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.

Previous findings from our laboratory demonstrated that some clonally expanded cerebrospinal fluid (CSF) B cells from MS patients exhibit diminished mutation targeting patterns in comparison to typical B cells selected in the context of germinal centers (GCs). In order to determine whether the overall CSF B cell repertoires adhered to mutation patterns typical of GC-selected B cells, we analyzed the immunoglobulin repertoires from CSF B cells of 8 MS patients for mutation characteristics typical of GC-derived B cells. Mutation targeting was preserved. Thus, clonal expansion of some CSF B cells may occur independently of GC, but the CSF B cell pool is governed by typical GC selection. Interestingly, the heavy chain CDR3's of CSF B cells from MS patients had a net acidic charge, similar to GC-derived B cells, but a tendency towards longer CDR3's, consistent with autoreactive B cells. How these findings may support current hypotheses regarding the origin of CSF B cells is discussed.

Spina bifida, or failure of the vertebrae to close at the midline, is a common congenital malformation in humans that is often synonymous with neural tube defects (NTDs). However, it is likely that other etiologies exist. Genetic disruption of platelet-derived growth factor receptor (PDGFR) ␣ results in spina bifida, but the underlying mechanism has not been identified. To elucidate the cause of this birth defect in PDGFR␣ mutant embryos, we examined the developmental processes involved in vertebrae formation. Exposure of chick embryos to the PDGFR inhibitor imatinib mesylate resulted in spina bifida in the absence of NTDs. We next examined embryos with a tissue-specific deletion of the receptor. We found that loss of the receptor from chondrocytes did not recapitulate the spina bifida phenotype. By contrast, loss of the receptor from all sclerotome and dermatome derivatives or disruption of PDGFR␣-driven phosphatidyl-inositol 3Ј kinase (PI3K) activity resulted in spina bifida. Furthermore, we identified a migration defect in the sclerotome as the cause of the abnormal vertebral development. We found that primary cells from these mice exhibited defects in PAK1 activation and paxillin localization. Taken together, these results indicate that PDGFR␣ downstream effectors, especially PI3K, are essential for cell migration of a somite-derived dorsal mesenchyme and disruption of receptor signaling in these cells leads to spina bifida.