An approach for controlling the timing and order of engineered mutations in mice

Significant advances in our understanding of normal development and disease have been facilitated by engineered mice in which genes can be altered in a spatially, temporally, or cell type restricted manner using site specific recombinase systems like Cre‐loxP or Flp‐frt. In many circumstances it is important to understand how interactions between multiple genes influence a given phenotype. Robust approaches for precisely controlling multiple genetic alterations independently are limited, however, thus the impact of mutation order and timing on phenotype is generally unknown. Here we describe and validate a novel Gt(ROSA)26Sor targeted transgene allowing precise control over the order and timing of multiple genetic mutations in the mouse. The transgene expresses an optimized, Flp‐estrogen receptor fusion protein (Flpo‐ERT2) under the control of a loxP‐stop‐loxP cassette. In this system, genes modified by loxP sites are altered first upon expression of Cre. Cre also eliminates the loxP‐stop‐loxP cassette, permitting widespread expression of Flpo‐ERT2. Because of the estrogen receptor fusion, Flp activity remains inert until administration of tamoxifen, allowing genes modified by frt sites to be modified subsequently with controllable timing. This mouse transgene will be useful in a wide variety of applications where independent control of different mutations in the mouse is desirable.     

A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β‐catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock‐in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi‐cistronic targeting cassette at the 3′ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock‐in allele expresses a bright fluorescent reporter (3xNLS‐SGFP2) and a doxycycline‐inducible driver for lineage tracing (rtTA3). We show that the Axin2^{P2A‐rtTA3‐T2A‐3xNLS‐SGFP2} strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.     

Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Cancer‐associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β‐catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF‐activated PTEN‐deficient mouse melanoma was significantly suppressed in vivo after blocking β‐catenin signaling in CAFs. Further analysis revealed that expression of phospho‐Erk1/2 and phospho‐Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial–mesenchymal transition (EMT)‐like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF‐MEK‐ERK signaling cascade in BRAF‐activated melanoma and may offer a new approach to abrogate host‐dependent drug resistance in targeted therapy.     

Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.     

Salvianolic acid B‐induced microRNA‐152 inhibits liver fibrosis by attenuating DNMT1‐mediated Patched1 methylation

Epithelial‐mesenchymal transition (EMT) was reported to be involved in the activation of hepatic stellate cells (HSCs), contributing to the development of liver fibrosis. Epithelial‐mesenchymal transition can be promoted by the Hedgehog (Hh) pathway. Patched1 (PTCH1), a negative regulatory factor of the Hh signalling pathway, was down‐regulated during liver fibrosis and associated with its hypermethylation status. MicroRNAs (miRNAs) are reported to play a critical role in the control of various HSCs functions. However, miRNA‐mediated epigenetic regulations in EMT during liver fibrosis are seldom studied. In this study, Salvianolic acid B (Sal B) suppressed the activation of HSCs in CCl₄‐treated mice and mouse primary HSCs, leading to inhibition of cell proliferation, type I collagen and alpha‐smooth muscle actin. We demonstrated that the antifibrotic effects caused by Sal B were, at least in part, via inhibition of EMT and the Hh pathway. In particular, up‐regulation of PTCH1 was associated with decreased DNA methylation level after Sal B treatment. Accordingly, DNA methyltransferase 1 (DNMT1) was attenuated by Sal B in vivo and in vitro. The knockdown of DNMT1 in Sal B‐treated HSCs enhanced PTCH1 expression and its demethylation level. Interestingly, increased miR‐152 in Sal B‐treated cells was responsible for the hypomethylation of PTCH1 by Sal B. As confirmed by the luciferase activity assay, DNMT1 was a direct target of miR‐152. Further studies showed that the miR‐152 inhibitor reversed Sal B‐mediated PTCH1 up‐regulation and DNMT1 down‐regulation. Collectively, miR‐152 induced by Sal B, contributed to DNMT1 down‐regulation and epigenetically regulated PTCH1, resulting in the inhibition of EMT in liver fibrosis.     

Epithelial‐to‐mesenchymal transition in epicardium is independent of snail1

The epicardium is the outer epithelial covering the heart. This tissue undergoes an epithelial‐to‐mesenchymal transition (EMT) to generate mesenchymal epicardial‐derived cells (EPDCs) that populate the extracellular matrix of the subepicardium and contribute to the development of the coronary vessels and cardiac interstitial cells. Although epicardial EMT plays a crucial role in heart development, the molecular regulation of this process is incompletely understood. Here we examined the possible role of the EMT regulator Snail1 in this process. Snail1 is expressed in the epicardium and EPDCs during mouse cardiac development. To determine the function of Snail1 in epicardial EMT, we deleted Snail1 in the epicardium using Wt1‐ and Tbx18‐Cre drivers. Unexpectedly, epicardial‐specific Snail1 mutants are viable and fertile and do not display any obvious morphological or functional cardiac abnormalities. Molecular analysis of these mice reveals that epicardial EMT occurs normally, and epicardial derivatives are established in these mutants. We conclude that Snail1 is not required for the initiation and progression of embryonic epicardial EMT. genesis 51:32–40, 2013. © 2012 Wiley Periodicals, Inc.     

