Tracing the origin of alveolar stem cells in lung repair and regeneration.

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

Dynamics of Endothelial Cell Generation and Turnover in Arteries During Homeostasis and Diseases.

BACKGROUND: Endothelial cell (EC) generation and turnover by self-proliferation contributes to vascular repair and regeneration. The ability to accurately measure the dynamics of EC generation would advance our understanding of cellular mechanisms of vascular homeostasis and diseases. However, it is currently challenging to evaluate the dynamics of EC generation in large vessels such as arteries because of their infrequent proliferation. METHODS: By using dual recombination systems based on Cre-loxP and Dre-rox, we developed a genetic system for temporally seamless recording of EC proliferation in vivo. We combined genetic recording of EC proliferation with single-cell RNA sequencing and gene knockout to uncover cellular and molecular mechanisms underlying EC generation in arteries during homeostasis and disease. RESULTS: Genetic proliferation tracing reveals that ≈3% of aortic ECs undergo proliferation per month in adult mice during homeostasis. The orientation of aortic EC division is generally parallel to blood flow in the aorta, which is regulated by the mechanosensing protein Piezo1. Single-cell RNA sequencing analysis reveals 4 heterogeneous aortic EC subpopulations with distinct proliferative activity. EC cluster 1 exhibits transit-amplifying cell features with preferential proliferative capacity and enriched expression of stem cell markers such as Sca1 and Sox18. EC proliferation increases in hypertension but decreases in type 2 diabetes, coinciding with changes in the extent of EC cluster 1 proliferation. Combined gene knockout and proliferation tracing reveals that Hippo/vascular endothelial growth factor receptor 2 signaling pathways regulate EC proliferation in large vessels. CONCLUSIONS: Genetic proliferation tracing quantitatively delineates the dynamics of EC generation and turnover, as well as EC division orientation, in large vessels during homeostasis and disease. An EC subpopulation in the aorta exhibits more robust cell proliferation during homeostasis and type 2 diabetes, identifying it as a potential therapeutic target for vascular repair and regeneration.

Genetic Lineage Tracing of Pericardial Cavity Macrophages in the Injured Heart.

BACKGROUND: Macrophages play an important role in cardiac repair after myocardial infarction (MI). In addition to the resident macrophages and blood-derived monocytes, Gata6+ cavity macrophages located in the pericardial space were recently reported to relocate to the injured myocardium and prevent cardiac fibrosis. However, there is no direct genetic evidence to support it. METHODS: We used dual recombinases (Cre and Dre) to specifically label Gata6+ pericardial macrophages (GPCMs) in vivo. For functional study, we generated genetic systems to specifically ablate GPCMs by induced expression of DTR (diphtheria toxin receptor) or knockout of Gata6 (GATA binding protein 6) gene in GPCMs. We used these genetic systems to study GPCMs in pericardium intact MI model. RESULTS: Dual recombinases-mediated genetic system targeted GPCMs specifically and efficiently. Lineage tracing study revealed accumulation of GPCMs on the surface of MI heart without deep penetration into the myocardium. We did not detect significant change of cardiac fibrosis or function of MI hearts after cell ablation or Gata6 knockout in GPCMs. CONCLUSIONS: GPCMs minimally invade the injured heart after MI. Nor do they prevent cardiac fibrosis and exhibit reparative function on injured heart. This study also underlines the importance of using specific genetic tool for studying in vivo cell fates and functions.

Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

BACKGROUND: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. METHODS: To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. RESULTS: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. CONCLUSIONS: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

A genetic system for tissue-specific inhibition of cell proliferation.

Cellular proliferation is a basic process during organ development, tissue homeostasis and 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 report a new genetic system that allows cell proliferation to be inhibited in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that enable 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 (i.e. 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.

Single protein encapsulated SN38 for tumor-targeting treatment.

BACKGROUND: The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2-8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug's efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity. METHODS: Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 (FDA IND #, 152154), which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models. RESULTS: The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUC0-∞ of 0.05 and 4.5 nmol × h/mL for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 90:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 19 and 28 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.5:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX's insensitivity to SK-LMS-1 with high toxicity. CONCLUSION: SPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX.

Seamless Genetic Recording of Transiently Activated Mesenchymal Gene Expression in Endothelial Cells During Cardiac Fibrosis.

