Migration of lymphatic endothelial cells and lymphatic vascular development in the craniofacial region of embryonic mice.

Lymphatic development in mice is initiated in the trunk at embryonic day (E) 9.5. This study aimed to examine the origin of craniofacial lymphatic endothelial cells (LECs) and the developmental process of lymphatic vessels in the mouse craniofacial region. Serial sections from ICR mouse embryos at E9.5-E14.5 were immunolabeled with LEC and venous endothelial cell (VEC) markers. These markers included prospero homeobox protein 1 (Prox1), vascular endothelial growth factor receptor 3 (Vegfr3), lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and C-C motif chemokine 2 (Ccl21) for LEC, and COUP transcription factor 2 (CoupTF2) and endomucin (Emcn) for VEC. LECs were monitored as an index in Prox1/Vegfr3 double-positive cells using three-dimensional analysis because LECs express Prox1 and Vegfr3 ab initio during lymphatic vascular development. LECs appeared in VECs of the lateral walls of cardinal veins (CVs) at E9.5. These LECs were dichotomized into LEC populations that formed lymph sacs close to CVs and were scattered in the surrounding CVs. The scattered LECs formed cellular streams and extended from the trunk to the mandibular arches at E10.5 - E11.5. In the mandibular arches, individual LECs aggregated, and formed lymph sacs and tubular lymphatic vessels at E11.5-E14.5. Expression of the LEC marker proteins Lyve1 and Ccl21 in LECs changed during craniofacial lymphatic vascular development. Collectively, these findings suggest that craniofacial LECs originate from CVs of the trunk and migrate into the mandibular arches. Additionally, we found that craniofacial lymphatic vessels are formed according to morphogenesis of individual LECs that migrate from CVs.

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