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Genetic lineage tracing unravels cell fate and plasticity in development, tissue homeostasis, and diseases. However, it remains technically challenging to trace temporary or transient cell fate, such as epithelial-to-mesenchymal transition (EMT) in tumor metastasis. Here, we generated a genetic fate-mapping system for temporally seamless tracing of transient cell fate. Highlighting its immediate application, we used it to study EMT gene activity from the local primary tumor to a distant metastatic site in vivo. In a spontaneous breast-to-lung metastasis model, we found that primary tumor cells activated vimentin and N-cadherin in situ, but only N-cadherin was activated and functionally required during metastasis. Tumor cells that have ever expressed N-cadherin constituted the majority of metastases in lungs, and functional deletion of N-cad significantly reduced metastasis. The seamless genetic recording system described here provides an alternative way for understanding transient cell fate and plasticity in biological processes., This study was supported by the National Key Research & Development Program of China (grant nos. 2019YFA011040, 2019YFA080200, 2018YFA010810, 2018YFA0107900, 2017YFC1001303, and 2016YFC1300600), Strategic Priority Research Program of the Chinese Academy of Sciences (CAS, grant nos. XDB19000000 and XDA16010507), National Science Foundation of China (grant nos. 31730112, 91849202, 31625019, 31730112 and 31900625), Key Project of Frontier Sciences of CAS (grant no. QYZDB-SSW-SMC003), International Cooperation Fund of CAS, Shanghai Science and Technology Commission (grant nos. 19JC1415700, 19YF1455300, 19ZR1479800, and 20QC1401000), China Postdoctoral Science Foundation (grant nos. 2018M640430 and 2019M660100), National Postdoctoral Program for Innovative Talents (grant nos. BX20180338 and BX20190343), the Pearl River Talent Recruitment Program of Guagdong Province (grant no. 2017ZT07S347), Royal Society-Newton Advanced Fellowship, and Sanofi-SIBS Fellowship.

Background: Mutations in low-density lipoprotein (LDL) receptor ( LDLR ) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease. Methods: The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, Ldlr E208X , based on a relevant familial hypercholesterolemia–related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into Ldlr E208X mice. Results: We found that homogeneous Ldlr E208X mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration. Conclusions: Our work shows that in vivo AAV-CRISPR/Cas9–mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.

Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre−, Cre−Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.

Cellular proliferation is a basic process during organ development, tissue homeostasis, and/or disease progression. Likewise, after injury typically multiple cell lineages respond to various cues and proliferate to initiate repair and/or remodeling of the injured tissue. Unravelling the specific role of proliferation of one cell type and its lineage in the context of the whole organism during tissue regeneration and/or disease progression would provide valuable information on these processes. Here we reported a new genetic system to inhibit cell proliferation in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that allow for the experimentally-induced permanent cell cycle arrest of specific cell lineages of interest, while genetically marking these cells. This system allows for the inhibition of pathogenic cell proliferation. We found that cardiac fibroblast proliferation inhibition significantly reduced scar formation, and promoted neovascularization and cardiomyocyte survival. Additionally, we found that inhibition of one type of cell proliferation (namely, hepatocytes) induces the lineage conversion of another type cells (ie, ductal cells) during tissue regeneration. These results validate the use of ip21-GFP mice as a new genetic tool for cell lineage-specific inhibition of cell proliferation in vivo.

Site‐specific recombinase‐mediated genetic technology, such as inducible Cre‐loxP recombination (CreER), is widely used for in vivo genetic manipulation with temporal control. The Cre‐loxP technology improves our understanding on the in vivo function of specific genes in organ development, tissue regeneration, and disease progression. However, inducible CreER often remains inefficient in gene deletion. In order to improve the efficiency of gene manipulation, we generated a self‐cleaved inducible CreER (sCreER) that switches inducible CreER into a constitutively active Cre by itself. We generated endocardial driver Npr3‐sCreER and fibroblast driver Col1a2‐sCreER, and compared them with conventional Npr3‐CreER and Col1a2‐CreER, respectively. For easy‐to‐recombine alleles such as R26‐tdTomato, there was no significant difference in recombination efficiency between sCreER and the conventional CreER. However, for alleles that were relatively inert for recombination such as R26‐Confetti, R26‐LZLT, R26‐GFP, or VEGFR2 (flox/flox) alleles, sCreER showed a significantly higher efficiency in recombination compared with conventional CreER in endocardial cells or fibroblasts. Compared with conventional CreER, sCreER significantly enhances the efficiency of recombination to induce gene expression or gene deletion, allowing temporal yet effective in vivo genomic modification for studying gene function in specific cell lineages.

