SUMMARYThe basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.

Abstract-The epicardium plays an essential role in coronary artery formation and myocardial development, but signals controlling the development and differentiation of this tissue are not well understood. To investigate the role of platelet-derived growth factor receptor (PDGFR)␤ in development of epicardial-derived vascular smooth muscle cells (VSMCs), we examined PDGFR␤ Ϫ/Ϫ and PDGFR␤ epicardial mutant hearts. We found that PDGFR␤ Ϫ/Ϫ hearts failed to form dominant coronary vessels on the ventral heart surface, had a thinned myocardium, and completely lacked coronary VSMCs (cVSMCs). This constellation of defects was consistent with a primary defect in the epicardium. To verify that these defects were specific to epicardial derivatives, we generated mice with an epicardial deletion of PDGFR␤ that resulted in reduced cVSMCs distal to the aorta. The regional absence of cVSMCs suggested that cVSMCs could arise from 2 sources, epicardial and nonepicardial, and that both were dependent on PDGFR␤. In the absence of PDGFR␤ signaling, epicardial cells adopted an irregular actin cytoskeleton, leading to aberrant migration of epicardial cells into the myocardium in vivo. In addition, PDGF receptor stimulation promoted epicardial cell migration, and PDGFR␤-driven phosphoinositide 3Ј-kinase signaling was critical for this process. Our data demonstrate that PDGFR␤ is required for the formation of 2 distinct cVSMC populations and that loss of PDGFR␤-PI3K signaling disrupts epicardial cell migration. , and past data have demonstrated that many coronary VSMCs (cVSMCs) are derived from the embryonic epicardium. 2,3 Whereas several genes have been identified that are essential for the formation, attachment, and spreading of the epicardium, few genes have been identified that are essential during epithelial-to-mesenchymal transition (EMT) and subsequent differentiation into cVSMCs and cardiac fibroblasts.Platelet-derived growth factor receptor (PDGFR) tyrosine kinases are 1 family of signaling proteins that are potentially involved in epicardial cell function. Analyses in the mouse have shown that PDGFR␤ signaling promotes proliferation and migration of VSMCs in multiple vascular beds including the heart. 4 -8 Therefore, we investigated the function of PDGFR␤ signaling during epicardial development. We have examined PDGFR␤ Ϫ/Ϫ , epicardial-specific PDGFR␤ mutant, and PDGFR␤ signaling-deficient embryos. We discovered that epicardial deletion resulted in the absence of cVSMCs distal to the aorta and that PDGFR␤ signaling through phosphoinositide 3Ј-kinase (PI3K) was required for proper cytoskeletal organization in epicardial cells. Our results designate PDGF receptor signaling as another growth factor system involved in epicardial development.

SUMMARY Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occuring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a 7-gene expression signature. Target efficacies were validated in vivo, and mechanism of action studies uncovered new cancer cell biology.

Tcf21 is a class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21iCre) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.