MiR‐552 promotes the proliferation,migration and EMT of hepatocellular carcinoma cells by inhibiting AJAP1 expression

Our goal was to explore the function of miR‐552 and its potential target AJAP1 in hepatocellular carcinoma (HCC) oncogenesis and progression. In this study, bioinformatics analysis was performed to detect abnormally expressed miRNAs. The relationship between miR‐552 and AJAP1 was validated using luciferase reporter assays. RT‐qPCR and Western blot assays were applied to explore the expression level of miR‐552, AJAP1 and epithelial‐mesenchymal transition (EMT) markers. HCC cell proliferation was examined using CCK8 assays, while migration and invasion were investigated using Transwell assays. Nude mouse tumourigenesis models were established to facilitate observation of HCC progression in vivo. Finally, prognostic analysis was performed to discover how the prognosis of HCC patients correlated with miR‐552 and AJAP1 expression. MiR‐552 overexpression in HCC cells promoted HCC cell migration, invasion and EMT by targeting/suppressing AJAP1. Poorer prognosis appeared in HCC patients with higher miR‐552 expression or lower AJAP1 levels. Our findings suggested that miR‐552 promotes HCC oncogenesis and progression by inhibiting AJAP1 expression.     

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.     

Glucose promotes epithelial‐mesenchymal transitions in bladder cancer by regulating the functions of YAP1 and TAZ

Glucose levels and type 2 diabetes (T2D) are both associated with tumorigenesis and epithelial‐mesenchymal transitions (EMTs). EMTs facilitate bladder cancer (BC) metastasis development, but the mechanism by which high‐glucose levels promote these EMTs in BC remains unclear. Therefore, we sought to elucidate the mechanism underlying EMT promotion due to increased glucose levels. T24 and UMUC‐3 cells were cultured in media containing different glucose concentrations. YAP1, TAZ, GLUT1 and EMT‐associated marker expression was analysed via Western blotting and qPCR. BC cell proliferation and invasion were assessed using MTT and Transwell assays, respectively. A xenograft nude mouse model of diabetes was used to evaluate tumour growth and metastasis in vivo. T2D was positively associated with pathologic grade (P = .016) and TNM stage (P < .001) in BC. High glucose triggered BC cell proliferation and invasion in both in vitro and in vivo conditions. High‐glucose levels also promoted EMTs in BC cells and increased YAP1 and TAZ expression. YAP1 or TAZ knockdown altered EMT marker expression and decreased GLUT1 expression. Overall, our results suggest that high‐glucose levels promote EMTs in BC cells via YAP1 and TAZ regulation. These effector molecules may be promising therapeutic targets for BC cases comorbid with T2D.     

Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3‐GFP, pOBCol3.6‐GFP, and DMP1‐GFP), similar to the endogenous proteins, are also expressed by bone‐forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP‐Cerulean/DMP1‐Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24‐258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1‐Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP‐Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.     

Oridonin inhibits the migration and epithelial‐to‐mesenchymal transition of small cell lung cancer cells by suppressing FAK‐ERK1/2 signalling pathway

Small cell lung cancer (SCLC) is a severe malignant with high morbidity; however, few effective and secure therapeutic strategy is used in current clinical practice. Oridonin is a small molecule from the traditional Chinese herb Rabdosia rubescens. This study mainly aimed to investigate the role of oridonin on inhibiting the process of H1688, a kind of small cell lung cancer cells from human. Oridonin could suppress H1688 cell proliferation and induce their apoptosis in a high dosage treatment (20 μmol/L). Meanwhile, cell migration was suppressed by oridonin (5 and 10 μmol/L) that did not affect cell proliferation and apoptosis. The expression level of E‐cadherin was significantly increased, and the expression of vimentin, snail and slug was reduced after administration of oridonin. These expression changes were associated with the suppressed integrin β1, phosphorylation of focal adhesion kinase (FAK) and ERK1/2. In addition, oridonin (5 and 10 mg/kg) inhibited tumour growth in a nude mouse model; however, HE staining revealed a certain degree of cytotoxicity in hepatic tissue after treatment oridonin (10 mg/kg). Furthermore, the concentration of alanine aminotransferase (ALP) was significantly increased and lactate dehydrogenase (LDH) was reduced after oridonin treatment (10 mg/kg). Immunohistochemical analysis further revealed that oridonin increased E‐cadherin expression and reduced vimentin and phospho‐FAK levels in vivo. These findings indicated that oridonin can inhibit the migration and epithelial‐to‐mesenchymal transition (EMT) of SCLC cells by suppressing the FAK‐ERK1/2 signalling pathway. Thus, oridonin may be a new drug candidate to offer an effect of anti‐SCLC with relative safety.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.