Wenjuan Pu

Yap1 Is Required for Endothelial to Mesenchymal Transition of the Atrioventricular Cushion

Background: YAP1 regulates EMT and cell proliferation. Results: Deletion of YAP1 in endocardial cells reduces EMT of endocardial cells, and causes cardiac cushion defect. Conclusion: YAP1 is required for cardiac cushion development. Significance: This is the first in vivo genetic evidence for YAP1 function in EMT. Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart diseases. Normally, heart valve mesenchyme is formed from an endothelial to mesenchymal transition (EMT) of endothelial cells of the endocardial cushions. Yes-associated protein 1 (YAP1) has been reported to regulate EMT in vitro, in addition to its known role as a major regulator of organ size and cell proliferation in vertebrates, leading us to hypothesize that YAP1 is required for heart valve development. We tested this hypothesis by conditional inactivation of YAP1 in endothelial cells and their derivatives. This resulted in markedly hypocellular endocardial cushions due to impaired formation of heart valve mesenchyme by EMT and to reduced endocardial cell proliferation. In endothelial cells, TGFβ induces nuclear localization of Smad2/3/4 complex, which activates expression of Snail, Twist1, and Slug, key transcription factors required for EMT. YAP1 interacts with this complex, and loss of YAP1 disrupts TGFβ-induced up-regulation of Snail, Twist1, and Slug. Together, our results identify a role of YAP1 in regulating EMT through modulation of TGFβ-Smad signaling and through proliferative activity during cardiac cushion development.

Endocardium Minimally Contributes to Coronary Endothelium in the Embryonic Ventricular Free Walls

RATIONALE There is persistent uncertainty regarding the developmental origins of coronary vessels, with 2 principal sources suggested as ventricular endocardium or sinus venosus (SV). These 2 proposed origins implicate fundamentally distinct mechanisms of vessel formation. Resolution of this controversy is critical for deciphering the programs that result in the formation of coronary vessels and has implications for research on therapeutic angiogenesis. OBJECTIVE To resolve the controversy over the developmental origin of coronary vessels. METHODS AND RESULTS We first generated nuclear factor of activated T cells (Nfatc1)-Cre and Nfatc1-Dre lineage tracers for endocardium labeling. We found that Nfatc1 recombinases also label a significant portion of SV endothelial cells in addition to endocardium. Therefore, restricted endocardial lineage tracing requires a specific marker that distinguishes endocardium from SV. By single-cell gene expression analysis, we identified a novel endocardial gene natriuretic peptide receptor 3 (Npr3). Npr3 is expressed in the entirety of the endocardium but not in the SV. Genetic lineage tracing based on Npr3-CreER showed that endocardium contributes to a minority of coronary vessels in the free walls of embryonic heart. Intersectional genetic lineage tracing experiments demonstrated that endocardium minimally contributes to coronary endothelium in the embryonic ventricular free walls. CONCLUSIONS Our study suggested that SV, but not endocardium, is the major origin for coronary endothelium in the embryonic ventricular free walls. This work thus resolves the recent controversy over the developmental origin of coronary endothelium, providing the basis for studying coronary vessel formation and regeneration after injury.

Genetic targeting of sprouting angiogenesis using Apln-CreER

Under pathophysiological conditions in adults, endothelial cells (ECs) sprout from pre-existing blood vessels to form new ones by a process termed angiogenesis. During embryonic development, Apelin (APLN) is robustly expressed in vascular ECs. In adult mice, however, APLN expression in the vasculature is significantly reduced. Here we show that APLN expression is reactivated in adult ECs after ischaemia insults. In models of both injury ischaemia and tumor angiogenesis, we find that Apln-CreER genetically labels sprouting but not quiescent vasculature. By leveraging this specific activity, we demonstrate that abolishment of the VEGF–VEGFR2 signalling pathway as well as ablation of sprouting ECs diminished tumour vascularization and growth without compromising vascular homeostasis in other organs. Collectively, we show that Apln-CreER distinguishes sprouting vessels from stabilized vessels in multiple pathological settings. The Apln-CreER line described here will greatly aid future mechanistic studies in both vascular developmental biology and adult vascular diseases.

