Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.     

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.     

基因谱系示踪技术揭示小鼠Tbx18+心脏祖细胞向心系细胞分化的潜能

心脏祖细胞(cardiac progenitor cells,CPCs)的研究对阐明先天性心脏病的机制及治疗心血管疾病具有重要意义.哺乳动物的心脏组织由多种不同CPCs分化形成.转录因子Tbx18在发育中的心外膜中表达,对心脏的发育形成起重要的调节作用.为了在组织及活体细胞水平检测和阐明Tbx18+CPC的分化潜能,应用Cre-LoxP系统建立Tbx18+CPCs基因命运谱系示踪模型:Tbx18-Cre/Rosa26R-EYFP和Tbx18-Cre/Rosa26R-LacZ双杂合基因敲入小鼠.该双杂合基因敲入小鼠通过Cre的表达能有效地示踪Tbx18+细胞在胚胎和成年小鼠中的分化命运.Tbx18-Cre/Rosa26R-EYFP双杂合小鼠心脏能非常容易地利用流式细胞分选系统(FACS)分离出YFP+细胞,也可在倒置共聚焦显微镜下观察.应用X-gal染色分析其表达模式,揭示Tbx18命运谱系参与心房肌、室间隔、心室肌、冠状动脉、瓣膜等的形成.应用免疫荧光技术初步揭示Tbx18+CPCs向心脏肌钙蛋白T(cTNT)阳性心肌细胞和平滑肌肌球蛋白重链11(MYH11)阳性血管平滑肌细胞分化的潜能.心脏是一个由多种肌肉和非肌肉组织细胞构成的复杂器官.推测Tbx18可能在心脏祖细胞向肌源性细胞分化的信号通路中起重要调节作用.在上述研究中应用基因谱系示踪技术,验证Tbx18可作为一类CPCs的标志,为更深入揭示心脏祖细胞向心系细胞的分化潜能打下基础.     

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.     

A series of Cre‐ERT2 drivers for manipulation of the skeletal muscle lineage

We report the generation of five mouse strains with the tamoxifen‐inducible Cre (Cre‐ER^T2; CE) gene cassette knocked into the endogenous loci of Pax3, Myod1, Myog, Myf6, and Myl1, collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R^LacZ, during embryogenesis and show that they direct tightly controlled tamoxifen‐inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen‐inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock‐in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. Although we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology. genesis 52:759–770, 2014. © 2014 Wiley Periodicals, Inc.     

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells

Abstract. Objectives: Previously, we have found that the ClC‐3 chloride channel is involved in endothelin‐1 (ET‐1)‐induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC‐3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC‐3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET‐1‐induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC‐3 protein. Silence of ClC‐3 by siRNA inhibited expression of ClC‐3 protein, prevented an increase in BrdU incorporation and cell number induced by ET‐1. Silence of ClC‐3 also caused cell cycle arrest in G₀/G₁ phase and prevented the cells’ progression from G₁ to S phase. Knockdown of ClC‐3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin‐dependent kinase inhibitors (CDKIs) p27^KIP and p21^CIP expression. Furthermore, ClC‐3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase‐3β (GSK‐3β) induced by ET‐1. Conclusion: Silence of ClC‐3 protein effectively suppressed phosphorylation of the Akt/GSK‐3β signal pathway, resulting in down‐regulation of cyclin D1 and cyclin E, and up‐regulation of p27^KIP and p21^CIP. In these BASMCs, integrated effects lead to cell cycle G₁/S arrest and inhibition of cell proliferation.     

Generation of mouse conditional and null alleles of the type III sodium‐dependent phosphate cotransporter PiT‐1

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter‐1 (PiT‐1), was shown to be required. To determine the in vivo role of PiT‐1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT‐1^flox, which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre‐mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT‐1^{Δe3,4/Δe3,4} embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT‐1 mouse alleles have been successfully generated and PiT‐1 has a necessary, nonredundant role in embryonic development. genesis 47:858–863, 2009. © 2009 Wiley‐Liss, Inc.     

Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: Characterization of Tnnt3tm2a(KOMP)Wtsi mice

The troponin complex, which consists of three regulatory proteins (troponin C, troponin I, and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue‐specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3^lacZ/+ mice are smaller than their WT littermates throughout development but do not display any gross phenotypes. Tnnt3^lacZ/lacZ embryos are smaller than heterozygotes and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3^lacZ/lacZ liver and kidney, which was not present in Tnnt3^lacZ/+ or WT, but no other gross tissue abnormalities. X‐gal staining for Tnnt3 promoter‐driven lacZ transgene expression revealed positive staining in skeletal muscle and diaphragm and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional‐inducible gene deletion approach genesis 51:667–675. © 2013 Wiley Periodicals, Inc.     

Development of a general‐purpose method for cell purification using Cre/loxP‐mediated recombination

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general‐purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock‐in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP‐mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1‐Cre‐transgenic (tg) embryos almost equally as efficiently as from Nr5a1‐hCD271‐tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh‐Cre‐tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. genesis 53:387–393, 2015. © 2015 Wiley Periodicals, Inc.     

