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1.
Development ; 147(4)2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-31988189

RESUMO

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.

2.
EMBO J ; 39(4): e102675, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-31943281

RESUMO

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 VEGFR2flox/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.

3.
J Biol Chem ; 295(3): 690-700, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31771978

RESUMO

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.

4.
Circulation ; 141(1): 67-79, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31779484

RESUMO

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, LdlrE208X, 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 LdlrE208X mice. RESULTS: We found that homogeneous LdlrE208X 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.

5.
Cell Stem Cell ; 26(1): 81-96.e4, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31883835

RESUMO

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+ VSCs in the adventitial layer of artery walls. After severe injury, Sca1+ VSCs migrate into the medial layer and generate de novo SMCs, which subsequently expand more efficiently compared with pre-existing smooth muscle. Genetic lineage tracing using dual recombinases distinguished a Sca1+PDGFRa+ VSC subpopulation that generates SMCs, and genetic ablation of Sca1+ VSCs or specific knockout of Yap1 in Sca1+ VSCs significantly impaired artery repair. These findings provide genetic evidence of a bona fide Sca1+ VSC population that produces SMCs and delineates their critical role in vessel repair.

6.
Nat Commun ; 10(1): 4158, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519886

RESUMO

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.


Assuntos
Sobrevivência Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Desenvolvimento Embrionário/genética , Feminino , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Inflamação/genética , Inflamação/metabolismo , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação/genética
8.
Circ Res ; 125(3): 343-355, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31185811

RESUMO

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.

9.
Nat Genet ; 51(4): 766, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30846878

RESUMO

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.

10.
Nat Genet ; 51(4): 728-738, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30778223

RESUMO

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.


Assuntos
Bronquíolos/fisiologia , Líquido da Lavagem Broncoalveolar/citologia , Pulmão/fisiologia , Células-Tronco Multipotentes/fisiologia , Regeneração/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Células Epiteliais/fisiologia , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
11.
Stem Cell Reports ; 12(3): 624-638, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30773487

RESUMO

Elucidation of the role of different cell lineages in the liver could offer avenues to drive liver regeneration. Previous studies showed that SOX9+ hepatocytes can differentiate into ductal cells after liver injuries. It is unclear whether SOX9+ hepatocytes are uni- or bipotent progenitors at a single-cell level during liver injury. Here, we developed a genetic tracing system to delineate the lineage potential of SOX9+ hepatocytes during liver homeostasis and regeneration. Fate-mapping data showed that these SOX9+ hepatocytes respond specifically to different liver injuries, with some contributing to a substantial number of ductal cells. Clonal analysis demonstrated that a single SOX9+ hepatocyte gives rise to both hepatocytes and ductal cells after liver injury. This study provides direct evidence that SOX9+ hepatocytes can serve as bipotent progenitors after liver injury, producing both hepatocytes and ductal cells for liver repair and regeneration.

13.
Cell Rep ; 25(5): 1241-1254.e5, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30380415

RESUMO

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.


Assuntos
Receptores de Apelina/metabolismo , Vasos Sanguíneos/patologia , Terapia de Alvo Molecular , Neoplasias/patologia , Envelhecimento/metabolismo , Animais , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Hipóxia Celular , Proliferação de Células , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Transdução de Sinais , Hipóxia Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Nat Protoc ; 13(10): 2217-2246, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30250288

RESUMO

Unraveling the fates of resident stem cells during tissue regeneration is an important objective in clinical and basic research. Genetic lineage tracing based on Cre-loxP recombination provides an effective strategy for inferring cell fate and cell conversion in vivo. However, the determination of the exact fates of resident stem cells or their derivatives in disease states and during tissue regeneration remains controversial in many fields of study, partly because of technical limitations associated with Cre-based lineage tracing, such as, for example, off-target labeling. Recently, we generated a new lineage-tracing platform we named DeaLT (dual-recombinase-activated lineage tracing) that uses the Dre-rox recombination system to enhance the precision of Cre-mediated lineage tracing. Here, we describe as an example a detailed protocol using DeaLT to trace the fate of c-Kit+ cardiac stem cells and their derivatives, in the absence of any interference from nontarget cells such as cardiomyocytes, during organ homeostasis and after tissue injury. This lineage-tracing protocol can also be used to delineate the fate of resident stem cells of other organ systems, and takes ~10 months to complete, from mouse crossing to final tissue analysis.


Assuntos
Rastreamento de Células/métodos , Miocárdio/citologia , Proteínas Proto-Oncogênicas c-kit/análise , Células-Tronco/citologia , Animais , Linhagem da Célula , Feminino , Técnicas de Introdução de Genes/métodos , Técnicas de Genotipagem/métodos , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-kit/genética , Recombinação Genética , Células-Tronco/metabolismo , Células-Tronco/patologia
15.
Circ Res ; 123(1): 86-99, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29764841

RESUMO

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.


Assuntos
Vasos Sanguíneos , Encéfalo/irrigação sanguínea , Vasos Coronários , Marcação de Genes/métodos , Animais , Barreira Hematoencefálica , Hipóxia Celular , Células Endoteliais , Técnicas de Inativação de Genes , Hibridização In Situ/métodos , Camundongos , Neovascularização Fisiológica , Especificidade de Órgãos , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
16.
Circulation ; 138(8): 793-805, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-29700121

RESUMO

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.


Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Linhagem da Célula , Rastreamento de Células/métodos , Coração/embriologia , Integrases/genética , Células-Tronco Embrionárias Murinas/fisiologia , Miócitos Cardíacos/fisiologia , Células-Tronco Adultas/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Escherichia coli/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Fenótipo , Recombinases/genética , Regeneração , Transdução de Sinais
17.
Circ Res ; 122(7): 984-993, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29374073

RESUMO

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.


Assuntos
Linhagem da Célula/genética , Endocárdio/citologia , Endotélio Vascular/citologia , Neovascularização Fisiológica , Transplante de Células-Tronco/métodos , Animais , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/terapia , Linhagem Celular , Transdiferenciação Celular , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Endocárdio/metabolismo , Endotélio Vascular/metabolismo , Humanos , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo
18.
Nat Med ; 23(12): 1488-1498, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29131159

RESUMO

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.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Proteínas de Escherichia coli/metabolismo , Integrases/metabolismo , Recombinases/metabolismo , Células-Tronco/citologia , Animais , Plasticidade Celular/genética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Imagem Molecular/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Recombinação Genética/genética , Sensibilidade e Especificidade , Células-Tronco/metabolismo
19.
Cell Res ; 27(9): 1157-1177, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28809397

RESUMO

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, TGFß signaling, the key mediator of fibroblasts activation, was highly upregulated in the EFE-like tissues. Targeting TGFß 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.


Assuntos
Fibroelastose Endocárdica/patologia , Fibroblastos/patologia , Pericárdio/patologia , Animais , Animais Recém-Nascidos , Linhagem da Célula , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Fibroelastose Endocárdica/metabolismo , Endocárdio/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibroblastos/metabolismo , Transplante de Coração , Integrases/metabolismo , Mesoderma , Camundongos , Modelos Biológicos , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
20.
Nat Commun ; 8(1): 87, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729659

RESUMO

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.


Assuntos
Cardiomiopatias/embriologia , Ventrículos do Coração/embriologia , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cardiomiopatias/congênito , Cardiomiopatias/metabolismo , Linhagem da Célula , Coração/embriologia , Coração/crescimento & desenvolvimento , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Organogênese , Precursores de Proteínas/metabolismo , Proteínas Repressoras/metabolismo
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