BACKGROUND: Cardiac fibrosis is a lethal outcome of excessive formation of myofibroblasts that are scar-forming cells accumulated after heart injury. It has been reported that cardiac endothelial cells (ECs) contribute to a substantial portion of myofibroblasts through endothelial to mesenchymal transition (EndoMT). Recent lineage tracing studies demonstrate that myofibroblasts are derived from the expansion of resident fibroblasts rather than from the transdifferentiation of ECs. However, it remains unknown whether ECs can transdifferentiate into myofibroblasts reversibly or EndoMT genes were just transiently activated in ECs during cardiac fibrosis. METHODS: By using the dual recombination technology based on Cre-loxP and Dre-rox, we generated a genetic lineage tracing system for tracking EndoMT in cardiac ECs. We used it to examine if there is transiently activated mesenchymal gene expression in ECs during cardiac fibrosis. Activation of the broadly used marker gene in myofibroblasts, αSMA (α-smooth muscle actin), and the transcription factor that induces epithelial to mesenchymal transition, Zeb1 (zinc finger E-box-binding homeobox 1), was examined. RESULTS: The genetic system enables continuous tracing of transcriptional activity of targeted genes in vivo. Our genetic fate mapping results revealed that a subset of cardiac ECs transiently expressed αSMA and Zeb1 during embryonic valve formation and transdifferentiated into mesenchymal cells through EndoMT. Nonetheless, they did not contribute to myofibroblasts, nor transiently expressed αSMA or Zeb1 after heart injury. Instead, expression of αSMA was activated in resident fibroblasts during cardiac fibrosis. CONCLUSIONS: Mesenchymal gene expression is activated in cardiac ECs through EndoMT in the developing heart, but ECs do not transdifferentiate into myofibroblasts, nor transiently express some known mesenchymal genes during homeostasis and fibrosis in the adult heart. Resident fibroblasts that are converted to myofibroblasts by activating mesenchymal gene expression are the major contributors to cardiac fibrosis.

Genetic Tracing Identifies Early Segregation of the Cardiomyocyte and Nonmyocyte Lineages.

RATIONALE: The developing heart is composed of cardiomyocytes and noncardiomyocytes since the early stage. It is generally believed that noncardiomyocytes including the cardiac progenitors contribute to new cardiomyocytes of the looping heart. However, it remains unclear what the cellular dynamics of nonmyocyte to cardiomyocyte conversion are and when the lineage segregation occurs during development. It also remains unknown whether nonmyocyte to cardiomyocyte conversion contributes to neonatal heart regeneration. OBJECTIVE: We quantify the lineage conversion of noncardiomyocytes to cardiomyocytes in the embryonic and neonatal hearts and determine when the 2 cell lineages segregate during heart development. Moreover, we directly test if nonmyocyte to cardiomyocyte conversion contributes to neonatal heart regeneration. METHODS AND RESULTS: We generated a dual genetic lineage tracing strategy in which cardiomyocytes and noncardiomyocytes of the developing heart could be simultaneously labeled by 2 orthogonal recombination systems. Genetic fate mapping showed that nonmyocyte to cardiomyocyte conversion peaks at E8.0 (embryonic day) to E8.5 and gradually declines at E9.5 and E10.5. Noncardiomyocytes do not generate any cardiomyocyte at and beyond E11.5 to E12.5. In the neonatal heart, noncardiomyocytes also do not contribute to any new cardiomyocyte in homeostasis or after injury. CONCLUSIONS: Noncardiomyocytes contribute to new cardiomyocytes of the developing heart at early embryonic stage before E11.5. The noncardiomyocyte and cardiomyocyte lineage segregation occurs between E10.5 and E11.5, which is maintained afterward even during neonatal heart regeneration.

Genetic Fate Mapping Defines the Vascular Potential of Endocardial Cells in the Adult Heart.

RATIONALE: Endocardium is the major source of coronary endothelial cells (ECs) in the fetal and neonatal hearts. It remains unclear whether endocardium in the adult stage is also the main origin of neovascularization after cardiac injury. OBJECTIVE: To define the vascular potential of adult endocardium in homeostasis and after cardiac injuries by fate-mapping studies. METHODS AND RESULTS: We generate an inducible adult endocardial Cre line (Npr3 [natriuretic peptide receptor C]-CreER) and show that Npr3-CreER efficiently and specifically labels endocardial cells but not coronary blood vessels in the adult heart. The adult endocardial cells do not contribute to any vascular ECs during cardiac homeostasis. To examine the formation of blood vessels from endocardium after injury, we generate 4 cardiac injury models with Npr3-CreER mice: myocardial infarction, myocardial ischemia-reperfusion, cryoinjury, and transverse aortic constriction. Lineage tracing experiments show that adult endocardium minimally contributes to coronary ECs after myocardial infarction. In the myocardial ischemia-reperfusion, cryoinjury, or transverse aortic constriction models, adult endocardial cells do not give rise to any vascular ECs, and they remain on the inner surface of myocardium that connects with lumen circulation. In the myocardial infarction model, very few endocardial cells are trapped in the infarct zone of myocardium shortly after ligation of coronary artery, indicating the involvement of endocardial entrapment during blood vessels formation. When these adult endocardial cells are relocated and trapped in the infarcted myocardium by transplantation or myocardial constriction model, very few endocardial cells survive and gain vascular EC properties, and their contribution to neovascularization in the injured myocardium remains minimal. CONCLUSIONS: Unlike its fetal or neonatal counterpart, adult endocardium naturally generates minimal, if any, coronary arteries or vascular ECs during cardiac homeostasis or after injuries.

