Co-development of epithelium and vasculature in early developing submandibular gland. Co-development of epithelium and vasculature in early developing submandibular gland. (A) Mouse embryonic salivary glands were excised and subjected to ICC and confocal imaging to detect CD31+ (cyan) endothelial cells, shown superimposed on a brightfield image. (B) Single confocal images of the glands without brightfield are shown at higher magnification. A CD31+ semi-discontinuous vasculature with some isolated endothelial islands (white arrow) was observed in E11.5-E12.5 mesenchyme (M) that was largely isolated from the emerging epithelial bud (E). In E13-E13.5 glands, a largely continuous vessel network (yellow arrow) was observed that progressively surrounds the epithelium and is distinct from the developing nerves (Tubb3+, red). Note that the CD31+ vessels penetrate into the maturing epithelial clefts at E13 (white arrowhead). (C) CD31+ vessels persist in E12.5 glands cultured ex vivo (shown here after 6 h and after 24 h). CD31+ endothelial cells also express VEGFR2 (red) and collagen IV (green). Because collagen IV is incorporated into the basement membrane of both the endothelial cells and the epithelial cells, it defines the boundary between the epithelium and mesenchyme. CD31+/VEGFR2+/collagen IV+ endothelial cells are found adjacent to the initiating epithelial clefts at E12+6 h growth (arrowheads in upper panels) and ingress into maturing clefts after 24 h of culture (arrowheads in lower panels). (D) Schematic summarizing co-development of the primary epithelial bud with vasculature in the early submandibular gland. Endothelial cells comprising discontinuous vasculature are found in mesenchyme at E12/E12.5. Developing vessels subsequently undergo elongation and maturation concomitant with branching of the primary epithelial bud. Hae Ryong Kwon et al. Development 2017;144:211-220 © 2017. Published by The Company of Biologists Ltd