Rapid regeneration of smooth muscle after vascular injury is essential for maintaining arterial function. The existence and putative roles of resident vascular stem cells (VSCs) in artery repair are controversial, and vessel regeneration is thought to be mediated by proliferative expansion of pre-existing smooth muscle cells (SMCs). Here, we performed cell fate mapping and single-cell RNA sequencing to identify Sca1

Summary The use of the dual recombinase-mediated intersectional genetic approach involving Cre-loxP and Dre-rox has significantly enhanced the precision of in vivo lineage tracing, as well as gene manipulation. However, this approach is limited by the small number of Dre recombinase driver constructs available. Here, we developed more than 70 new intersectional drivers to better target diverse cell lineages. To highlight their applicability, we used these new tools to study the in vivo adipogenic fate of perivascular progenitors, which revealed that PDGFRa+ but not PDGFRa–PDGFRb+ perivascular cells are the endogenous progenitors of adult adipocytes. In addition to lineage tracing, we used members of this new suite of drivers to more specifically knock out genes in complex tissues, such as white adipocytes and lymphatic vessels, that heretofore cannot be selectively targeted by conventional Cre drivers alone. In summary, these new transgenic tools expand the intersectional genetic approach while enhancing its precision.

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.

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.

The ubiquitination status of RIPK1 is considered to be critical for cell fate determination. However, the in vivo role for RIPK1 ubiquitination remains undefined. Here we show that mice expressing RIPK1K376R which is defective in RIPK1 ubiquitination die during embryogenesis. This lethality is fully rescued by concomitant deletion of Fadd and Ripk3 or Mlkl. Mechanistically, cells expressing RIPK1K376R are more susceptible to TNF-α induced apoptosis and necroptosis with more complex II formation and increased RIPK1 activation, which is consistent with the observation that Ripk1K376R/K376R lethality is effectively prevented by treatment of RIPK1 kinase inhibitor and is rescued by deletion of Tnfr1. However, Tnfr1−/− Ripk1K376R/K376R mice display systemic inflammation and die within 2 weeks. Significantly, this lethal inflammation is rescued by deletion of Ripk3. Taken together, these findings reveal a critical role of Lys376-mediated ubiquitination of RIPK1 in suppressing RIPK1 kinase activity–dependent lethal pathways during embryogenesis and RIPK3-dependent inflammation postnatally., RIPK1 integrates signals that drive both NF-κB activation and cell death pathways. Here Zhang et al. generate RIPK1 knock-in mice lacking a major ubiquitination site and demonstrate that this modification is important to suppress cell death during embryogenesis and inflammation postnatally.

With hybrid access model in heterogeneous networks (HetNets), a capacity efficient resource allocation strategy is proposed in this paper. Combining with millimeter wave (mmWave) and Sub-6GHz, we divide the optimization of system capacity into two Sub- optimization problems. Firstly, this paper proposes a resource allocation algorithm based on the maximum interference free group (MIFG) for the users in mmWave Pico BS (PBS). The capacity of each PBS is maximized by grouping users and maximizing the utilization of resource block (RB). Secondly, to ensure the service quality of users in Macro BS (MBS), a twostage QoS guaranteed greedy pair (TQGP) algorithm is proposed in this paper. Simulation results indicate that the proposed capacity efficient resource allocation strategy can improve the system capacity with user QoS guaranteed.