Genetic lineage tracing identifies endocardial origin of liver vasculature

The hepatic vasculature is essential for liver development, homeostasis and regeneration, yet the developmental program of hepatic vessel formation and the embryonic origin of the liver vasculature remain unknown. Here we show in mouse that endocardial cells form a primitive vascular plexus surrounding the liver bud and subsequently contribute to a substantial portion of the liver vasculature. Using intersectional genetics, we demonstrate that the endocardium of the sinus venosus is a source for the hepatic plexus. Inhibition of endocardial angiogenesis results in reduced endocardial contribution to the liver vasculature and defects in liver organogenesis. We conclude that a substantial portion of liver vessels derives from the endocardium and shares a common developmental origin with coronary arteries.

Endocardium Contributes to Cardiac Fat

RATIONALE Unraveling the developmental origin of cardiac fat could offer important implications for the treatment of cardiovascular disease. The recent identification of the mesothelial source of epicardial fat tissues reveals a heterogeneous origin of adipocytes in the adult heart. However, the developmental origin of adipocytes inside the heart, namely intramyocardial adipocytes, remains largely unknown. OBJECTIVE To trace the developmental origin of intramyocardial adipocytes. METHODS AND RESULTS In this study, we identified that the majority of intramyocardial adipocytes were restricted to myocardial regions in close proximity to the endocardium. Using a genetic lineage tracing model of endocardial cells, we found that Nfatc1(+) endocardial cells contributed to a substantial number of intramyocardial adipocytes. Despite the capability of the endocardium to generate coronary vascular endothelial cells surrounding the intramyocardial adipocytes, results from our lineage tracing analyses showed that intramyocardial adipocytes were not derived from coronary vessels. Nevertheless, the endocardium of the postnatal heart did not contribute to intramyocardial adipocytes during homeostasis or after myocardial infarction. CONCLUSIONS Our in vivo fate-mapping studies demonstrated that the developing endocardium, but not the vascular endothelial cells, gives rise to intramyocardial adipocytes in the adult heart.

Preexisting endothelial cells mediate cardiac neovascularization after injury

The mechanisms that promote the generation of new coronary vasculature during cardiac homeostasis and after injury remain a fundamental and clinically important area of study in the cardiovascular field. Recently, it was reported that mesenchymal-to-endothelial transition (MEndoT) contributes to substantial numbers of coronary endothelial cells after myocardial infarction. Therefore, the MEndoT has been proposed as a paradigm mediating neovascularization and is considered a promising therapeutic target in cardiac regeneration. Here, we show that preexisting endothelial cells mainly beget new coronary vessels in the adult mouse heart, with essentially no contribution from other cell sources through cell-lineage transdifferentiation. Genetic-lineage tracing revealed that cardiac fibroblasts expand substantially after injury, but do not contribute to the formation of new coronary blood vessels, indicating no contribution of MEndoT to neovascularization. Moreover, genetic-lineage tracing with a pulse-chase labeling strategy also showed that essentially all new coronary vessels in the injured heart are derived from preexisting endothelial cells, but not from other cell lineages. These data indicate that therapeutic strategies for inducing neovascularization should not be based on targeting presumptive lineage transdifferentiation such as MEndoT. Instead, preexisting endothelial cells appear more likely to be the therapeutic target for promoting neovascularization and driving heart regeneration after injury.

Enhancing the precision of genetic lineage tracing using dual recombinases

The Cre–loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre–rox recombination system to enhance the precision of conventional Cre–loxP-mediated lineage tracing. The Dre–rox system permits rigorous control of Cre–loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates—the contribution of c-Kit+ cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9+ hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre–loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.

