Christine Biben

Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7−/− mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7−/− mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7−/− heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

An Nkx2-5/Bmp2/Smad1 Negative Feedback Loop Controls Heart Progenitor Specification and Proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

Cardiac Septal and Valvular Dysmorphogenesis in Mice Heterozygous for Mutations in the Homeobox Gene Nkx2-5

Heterozygous mutations in the cardiac homeobox gene, NKX2-5, underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for Nkx2-5–null alleles were assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of Nkx2-5 heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the Nkx2-5 heterozygous phenotype. Our data demonstrate that the complex effects of Nkx2-5 haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human NKX2-5 mutations may be modulated by interacting alleles.

Adult Cardiac-Resident MSC-like Stem Cells with a Proepicardial Origin

Colony-forming units - fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

Hop is an unusual homeobox gene that modulates cardiac development.

Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

DAN is a secreted glycoprotein related to Xenopus cerberus

We report that DAN, a potential cell cycle regulator and tumour suppressor, is a secreted glycoprotein related to Xenopus cerberus. DAN, cerberus, its mouse relative Cer-1/cer-l/Cerberus-like/Cerr1, and the recently described factor DRM/Gremlin, appear to be members of the cystine knot superfamily, which includes TGFbetas and BMPs. Like cerberus and mCer-1, DAN-induced cement glands as well as markers of anterior neural tissue and endoderm in Xenopus animal cap assays, features of BMP signalling blockade. During mouse embryogenesis, Dan was expressed from E8.5 in cranial mesenchyme and somites, then later in limb and facial mesenchyme. The pattern in somites was highly dynamic, with transcripts initially localized to the caudal half of the nascent epithelial somite, then, after maturation, to sclerotomal cells adjacent to the neural tube. Dan was also expressed in the developing myotome. The expression domains include sites in which BMP inhibition is known to be important for development. Thus, DAN appears to be a secreted factor belonging to the cystine knot superfamily, and one of a growing number of antagonists acting to modulate BMP signalling during development.

epicardin: A novel basic helix-loop-helix transcription factor gene expressed in epicardium, branchial arch myoblasts, and mesenchyme of developing lung, gut, kidney, and gonads

We report the cloning, chromosomal localization, and analysis of the expression pattern of epicardin, a member of the basic helix‐loop‐helix (bHLH) family of transcription factors. Within its bHLH domain, the human and murine epicardin genes were most similar to paraxis, a bHLH gene important for segmentation of embryonic paraxial mesoderm. In situ hybridization studies revealed strong epicardin expression in murine embryos at 9.5 days postcoitum (dpc) in a region of the septum transversum at the base of the heart known as the proepicardial organ. This mesenchymal structure extends villous projections from which epicardial precursor cells emerge and migrate out over the surface of the myocardium. Strong expression was seen in individual migratory cells and clusters at 9.5 dpc and in a continuous epicardial cell layer in more mature hearts. Also from 9.5 dpc, epicardin transcripts were seen in endocardial cushions of the atrioventricular canal and outflow tract, in skeletal myoblasts within branchial arches and in condensing mesenchyme of gut, kidney, urinary tract, gonads, spleen, and lung. Northern analysis showed that expression persisted in mature visceral organs and heart, but was transient in skeletal muscle. The central role played by bHLH factors in pathways for tissue determination in the embryo suggests a function for epicardin in specification of select mesodermal cell populations associated with heart, cranial skeletal muscle, gut, and urogenital system. Dev. Dyn. 1998;213:105–113.

EXPRESSION OF NK-2 CLASS HOMEOBOX GENE NKX2-6 IN FOREGUT ENDODERM AND HEART

NK-2 class homeobox genes are candidate patterning and lineage regulators in diverse organisms. We report here the embryonic expression pattern of murine member, Nkx2-6. In keeping with its vertebrate relatives, Nkx2-6 was transcribed in ventrolateral embryonic structures. Expression was first detected at E8.0 in endodermal walls of the foregut pocket, tissue destined to become pharyngeal floor. From E8.0-10.5, transcripts were concentrated in pharyngeal pouches and juxtaposed arch ectoderm and mesoderm, as well as in more caudal gut segments. Expression was also seen at opposite poles of the developing heart from E8-8.5 in posterior myocardial progenitors, then sinus venosa and dorsal pericardium, and from E9.5 in outflow tract myocardium.