Identification of cellular surface markers that distinguish tumorous from normal vasculature is important for the development of tumor vessel-targeted therapy. Here, we show that Apj, a G protein-coupled receptor, is highly enriched in tumor endothelial cells but absent from most endothelial cells of adult tissues in homeostasis. By genetic targeting using Apj-CreER and Apj-DTRGFP-Luciferase, we demonstrated that hypoxia-VEGF signaling drives expansion of Apj

Characterizing the stem cells responsible for lung repair and regeneration is important for the treatment of pulmonary diseases. Recently, a unique cell population located at the bronchioalveolar-duct junctions has been proposed to comprise endogenous stem cells for lung regeneration. However, the role of bronchioalveolar stem cells (BASCs) in vivo remains debated, and the contribution of such cells to lung regeneration is not known. Here we generated a genetic lineage-tracing system that uses dual recombinases (Cre and Dre) to specifically track BASCs in vivo. Fate-mapping and clonal analysis showed that BASCs became activated and responded distinctly to different lung injuries, and differentiated into multiple cell lineages including club cells, ciliated cells, and alveolar type 1 and type 2 cells for lung regeneration. This study provides in vivo genetic evidence that BASCs are bona fide lung epithelial stem cells with deployment of multipotency and self-renewal during lung repair and regeneration.

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.

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.

Aiming at reducing the overhead of codebook design and improving the performance of massive multiple-input multiple-output (MIMO) systems, a novel two-stage codebook design is presented in this paper. Using the householder-transform, which perturbs the first-codebook beam direction, the second-codebook can be easily obtained. In addition, the second-codebook maintains the properties of the first-codebook and further simplifies the modality of perturbation. Then we introduce a self-adaptive algorithm for codebook selection to maximize the system performance. The self-adaptive algorithm dynamically chooses the optimal codeword of a two-stage codebook then feeds back one of the codewords to the transmitter, and it reduces feedback overhead and avoids the inaccurate channel matching caused by only using the second-codebook. Specifically, the algorithm increases the acceleration of codebook selection and reduces the complexity and blindness. The simulation results suggest that the proposed perturbation-based codebook design can improve the system bit error rate (BER) performance and can be applied to the case of multi-stream.

Hybrid beamforming (HBF) has been widely accepted to offer a compromise between cost and system performance in millimeter wave (mmWave) systems, while its performance is highly dependent on the corresponding computation complexity. Using an assumption of orthogonal constraint and phase extraction, an effective HBF algorithm is presented in this paper, which consists of a digital beamformer (DBF) stage and an analog beamformer (ABF) stage. Firstly, the optimal DBF with orthogonal constraint is derived for a given RF beamformer. Secondly, when the orthogonal property of the digital beamformer is enabled, the analog beamformer can be developed by extracting the phases of the unconstraint solution of the optimization in RF domain. Specifically, we consider the case of that the number of RF chains is equal to the number of data streams, which can further improve the energy efficiency of mmWave systems. Simulation results show that our proposed algorithm can effectively approach the full digital beamformer performance limit.

In the version of this article initially published, the following grant numbers and recipients were missing from the Acknowledgements: XDB19000000 to H.J. and B.Z.; 81430066 and 31621003 to H.J.; 2017YFA0505500 to H.J.; and 15XD1504000 to H.J. The errors have been corrected in the HTML and PDF versions of the article.

Summary Identification of cellular surface markers that distinguish tumorous from normal vasculature is important for the development of tumor vessel-targeted therapy. Here, we show that Apj, a G protein-coupled receptor, is highly enriched in tumor endothelial cells but absent from most endothelial cells of adult tissues in homeostasis. By genetic targeting using Apj-CreER and Apj-DTRGFP-Luciferase, we demonstrated that hypoxia-VEGF signaling drives expansion of Apj+ tumor vessels and that targeting of these vessels, genetically and pharmacologically, remarkably inhibits tumor angiogenesis and restricts tumor growth. These in vivo findings implicate Apj+ vessels as a key driver of pathological angiogenesis and identify Apj+ endothelial cells as an important therapeutic target for the anti-angiogenic treatment of tumors.

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.

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., Apelin expression is robust in embryonic but not in adult endothelial cells (ECs), where it can be reactivated by hypoxia. Liu et al. show that apelin-driven expression of Cre recombinase in mice can be used for labelling of, or gene ablation in, sprouting but not quiescent ECs in pathologies characterized by hypoxia.

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.