Identification of a hybrid myocardial zone in the mammalian heart after birth

Noncompaction cardiomyopathy is characterized by the presence of extensive trabeculations, which could lead to heart failure and malignant arrhythmias. How trabeculations resolve to form compact myocardium is poorly understood. Elucidation of this process is critical to understanding the pathophysiology of noncompaction disease. Here we use genetic lineage tracing to mark the Nppa+ or Hey2+ cardiomyocytes as trabecular and compact components of the ventricular wall. We find that Nppa+ and Hey2+ cardiomyocytes, respectively, from the endocardial and epicardial zones of the ventricular wall postnatally. Interposed between these two postnatal layers is a hybrid zone, which is composed of cells derived from both the Nppa+ and Hey2+ populations. Inhibition of the fetal Hey2+ cell contribution to the hybrid zone results in persistence of excessive trabeculations in postnatal heart. Our findings indicate that the expansion of Hey2+ fetal compact component, and its contribution to the hybrid myocardial zone, are essential for normal formation of the ventricular walls.Fetal trabecular muscles in the heart undergo a poorly described morphogenetic process that results into a solidified compact myocardium after birth. Tian et al. show that cardiomyocytes in the fetal compact layer also contribute to this process, forming a hybrid myocardial zone that is composed of cells derived from both trabecular and compact layers.

Mfsd2a + hepatocytes repopulate the liver during injury and regeneration

Hepatocytes are functionally heterogeneous and are divided into two distinct populations based on their metabolic zonation: the periportal and pericentral hepatocytes. During liver injury and regeneration, the cellular dynamics of these two distinct populations remain largely elusive. Here we show that major facilitator super family domain containing 2a (Mfsd2a), previously known to maintain blood–brain barrier function, is a periportal zonation marker. By genetic lineage tracing of Mfsd2a+ periportal hepatocytes, we show that Mfsd2a+ population decreases during liver homeostasis. Nevertheless, liver regeneration induced by partial hepatectomy significantly stimulates expansion of the Mfsd2a+ periportal hepatocytes. Similarly, during chronic liver injury, the Mfsd2a+ hepatocyte population expands and completely replaces the pericentral hepatocyte population throughout the whole liver. After injury recovery, the adult liver re-establishes the metabolic zonation by reprogramming the Mfsd2a+-derived hepatocytes into pericentral hepatocytes. The evidence of entire zonation replacement during injury increases our understanding of liver biology and disease.

Fibroblasts in an endocardial fibroelastosis disease model mainly originate from mesenchymal derivatives of epicardium

Endocardial fibroelastosis (EFE) refers to the thickening of the ventricular endocardium as a result of de novo deposition of subendocardial fibrous tissue layers during neonatal heart development. The origin of EFE fibroblasts is proposed to be postnatal endocardial cells that undergo an aberrant endothelial-to-mesenchymal transition (EndMT). Genetic lineage tracing of endocardial cells with the inducible endocardial Cre line Npr3-CreER and the endothelial cell tracing line Cdh5-CreER on an EFE-like model did not reveal any contribution of neonatal endocardial cells to fibroblasts in the EFE-like tissues. Instead, lineage tracing of embryonic epicardium by Wt1-CreER suggested that epicardium-derived mesenchymal cells (MCs) served as the major source of EFE fibroblasts. By labeling MCs using Sox9-CreER, we confirmed that MCs of the embryonic heart expand and contribute to the majority of neonatal EFE fibroblasts. During this pathological process, TGFβ signaling, the key mediator of fibroblasts activation, was highly upregulated in the EFE-like tissues. Targeting TGFβ signaling by administration of its antagonist bone morphogenetic protein 7 effectively reduced fibroblast accumulation and tissue fibrosis in the EFE-like model. Our study provides genetic evidence that excessive fibroblasts in the EFE-like tissues mainly originate from the epicardium-derived MCs through epicardial to mesenchymal transition (EpiMT). These EpiMT-derived fibroblasts within the EFE-like tissues could serve as a potential therapeutic target.