Abstract Lymphatic vessels play a role in several physiological and pathological processes including tissue fluid homeostasis, dietary fat absorption, immunosurveillance, and immunomodulation. During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of the transcription factor Prospero-related homeobox 1 ( PROX1). PROX1 is essential for lymphatic vascular network formation in mouse and zebrafish. The initiation of PROX1 expression precedes LEC sprouting and migration, serving as the definitive marker of specified LECs. Despite its crucial role in lymphatic development, the upstream regulation of PROX1 in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 expression in mice by binding to its promoter region. However, how the specificity of Prox1 expression to LECs is achieved remains to be studied in detail. In this study, we analysed evolutionary conservation and chromatin accessibility to identify enhancer sequences located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this genomic region results in impaired valve morphology and function. Overall, our results reveal the intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, while vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic vessels. Graphical Abstract

Multiplecis-regulatory elements controlprox1aexpression in distinct lymphatic vascular beds