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. 2024 Oct 10;15:8793. doi: 10.1038/s41467-024-53009-7

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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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Fig. 4 — A ISH revealing Shox2 downregulation in the cardiac sinus venosus (SV) (red arrowhead) and nodose ganglion of the vagus nerve (red asterisk) in GD^Δ/Δ embryos at E10.5. White arrow indicates normal Shox2 expression in the dorsal root ganglia (DRG) of GD^Δ/Δ embryos. Scale bar, 100 μm. B Quantitative PCR (qPCR) revealing depletion of Shox2 in GD^Δ/Δ hearts compared to WT controls at E11.5. Box plots indicate interquartile range, median, maximum/minimum values (bars). Dots represent individual data points. ****P < 0.0001; n.s., not significant (two-tailed, unpaired t-test). C Co-localization of SHOX2 (green), HCN4 (red) and NKX2-5 (blue) in hearts of GD^Δ/Δ and WT control embryos at E11.5. SHOX2 is lost in the HCN4-marked SAN pacemaker myocardium in absence of the gene desert (dashed outline). Nuclei are shown in gray. Scale bars, 50 μm. D SAN enhancer candidate regions in the gene desert interval (VS-250) essential for Shox2 expression in the SV⁵⁵. Top: Virtual 4 C (v4C) Shox2 promoter interaction signatures in embryonic hearts (HT) and limbs (FL) (gray) at E11.5 overlapped with HT (red) and FL (green) -specific subtraction profiles. Below: ATAC-seq tracks from embryonic hearts at E11.5 and SAN cells from sorted Hcn4-GFP mouse hearts at P0 (Supplementary Data 3)^46,66. Desert enhancers (DEs) (black) and putative SAN enhancer elements with distance to the Shox2 TSS in kb (+) are indicated. Cons, vertebrate conservation track by PhyloP. E Transgenic LacZ reporter validation in mouse embryos at E11.5. Left: the +325 element drives transgenic LacZ reporter expression exclusively in the SV. Right: the +325A subregion drives Shox2-overlapping SV activity, similar to +325B (Supplementary Fig. 5). The interval shared between +325A/B subregions contains a conserved core element (marked gray) that interacts with TBX5 in embryonic hearts at E12.5⁶⁷. “n” denotes fraction of biological replicates with reproducible results. Single numbers represent the total of transgenic embryos obtained, including those without staining. RA, right atrium. RV, right ventricle. OFT, outflow tract. Corresponding Vista Enhancer IDs (mm) are listed in Supplementary Table 5. Source data are provided in the Source Data file.