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. 2022 Jan 13;13:304. doi: 10.1038/s41467-021-27899-w

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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. 3 — A Spatial and temporal expression of Gbx2 in HACNS1 homozygous, chimpanzee ortholog line, and wild type E11-E12 embryos visualized by whole-mount in situ hybridization (ISH). Representative images are shown for each genotype at three fine-scale time points; see text and associated Source Data for details on staging. Magnified views of Gbx2 expression in limb buds are shown to the right of each embryo. Annotations of anatomical structures and developmental axes are indicated at the top right: FL forelimb, HL hindlimb, DI diencephalon, NT neural tube, PA pharyngeal arch, A anterior, P posterior. The arrows at the top far right indicate the anterior-posterior (A-P) and proximal-distal (Pr-D) axes for the magnified limb buds. Bottom right: Crown-rump lengths for all embryos assayed for Gbx2 mRNA expression by ISH. Each point indicates a single embryo. Colors denote each fine-scale time point (T1-T6). B Left: representative images of anterior, posterior, proximal, distal (top), and strong versus weak Gbx2 staining patterns (bottom). Anterior (A), posterior (P), and body wall (BW) domains are denoted on top left limb bud. Right: Gbx2 ISH staining pattern data across 6 developmental timepoints from each of three independent, blinded scorers (marked at top as counting replicates 1–3; see text and Fig. 3A for timepoint scheme and associated Source Data for annotations). The darkest shade for HACNS1 homozygous (dark green), chimpanzee ortholog line (olive), and wild type (teal) represents percentage of forelimbs or hindlimbs showing strong anterior and posterior limb bud staining. Medium-dark shade, as shown in the legend on the left, denotes strong anterior staining only, while the lightest shade denotes weak staining in any domain. Independent biological specimens were analyzed for n = 139 (wild type), n = 103 (chimpanzee ortholog line), and n = 106 (human ortholog line) forelimbs and n = 137 (wild type), n = 103 (chimpanzee ortholog line), and n = 102 (human ortholog line) hindlimbs. For body wall and pharyngeal arch scoring data see Supplementary Fig. 3A, B and associated Source Data.