Genetic Targeting of Organ-Specific Blood Vessels.

RATIONALE: Organs of the body require vascular networks to supply oxygen and nutrients and maintain physiological function. The blood vessels of different organs are structurally and functionally heterogeneous in nature. To more precisely dissect their distinct in vivo function in individual organs, without potential interference from off-site targets, it is necessary to genetically target them in an organ-specific manner. OBJECTIVE: The objective of this study was to generate a genetic system that targets vascular endothelial cells in an organ- or tissue-specific manner and to exemplify the potential application of intersectional genetics for precise, target-specific gene manipulation in vivo. METHODS AND RESULTS: We took advantage of 2 orthogonal recombination systems, Dre-rox and Cre-loxP, to create a genetic targeting system based on intersectional genetics. Using this approach, Cre activity was only detectable in cells that had expressed both Dre and Cre. Applying this new system, we generated a coronary endothelial cell-specific Cre (CoEC-Cre) and a brain endothelial cell-specific Cre (BEC-Cre). Through lineage tracing, gene knockout and overexpression experiments, we demonstrated that CoEC-Cre and BEC-Cre efficiently and specifically target blood vessels in the heart and brain, respectively. By deletion of vascular endothelial growth factor receptor 2 using BEC-Cre, we showed that vascular endothelial growth factor signaling regulates angiogenesis in the central nervous system and also controls the integrity of the blood-brain barrier. CONCLUSIONS: We provide 2 examples to illustrate the use of intersectional genetics for more precise gene targeting in vivo, namely manipulation of genes in blood vessels of the heart and brain. More broadly, this system provides a valuable strategy for tissue-specific gene manipulation that can be widely applied to other fields of biomedical research.

Machine Learning Methods in Drug Discovery.

The advancements of information technology and related processing techniques have created a fertile base for progress in many scientific fields and industries. In the fields of drug discovery and development, machine learning techniques have been used for the development of novel drug candidates. The methods for designing drug targets and novel drug discovery now routinely combine machine learning and deep learning algorithms to enhance the efficiency, efficacy, and quality of developed outputs. The generation and incorporation of big data, through technologies such as high-throughput screening and high through-put computational analysis of databases used for both lead and target discovery, has increased the reliability of the machine learning and deep learning incorporated techniques. The use of these virtual screening and encompassing online information has also been highlighted in developing lead synthesis pathways. In this review, machine learning and deep learning algorithms utilized in drug discovery and associated techniques will be discussed. The applications that produce promising results and methods will be reviewed.

Genetic Lineage Tracing of Nonmyocyte Population by Dual Recombinases.

BACKGROUND: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1+, Bmi1+, Isl1+, or Abcg2+ cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. METHODS: To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes. RESULTS: By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury. CONCLUSIONS: This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

GATA4 regulates Fgf16 to promote heart repair after injury.

Although the mammalian heart can regenerate during the neonatal stage, this endogenous regenerative capacity is lost with age. Importantly, replication of cardiomyocytes has been found to be the key mechanism responsible for neonatal cardiac regeneration. Unraveling the transcriptional regulatory network for inducing cardiomyocyte replication will, therefore, be crucial for the development of novel therapies to drive cardiac repair after injury. Here, we investigated whether the key cardiac transcription factor GATA4 is required for neonatal mouse heart regeneration. Using the neonatal mouse heart cryoinjury and apical resection models with an inducible loss of GATA4 specifically in cardiomyocytes, we found severely depressed ventricular function in the Gata4-ablated mice (mutant) after injury. This was accompanied by reduced cardiomyocyte replication. In addition, the mutant hearts displayed impaired coronary angiogenesis and increased hypertrophy and fibrosis after injury. Mechanistically, we found that the paracrine factor FGF16 was significantly reduced in the mutant hearts after injury compared with littermate controls and was directly regulated by GATA4. Cardiac-specific overexpression of FGF16 via adeno-associated virus subtype 9 (AAV9) in the mutant hearts partially rescued the cryoinjury-induced cardiac hypertrophy, promoted cardiomyocyte replication and improved heart function after injury. Altogether, our data demonstrate that GATA4 is required for neonatal heart regeneration through regulation of Fgf16, suggesting that paracrine factors could be of potential use in promoting myocardial repair.