Myocardin and microRNA‐1 modulate bladder activity through connexin 43 expression during post‐natal development

Overactive bladder (OAB) is a pervasive clinical problem involving alterations in both neurogenic and myogenic activity. While there has been some progress in understanding neurogenic inputs to OAB, the mechanisms controlling myogenic bladder activity are unclear. We report the involvement of myocardin (MYOCD) and microRNA‐1 (miR‐1) in the regulation of connexin 43 (GJA1), a major gap junction in bladder smooth muscle, and the collective role of these molecules during post‐natal bladder development. Wild‐type (WT) mouse bladders showed normal development from early post‐natal to adult including increases in bladder capacity and maintenance of normal sensitivity to cholinergic agents concurrent with down‐regulation of MYOCD and several smooth muscle cell (SMC) contractile genes. Myocardin heterozygous‐knockout mice exhibited reduced expression of Myocd mRNA and several SMC contractile genes concurrent with bladder SMC hypersensitivity that was mediated by gap junctions. In both cultured rat bladder SMC and in vivo bladders, MYOCD down‐regulated GJA1 expression through miR‐1 up‐regulation. Interestingly, adult myocardin heterozygous‐knockout mice showed normal increases in bladder and body weight but lower bladder capacity compared to WT mice. These results suggest that MYOCD down‐regulates GJA1 expression via miR‐1 up‐regulation, thereby contributing to maintenance of normal sensitivity and development of bladder capacity. J. Cell. Physiol. 228: 1819–1826, 2013. © 2013 Wiley Periodicals, Inc.     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

Mesenchymal stem cell‐loaded cardiac patch promotes epicardial activation and repair of the infarcted myocardium

Cardiac patch is considered a promising strategy for enhancing stem cell therapy of myocardial infarction (MI). However, the underlying mechanisms for cardiac patch repairing infarcted myocardium remain unclear. In this study, we investigated the mechanisms of PCL/gelatin patch loaded with MSCs on activating endogenous cardiac repair. PCL/gelatin patch was fabricated by electrospun. The patch enhanced the survival of the seeded MSCs and their HIF‐1α, Tβ4, VEGF and SDF‐1 expression and decreased CXCL14 expression in hypoxic and serum‐deprived conditions. In murine MI models, the survival and distribution of the engrafted MSCs and the activation of the epicardium were examined, respectively. At 4 weeks after transplantation of the cell patch, the cardiac functions were significantly improved. The engrafted MSCs migrated across the epicardium and into the myocardium. Tendency of HIF‐1α, Tβ4, VEGF, SDF‐1 and CXCL14 expression in the infarcted myocardium was similar with expression in vitro. The epicardium was activated and epicardial‐derived cells (EPDCs) migrated into deep tissue. The EPDCs differentiated into endothelial cells and smooth muscle cells, and some of EPDCs showed to have differentiated into cardiomyocytes. Density of blood and lymphatic capillaries increased significantly. More c‐kit⁺ cells were recruited into the infarcted myocardium after transplantation of the cell patch. The results suggest that epicardial transplantation of the cell patch promotes repair of the infarcted myocardium and improves cardiac functions by enhancing the survival of the transplanted cells, accelerating locality paracrine, and then activating the epicardium and recruiting endogenous c‐kit⁺ cells. Epicardial transplantation of the cell patch may be applied as a novel effective MI therapy.     

Msx1creERT2 knock‐In allele: A useful tool to target embryonic and adult cardiac valves

Summary : Heart valve development begins with the endothelial‐to‐mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate‐mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock‐in tamoxifen‐inducible Cre line was recently generated (Msx1^CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock‐in allele and track the Msx1‐expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1^CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling. genesis 53:337–345, 2015. © 2015 Wiley Periodicals, Inc.     

Generation and characterization of Csrp1 enhancer-driven tissue-restricted Cre-recombinase mice

Cell type-specific genetic modification using the LoxP/Cre system is a powerful tool for genetic analysis of distinct cell lineages. Because of the unique arterial smooth muscle-restricted expression of a 5.0 kb cysteine-rich protein (Csrp1) enhancer (Lilly et al.,2001, Dev Biol 240:531-547), we hypothesized that a transgenic Cre line would prove useful for the smooth muscle lineage-specific genetic manipulation. Here we describe a transgenic mouse line, ECsrp1(Cre), where Cre is initially specifically expressed in arterial smooth muscle cells. Use of the ROSA26R reporter allele confirmed that Cre-mediated recombination in vascular smooth muscle cells began at approximately E10.0 and was highly proficient. Subsequently, Cre is expressed in restricted skeletal and nonvascular smooth muscle lineages. This lineage tracing data is important for future conditional knockout studies to understand where and when Cre-mediated deletion occurs and where Cre-expressing daughter cells finally localize. Additionally, we crossed the ECsrp1(Cre) mice to the ROSA26(-eGFP-DTA) diphtheria toxin A-expressing mice to genetically ablate ECsrp1(Cre) expressing cells. This ECsrp1(Cre) transgenic line should thus prove useful for genetic analysis of diverse aspects of cardiovascular morphogenesis and as a general smooth muscle lineage deletor line.     

Optical induction of muscle contraction at the tissue scale through intrinsic cellular amplifiers

The smooth muscle cell is the principal component responsible for involuntary control of visceral organs, including vascular tonicity, secretion, and sphincter regulation. It is known that the neurotransmitters released from nerve endings increase the intracellular Ca²⁺ level in smooth muscle cells followed by muscle contraction. We herein report that femtosecond laser pulses focused on the diffraction‐limited volume can induce intracellular Ca²⁺ increases in the irradiated smooth muscle cell without neurotransmitters, and locally increased intracellular Ca²⁺ levels are amplified by calcium‐induced calcium‐releasing mechanisms through the ryanodine receptor, a Ca²⁺ channel of the endoplasmic reticulum. The laser‐induced Ca²⁺ increases propagate to adjacent cells through gap junctions. Thus, ultrashort‐pulsed lasers can induce smooth muscle contraction by controlling Ca²⁺, even with optical stimulation of the diffraction‐limited volume. This optical method, which leads to reversible and reproducible muscle contraction, can be used in research into muscle dynamics, neuromuscular disease treatment, and nanorobot control. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)