Generation of a tamoxifen inducible Tnnt2MerCreMer knock‐in mouse model for cardiac studies

Tnnt2, encoding thin‐filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2^MerCreMer/+) knock‐in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock‐in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2^MerCreMer/+ mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre‐LoxP technology. The Tnnt2^MerCreMer/+ mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury. genesis 53:377–386, 2015. © 2015 Wiley Periodicals, Inc.     

Reassessment of Isl1 and Nkx2-5 cardiac fate maps using a Gata4-based reporter of Cre activity

Isl1 and Nkx2-5-expressing cardiovascular progenitors play pivotal roles in cardiogenesis. Previously reported Cre-based fate-mapping studies showed that Isl1 progenitors contribute predominantly to the derivatives of the second heart field, and Nkx2-5 progenitors contributed mainly to the cardiomyocyte lineage. However, partial recombination of Cre reporter genes can complicate interpretation of Cre fate-mapping experiments. We found that a Gata4-based Cre-activated reporter was recombined by Isl1^Cre and Nkx2-5^Cre in a substantially broader domain than previously reported using standard Cre-activated reporters. The expanded Isl1 and Nkx2-5 cardiac fate maps were remarkably similar, and included extensive contributions to cardiomyocyte, endocardial, and smooth muscle lineages in all four cardiac chambers. These data indicate that Isl1 is expressed in progenitors of both primary and secondary heart fields, and that Nkx2-5 is expressed in progenitors of cardiac endothelium and smooth muscle, in addition to cardiomyocytes. These results have important implications for our understanding of cardiac lineage diversification in vivo, and for the interpretation of Cre-based fate maps.     

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ～ 84% of each lineage was EYFP⁺, and nearly all cells that come from Tie2‐expressing lineages are CD45⁺, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2⁺ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.     

EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

Eph receptor (Eph)‐ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4–ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5⁺ cardiac progenitor cells and in α‐MHC⁺ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5‐GFP⁺ cardiac progenitor cells, but not in α‐MHC‐GFP⁺ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL‐RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full‐length EphB4 or intracellular domain‐truncated EphB4 in EphB4‐null ES cells was established to investigate the role of EphB4‐forward signaling in ES cells. Interestingly, while full‐length EphB4 was able to restore the cardiac lineage development in EphB4‐null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor‐kinase‐dependent EphB4‐forward signaling plays a crucial role in the development of cardiac progenitor cells. J. Cell. Biochem. 116: 467–475, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.     

Identification and biological characterization of chicken embryonic cardiac progenitor cells

Objectives: Many kinds of cardiac progenitor cell populations have been identified, including c‐kit⁺, Nkx2.5⁺s and GATA4⁺ cells. However, these progenitors have limited ability to differentiate into different cardiac cell types. Recently, a new kind of cardiac progenitor cell named the multipotent Isl1⁺ cardiovascular progenitor (MICPs) has been identified, which also expresses Nkx2.5, GATA4, CD34 and Flk1. Materials and methods: In this study, we have isolated and characterized MICPs from chicken embryonic heart tissues using immunofluorescence and PCR. Results: Results shown that they express markers of cardiac progenitor cells, with high clonality. They have the ability to self‐renew and can give rise to three types of heart cell in vitro. Conclusions: Myocytes, smooth muscle cells and endothelial cells. Our work provides evidence for a developmental paradigm of the heart, that endothelial and muscle lineage diversification arises from multipotent cardiac progenitor cells. Existence of these cells provides a new opportunity for myocardial injury repair.     

Sox9‐expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons

Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell‐lineage tracing and gene‐function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9^CreERT2/+;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9‐expressing tissues. To determine the suitability of this mouse line for developmental stage‐specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9‐expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9‐expressing cells. genesis 48:635–644, 2010. © 2010 Wiley‐Liss, Inc.     

Direct Contact with Endoderm-Like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells

Rationale

Pluripotent stem cell–derived cardiac progenitor cells (CPCs) have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations.

Objective

Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells.

Method and Result

To test the hypothesis, we cocultured mouse embryonic stem (ES) cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1⁺ PDGFRa⁺ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS) cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5⁺ and Isl1⁺ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR⁺ PDGFRa⁺ CPCs from human ES cells.

Conclusions

Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.     

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ER^T2). In the background of the Cre reporter mouse strain Rosa26^lacZ, we show that the fusion CreER^T2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 Wiley‐Liss, Inc.