Fig. 2: CD44 splits the VE-Cadherin⁺ cells of the AGM into different populations.

a Immunofluorescence of VE-Cad (magenta) and CD44 (green) expression in a cross-section of the AGM region of a wild-type embryo at E10 (32 somite pairs). Images 1 and 2 show higher magnification of the areas highlighted in the main image, showing CD44 marking endothelial cells in the vascular wall and a haematopoietic cluster. Scale bars represents 25 μM. b FACS plots indicating percentage of cells expressing high levels of VE-Cad from dissected AGMs of wild-type embryos. The histograms indicate the percentage of VE-Cad^High cells positive for CD44 at both E9.5 (28 somite pairs) and E10.5 (35 somite pairs) compared with the FMO. c Percentage of CD44⁺ cells within the VE-Cad^High fraction, each data point represents an independent experiment and independent pooled litter of embryos, E9.5 n = 4 and E10.5 n = 5. We compared the average proportion of CD44⁺ cells at E9.5 (M = 22.13, SD = 4.85) to E10.5 (M = 30.88, SD = 5.08). Significance was determined by a Welch’s two-sample t-test, t(6.7) = 2.635, p-value = 0.0345. d Representative FACS plots of gating strategy and expression of VE-Cad and CD44 in the AGM region of wild-type embryos at E10 (30–34 somite pairs). Expression of Kit cell surface marker is highlighted for the CD44^Low population. Full gating strategy is shown in Supplementary Fig. 5a. e Mean FSC-A as an indication of cell size is plotted for each population (CD44^Neg, CD44^LowKit^Neg, CD44^LowKit^Pos and CD44^High) for five independent litters of E10 wild-type embryos (n = 5). Significance was determined by a one-way ANOVA, F(3, 16) = 142.01, p-value < 0.0001 followed by Tukey’s HSD post-hoc tests to determine where the significance lies *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SD. Source data are provided as a Source Data file. See also Supplementary Fig. 2.