Myoblasts and macrophages share molecular components that contribute to cell–cell fusion

Cell–cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and β1-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells

Hematopoietic Jagged1 is a fetal liver niche factor required for functional maturation and engraftment of fetal hematopoietic stem cells

Notch signaling is essential for the emergence of definitive hematopoietic stem cells (HSCs) in the embryo and their development in the fetal liver niche. However, how Notch signaling is activated and which fetal liver cell type provides the ligand for receptor activation in HSCs is unknown. Here we provide evidence that endothelial Jagged1 (Jag1) has a critical early role in fetal liver vascular development but is not required for hematopoietic function during fetal HSC expansion. We demonstrate that Jag1 is expressed in many hematopoietic cells in the fetal liver, including HSCs, and that its expression is lost in adult bone marrow HSCs. Deletion of hematopoietic Jag1 does not affect fetal liver development; however, Jag1-deficient fetal liver HSCs exhibit a significant transplantation defect. Bulk and single-cell transcriptomic analysis of HSCs during peak expansion in the fetal liver indicates that loss of hematopoietic Jag1 leads to the downregulation of critical hematopoietic factors such as GATA2, Mllt3, and HoxA7, but does not perturb Notch receptor expression. Ex vivo activation of Notch signaling in Jag1-deficient fetal HSCs partially rescues the functional defect in a transplant setting. These findings indicate a new fetal-specific niche that is based on juxtracrine hematopoietic Notch signaling and reveal Jag1 as a fetal-specific niche factor essential for HSC function

A Tie2-Notch1 signaling axis regulates regeneration of the endothelial bone marrow niche.

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.We thank Dr. Warren Pear for invaluable advice and for sharing the Notch1+/ΔTAD murine model system. We also thank Dr. Kishore Wary for sharing the Cdh5-CreERT2 mouse model. Drs. Jon Aster and Stephen Blacklow for advice and thoughtful discussion, Dr. Dawson Gerhardt for her help in generating the Notch1- ΔTAD plasmids and vector constructs, Dr. Jan Kitejewski for helpful advice on Notch mutant mice and Drs. Fotini Gounari and Linda Dagenstein of the University of Chicago transgenic mouse facility for help in maintaining the transgenic mouse colonies. The following cores at the University of Illinois at Chicago contributed to this study: RRC Histology Core and RRC Flow Cytometry Core. This study was funded by NIH grants 1R01HL134971 to KVP and 1R01HL136529 to DL.S

Trib2 suppresses tumor initiation in Notch-driven T-ALL

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes

Exploring the mycobacteriophage metaproteome: Phage genomics as an educational platform

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three - encoding tape-measure proteins, lysins, and minor tail proteins - are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education. © 2006 Hatfull et al

Myoblasts and macrophages share molecular components that contribute to cell-cell fusion.

Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and beta(1)-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells

Dlk1-Mediated Temporal Regulation of Notch Signaling Is Required for Differentiation of Alveolar Type II to Type I Cells during Repair

Summary: Lung alveolar type I cells (AT1) and alveolar type II cells (AT2) regulate the structural integrity and function of alveoli. AT1, covering ∼95% of the surface area, are responsible for gas exchange, whereas AT2 serve multiple functions, including alveolar repair through proliferation and differentiation into AT1. However, the signaling mechanisms for alveolar repair remain unclear. Here, we demonstrate, in Pseudomonas aeruginosa-induced acute lung injury in mice, that non-canonical Notch ligand Dlk1 (delta-like 1 homolog) is essential for AT2-to-AT1 differentiation. Notch signaling was activated in AT2 at the onset of repair but later suppressed by Dlk1. Deletion of Dlk1 in AT2 induced persistent Notch activation, resulting in stalled transition to AT1 and accumulation of an intermediate cell population that expressed low levels of both AT1 and AT2 markers. Thus, Dlk1 expression leads to precisely timed inhibition of Notch signaling and activates AT2-to-AT1 differentiation, leading to alveolar repair. : Finn et al. show that Notch signaling is activated in type II cells after alveolar injury but that subsequent Dlk1-mediated inhibition of Notch is required for complete type II-to-type I cell transition and alveolar repair. Thus, Dlk1 and Notch are potential therapeutic targets for treatment of lung injury. Keywords: lung, alveoli, progenitor type II cell, Notch, Dlk

Embryonic Stem Cell Differentiation to Functional Arterial Endothelial Cells through Sequential Activation of ETV2 and NOTCH1 Signaling by HIF1α

The generation of functional arterial endothelial cells (aECs) from embryonic stem cells (ESCs) holds great promise for vascular tissue engineering. However, the mechanisms underlying their generation and the potential of aECs in revascularizing ischemic tissue are not fully understood. Here, we observed that hypoxia exposure of mouse ESCs induced an initial phase of HIF1α-mediated upregulation of the transcription factor Etv2, which in turn induced the commitment to the EC fate. However, sustained activation of HIF1α in these EC progenitors thereafter induced NOTCH1 signaling that promoted the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the mouse hindlimb ischemia model. Furthermore, transplantation of aECs in mice showed engraftment in ischemic myocardium and restored cardiac function in contrast to ECs derived under normoxia. Thus, HIF1α activation of Etv2 in ESCs followed by NOTCH1 signaling is required for the generation aECs that are capable of arteriogenesis and revascularization of ischemic tissue