Extended Data Fig. 8: The role of macrophages in prenatal skin neurovascular development.

(a) UMAP showing clustered cell states in integrated data from embryonic immune cells⁵⁷ and prenatal skin myeloid cell subset. (b) Heatmap showing the correspondence (measured by Jaccard index) between embryonic immune cells (x-axis) and prenatal skin (y-axis) myeloid cell states for a logistic regression model trained on embryonic data⁵⁷ and projected onto prenatal skin myeloid cell subset. TML macrophage had the highest proportion prediction to Mac4 (embryonic brain microglia). (c) Gene set enrichment analysis results of over-expressed genes (wilcoxon, two-sided, Benjamini-Hochberg adjusted) in macrophage subsets (TML, Iron-recycling, LYVE1⁺ and MHCII⁺, macrophages). Each plot shows the top 10 enriched gene sets (using Gene Ontology Biological Process 2023 (left) and MSigDB Hallmark 2020 (right) databases). The x-axis shows the negative log₁₀ of the adjusted p-value (Fisher’s exact test, Benjamini-Hochberg correction for multiple testing); dot size is proportional to the number of genes associated with the gene set and colour represents the combined Enrichr score calculated within GSEApy. (d) Heatmap showing prediction probabilities from a logistic regression model trained on classes of reindeer fibroblasts (pro-regenerative, pro-inflammatory and mixed populations) (x-axis), projected onto prenatal skin fibroblasts grouped by age (y-axis). Colour scale indicates median prediction probabilities. (e) Dot plot showing the variance-scaled, mean expression (dot colour) and percent of expressing cells (dot size) of selected pro-regenerative and pro-fibrotic genes in prenatal skin fibroblasts by gestational age (grouped PCW). (f) Heatmap showing prediction probabilities from a logistic regression model trained on reindeer macrophage clusters (x-axis), projected onto prenatal skin macrophage subsets (y-axis). Colour scale indicates median prediction probabilities. (g) Percentage scratch width closure (y-axis) quantified over time (x-axis) for fibroblasts cultured with macrophages (green) or in isolation (black) in n = 3 independent experiments. Data represented as percentage values ±SD. Statistics were generated with two-way ANOVA with Tukey’s multiple comparisons test (p-values shown at 6hrs: 0.0480, 12hrs: 0.0035, 18hrs: 0.0001, 24hrs: 0.0042, 66hrs: 0.0118, 72hrs: <0.0001). (h) Circos plot visualisation of selected significant (adjusted p-value <0.05, significance calculated in CellphoneDB using empirical shuffling and FDR-adjusted) predicted interactions between TML macrophages and co-localising neural cells in prenatal skin. Arrows represent directionality of interactions (ligand to receptor); connections are coloured by sender cell type with width proportional to the CellphoneDB mean value for each ligand-receptor pair. (i) Heatmap of normalised (z-score) mean expression of angiogenesis gene modules in prenatal skin macrophages. CD7^hiP: CD7^high progenitors; CD7^loP: CD7^low progenitors; ErP, erythroid progenitors; GMP, granulocyte-monocyte progenitors; HSPC, haematopoietic stem and progenitor cells; LC, Langerhans cells; Mac1-4, macrophages 1-4; MkP, megakaryocte progenitors; YSMP, yolk-sac derived myeloid-biased progenitors. For details on statistics and reproducibility, see Methods.

Source Data