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. 2024 Aug 21;15:7154. doi: 10.1038/s41467-024-51328-3

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 5 — a TUNEL assay of Esco2^fl/fl limb buds (n = 3) and Esco2^fl/fl;Prrx1-Cre^Tg/0 limb buds (n = 3) from E9.5-E11.5 in the coronal plane. Scale bar: 100 µm. b Corrected total cell fluorescence (CTCF) of TUNEL+ signal between Esco2^fl/fl and Esco2^fl/fl;Prrx1-Cre^Tg/0 limbs at E9.5, E10.5 and E11.5. n = 4 for Esco2^fl/fl and Esco2^fl/fl;Prrx1-Cre^Tg/0 limb buds at E9.5, n = 5 for each genotype at E10.5, and n = 2 for each genotype at E11.5. In each box plot, the center line indicates the median, the edges of the box represent the first and third quartiles, and the whiskers extend to span a 1.5 interquartile range from the edges. Asterisk indicates a significant difference using a one-tailed, Student’s t-test (p ≤ 0.05; p = 0.271, p = 0.038, p = 0.034 for E9.5, E10.5, and E11.5 respectively). c Immunofluorescent staining of Esco2^fl/fl (n = 3) and Esco2^fl/fl;Prrx1-Cre^Tg/0 (n = 3) limb buds at E10.5 (coronal plane) with cleaved-caspase 3 (CC3) and PECAM1. All samples were co-stained with DAPI. Scale: 100 µm. d Immunofluorescent staining of Esco2^fl/fl (n = 3) and Esco2^fl/fl;Prrx1-Cre^Tg/0 (n = 3) limb buds at E9.5 (coronal plane), E10.5 (coronal plane) and E11.5 (transverse plane) with PECAM1. All samples were co-stained with DAPI. Scale: 200 µm. e Whole mount immunofluorescent staining of Esco2^fl/fl and Esco2^fl/fl;Prrx1-Cre^Tg/0 limb buds at E10.5. Scale: 20 mm. f Average blood vessel diameter (µm) of Esco2^fl/fl (n = 3) and Esco2^fl/fl;Prrx1-Cre^Tg/0 (n = 3) limb buds at E10.5. Littermate pairs are color matched by gray-scale. Data shown are the average measurements from each sample with mean ± SD. Black bar indicates median. Asterisk indicates a significant difference using a one-tailed, Student’s t-test (p ≤ 0.05; p = 0.013). g Number of branch points per sample of Esco2^fl/fl (n = 3) and Esco2^fl/fl;Prrx1-Cre^Tg/0 (n = 3) limb buds at E10.5. Littermate pairs are color matched by gray-scale. Data shown are the total measurements from each sample with mean ± SD. Asterisk indicates a significant difference using a one-tailed, Student’s t-test (p ≤ 0.05; p = 0.047). h Immunofluorescent staining of Esco2^fl/fl (n = 3) limb buds and Esco2^fl/fl;Prrx1-Cre^Tg/0 (n = 3) limb buds from E9.5–E11.5 with Anti-macrophage in the coronal plane. Scale: 100 µm. All samples were co-stained with DAPI. Magenta box delineates region with high density of macrophages, magnified in bottom right corner. Source data for 5b, 5f, and 5 g are provided with this paper.