Retrospective clonal analysis of the developing mouse heart

espanolEl tracto de salida cardiaco (OFT) esta compuesto por celulas endoteliales (EC), celulas de musculo liso (SMC), fibroblastos (Fbs) y cardiomiocitos (CM) que derivan de los progenitores de la cresta neural y del segundo campo cardiaco (SHF). El OFT esta recubierto por una capa de celulas mesoteliales arteriales (AMCs) continua al epicardio. El epicardio proviene del proepicardio. Sin embargo, el progenitor temprano de las AMCs esta desconocido. Mediante el uso de lineas Cre de mesotelio y de SHF, identificamos el origen de las AMCs en el mesodermo faringeo dorsal. Durante un analisis sistematico de los linajes cardiacos mediante el etiquetado clonal aleatorio en corazones de ratones en desarrollo, encontramos que las AMCs comparten una relacion clonal con las celulas de las grandes arterias. Los clones de AMCs eran diferentes segun su posicion en las grandes arterias. Por un lado, las AMCs distales en ambos vasos eran asociadas clonalmente con Fbs, SMC y EC. Por otro lado, las AMC proximales de la arteria pulmonar (AP) eran asociadas clonalmente con Fbs, SMC, CM y celulas endoteliales linfaticas (LEC) de la parte ventral del corazon. Sin embargo, estos clones no solapan con los clones linfaticos de la parte dorsal del corazon, lo que sugiere multiples origenes para los vasos linfaticos cardiacos. El trazado de los progenitores VEGFR3 + a diferentes estadios, demostro que los linfaticos dorsales derivan de vasos linfaticos preexistentes mientras que la formacion de linfaticos ventrales implica el reclutamiento tardio de celulas no linfaticas. Usando un abanico de lineas Cre especificas del SHF, Recapitulamos el desarrollo de los linfaticos ventrales exclusivamente. Esto sugiere que los progenitores del SHF se reclutan en la region mesotelial/sub-mesotelial de la AP. Transplantamos trozos de mesotelio / sub-mesotelilo de la AP de donantes fluorescentes a corazones de tipo salvaje a E14.5. El Tejido donante invadio el miocardio del recipiente, formando vasos linfaticos. La exploracion de senales moleculares involucradas en la formacion de ese nicho mostro que Raldh2 esta regulado a la baja a nivel transcripcional y proteico en las AMCs proximales. Ademas, la perdida de senalizacion del Acido Retinoico (AR) en las celulas mesoteliales resulto en vasos linfaticos inmaduros, mientras que un exceso de AR desencadeno una hiper-remodelacion linfatica. Estos datos muestran que parte de los linfaticos ventrales se forman mediante el reclutamiento de progenitores de SHF en un nicho vasculogenico specificado en condiciones de bajo niveles de Raldh EnglishThe 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 non-lymphatic 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. 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