Retrospective clonal analysis of the developing mouse heart

Abstract

The cardiac OFT is composed of endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts (Fbs) and Cardiomyocytes (CMs) known to derive from neural crest and second heart field (SHF) progenitors. The OFT is also covered by a mesothelial sheet of arterial mesothelial cells (AMCs) continuous with the epicardium. While the epicardium arises from the proepicardium, the early progenitor of AMCs is unknown. Using mesothelial and SHF related Cre lines we identified the origin of AMCs to be in the dorsal pharyngeal mesoderm. During a systematic analysis of cardiac lineages by random clonal labelling in developing mouse hearts, we found that AMCs shared a clonal relationship with cells of the great arteries. AMC clones were of different nature along the great arteries. On one hand, distal AMCs in both vessels were clonally related to Fbs, SMCs and ECs. On the other hand, the proximal AMCs of the Pulmonary artery (PA), were clonally related to Fbs, SMCs, CMs and lymphatic endothelial cells (LECs) of the ventral part of the heart. These clones however, were never found together with lymphatic clones of the dorsal part of the heart suggesting multiple origins for cardiac lymphatic vessels. Fate mapping of VEGFR3+ progenitors at different stages showed that whereas dorsal lymphatics derive from pre-existing lymphatic vessels, formation of ventral lymphatics involves the late recruitment of nonlymphatic cells. Using a range of SHF-specific Cre lines, we found that SHF-derived cells form ventral but not dorsal cardiac lymphatics. These observations suggest that SHF progenitors are recruited in the mesothelial-sub-mesothelial region of the PA. To test this hypothesis, we transplanted portions of PA mesothelial/sub-mesothelial layers from fluorescent donors into wild type E14.5 hearts. Donor tissue invaded the host myocardium, forming lymphatic vessels. The exploration of molecular cues involved in the formation of that niche showed that Raldh2 is downregulated at transcriptional and protein levels in proximal AMCs. Moreover, the loss of RA signalling in mesothelial cells resulted in immature lymphatic vessels while an excess of RA triggered lymphatic hyper-remodelling in the PA. These data show that part of the ventral lymphatics form by recruitment of SHF progenitors at a vasculogenic niche specified in a low Raldh2 environment at the base of the PA.