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. 2022 May 16;13:2690. doi: 10.1038/s41467-022-30416-2

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Immunostaining for CGRP, a marker of pulmonary neuroendocrine cells (PNECs) on sections from E18.5 Shh^cre/+;Rest^+/+, Rest^+/fl, and Rest^fl/fl lungs. DAPI stains DNA in blue. Scale bar, 50 μm. b–d Quantification of the average number of CGRP⁺ PNECs (b) and neuroepithelial bodies (NEBs) (≥5 PNECs) (c) per lung section from each embryo (n = 5 embryos with 37 sections counted for Rest^+/+ and 4 embryos with 30 sections counted for Rest^+/fl and Rest^fl/fl). Unpaired t-test, data represented as mean ± s.d. In (d), a scatterplot of all solitary PNECs and clusters of each embryo (n = 438 for Rest^+/+, 688 for Rest^fl/fl). Wilcoxon rank sum test, two-sided, on the combined data points of each genotype. e, Median number of PNECs per cluster for each embryo as in (d) (n = 5 embryos for Rest^+/+ and 4 embryos for Rest^fl/fl). Unpaired t-test, data represented as mean ± s.d. f Relative mRNA expression levels of NE markers and Rest in E18.5 Shh^cre/+;Rest^+/+, Rest^+/fl, and Rest^fl/fl lungs (n = 5 embryos for Rest^+/+ and 4 embryos for Rest^+/fl and Rest^fl/fl). Unpaired t-test, data represented as mean ± s.d. g Representative brightfield images of Chga RNAscope in situ hybridization (ISH, red signal) in E18.5 Shh^cre/+;Rest^+/+ and Rest^fl/fl lungs (hematoxylin counterstain). Scale bar, 50 μm. h Quantification of the average number of RNAscope signal per μm² of lung section in (g) (n = 3 embryos for Rest^+/+ and 4 embryos for Rest^fl/fl). Unpaired t-test, data represented as mean ± s.d. i, Representative images for Chga RNAscope ISH (orange) and E-cadherin immunofluorescence (red) in E18.5 Shh^cre/+;Rest^+/+ and Rest^fl/fl lungs. DAPI stains the DNA in blue. White arrows, NEBs. Scale bar, 100 μm. See also Supplementary Fig. 8. Source data are provided as a Source Data file.