) and TUNEL (g) signal intensities were calculated along the indicated region (lateral and central, grey box 1600 × 1000 µm²) for three biological repeats (yellow, blue, orange curves). h, The 4-HNE and TUNEL signal intensities at the central and lateral regions normalized to the total signal intensities. i, j, In ovo ferroptosis suppression impairs muscle remodelling in avian embryonic limb. Muscle fibre count (i) and distribution of entropy of the muscle fibre orientation (j) in embryonic limbs dissected from UAMC-3203-treated and vehicle control (DMSO)-treated embryos. Data represent mean ± s.d. of three biological repeats. The muscle fibre count and entropy of the muscle fibre orientations for limbs dissected from UAMC-3203-treated embryos are higher than those from the control embryos (* two-sided Wilcoxon rank-sum tests, P = 0.0495 and 0, respectively). Scale bars, 500 (upper panels in a), 300 (lower panels in a), 200 (b), 50 (c), 100 (d, e), and 400 (f, g) μm.

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