EBIC: an evolutionary-based parallel biclustering algorithm for pattern discovery.

Motivation: Biclustering algorithms are commonly used for gene expression data analysis. However, accurate identification of meaningful structures is very challenging and state-of-the-art methods are incapable of discovering with high accuracy different patterns of high biological relevance. Results: In this paper, a novel biclustering algorithm based on evolutionary computation, a sub-field of artificial intelligence, is introduced. The method called EBIC aims to detect order-preserving patterns in complex data. EBIC is capable of discovering multiple complex patterns with unprecedented accuracy in real gene expression datasets. It is also one of the very few biclustering methods designed for parallel environments with multiple graphics processing units. We demonstrate that EBIC greatly outperforms state-of-the-art biclustering methods, in terms of recovery and relevance, on both synthetic and genetic datasets. EBIC also yields results over 12 times faster than the most accurate reference algorithms. Availability and implementation: EBIC source code is available on GitHub at https://github.com/EpistasisLab/ebic. Supplementary information: Supplementary data are available at Bioinformatics online.

runibic: a Bioconductor package for parallel row-based biclustering of gene expression data.

Motivation: Biclustering is an unsupervised technique of simultaneous clustering of rows and columns of input matrix. With multiple biclustering algorithms proposed, UniBic remains one of the most accurate methods developed so far. Results: In this paper we introduce a Bioconductor package called runibic with parallel implementation of UniBic. For the convenience the algorithm was reimplemented, parallelized and wrapped within an R package called runibic. The package includes: (i) a couple of times faster parallel version of the original sequential algorithm, (ii) much more efficient memory management, (iii) modularity which allows to build new methods on top of the provided one and (iv) integration with the modern Bioconductor packages such as SummarizedExperiment, ExpressionSet and biclust. Availability and implementation: The package is implemented in R and is available from Bioconductor (starting from version 3.6) at the following URL http://bioconductor.org/packages/runibic with installation instructions and tutorial. Supplementary information: Supplementary data are available at Bioinformatics online.

Genetic tracing of hepatocytes in liver homeostasis, injury, and regeneration.

The liver possesses a remarkable capacity to regenerate after damage. There is a heated debate on the origin of new hepatocytes after injuries in adult liver. Hepatic stem/progenitor cells have been proposed to produce functional hepatocytes after injury. Recent studies have argued against this model and suggested that pre-existing hepatocytes, rather than stem cells, contribute new hepatocytes. This hepatocyte-to-hepatocyte model is mainly based on labeling of hepatocytes with Cre-recombinase delivered by the adeno-associated virus. However, the impact of virus infection on cell fate determination, consistency of infection efficiency, and duration of Cre-virus in hepatocytes remain confounding factors that interfere with the data interpretation. Here, we generated a new genetic tool Alb-DreER to label almost all hepatocytes (>99.5%) and track their contribution to different cell lineages in the liver. By "pulse-and-chase" strategy, we found that pre-existing hepatocytes labeled by Alb-DreER contribute to almost all hepatocytes during normal homeostasis and after liver injury. Virtually all hepatocytes in the injured liver are descendants of pre-existing hepatocytes through self-expansion. We concluded that stem cell differentiation is unlikely to be responsible for the generation of a substantial number of new hepatocytes in adult liver. Our study also provides a new mouse tool for more precise in vivo genetic study of hepatocytes in the field.

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.

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.

BinPacker: Packing-Based De Novo Transcriptome Assembly from RNA-seq Data.

High-throughput RNA-seq technology has provided an unprecedented opportunity to reveal the very complex structures of transcriptomes. However, it is an important and highly challenging task to assemble vast amounts of short RNA-seq reads into transcriptomes with alternative splicing isoforms. In this study, we present a novel de novo assembler, BinPacker, by modeling the transcriptome assembly problem as tracking a set of trajectories of items with their sizes representing coverage of their corresponding isoforms by solving a series of bin-packing problems. This approach, which subtly integrates coverage information into the procedure, has two exclusive features: 1) only splicing junctions are involved in the assembling procedure; 2) massive pell-mell reads are assembled seemingly by moving a comb along junction edges on a splicing graph. Being tested on both real and simulated RNA-seq datasets, it outperforms almost all the existing de novo assemblers on all the tested datasets, and even outperforms those ab initio assemblers on the real dog dataset. In addition, it runs substantially faster and requires less memory space than most of the assemblers. BinPacker is published under GNU GENERAL PUBLIC LICENSE and the source is available from: http://sourceforge.net/projects/transcriptomeassembly/files/BinPacker_1.0.tar.gz/download. Quick installation version is available from: http://sourceforge.net/projects/transcriptomeassembly/files/BinPacker_binary.tar.gz/download.