Fig. 5: Left–right asymmetry at the late-gastrulation stage.
a The strategy of laser capture microdissection of cell samples of E7.5 embryos. For the ectoderm and endoderm germ layers, the same Geo-seq strategy was applied as in Supplementary Fig. 1. The mesoderm germ layer was partitioned into MAL (anterior left mesoderm), MAR (anterior right mesoderm), MPL (posterior left mesoderm) and MPR (posterior right mesoderm) areas for sampling. Sampling areas are shown in histology images; scale bar, 60 μm. Three biologically independent sequencing replicates were prepared. b Corn plots showing the spatial pattern of expression of Pou3f1 and Sox7. Hollow circles indicate missing samples. c Heat map showing the differentially expressed genes (DEGs) of the left lateral mesoderm (n = 518) and right lateral mesoderm (n = 881) (one-sided test, p < 0.05, fold change >1.5). The enriched gene ontology (GO) terms for each group were listed on the right (p < 0.01). d The enrichment for target and response genes of development-related signaling pathways in the left and right mesoderm. Signaling activity: red, activating (A); green, inhibitory (I). The significance of −log10(FDR) value in each cell was calculated by one-sided Fisher’s exact test followed by Benjamini–Hochberg correction. e Deconvolution analysis inferred the proportion of left and right lateral mesoderm cell populations and visualized on a t-SNE plot. Cells are colored by inferred positions: MA-L, MP-L, MA-R, and MP-R. f t-SNE plots showing the distribution of Lefty2-expressing cells in the left mesoderm and Hand1- and Smad6-expressing cells in the right mesoderm. g Corn plots showing the distribution of Lefty2-expressing cells in the left mesoderm and Hand1- and Smad6-expressing cells in the right mesoderm. h RNAscope analysis validated the bilaterally asymmetric expression of Lefty2, Hand1, and Smad6 in the selected transverse sections (S-numbered, reference: (g)). The right panels summarize the quantified signal intensity and statistical results, data are presented as mean values ± S.E.M, n = 10 for Lefty2, n = 9 for Hand1, n = 5 for Smad6, n represents biologically independent samples subject to RNAscope analyses. Student’s t-test was performed, *** represents p-value < 0.001, the exact p-values for Lefty2, Hand1, Smad6 group were 0.00011, 1e−6, 0.00086, respectively.
