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Abstract Background Notch4 is a member of the Notch family of receptors that is primarily expressed in the vascular endothelial cells. Genetic deletion of Notch4 does not result in an overt phenotype in mice, thus the function of Notch4 remains poorly understood. Methods We examined the requirement for Notch4 in the development of breast cancer vasculature. Orthotopic transplantation of mouse mammary tumor cells wild type for Notch4 into Notch4 deficient hosts enabled us to delineate the contribution of host Notch4 independent of its function in the tumor cell compartment. Results Here, we show that Notch4 expression is required for tumor onset and early tumor perfusion in a mouse model of breast cancer. We found that Notch4 expression is upregulated in mouse and human mammary tumor vasculature. Moreover, host Notch4 deficiency delayed the onset of MMTV-PyMT tumors, wild type for Notch4, after transplantation. Vessel perfusion was decreased in tumors established in Notch4-deficient hosts. Unlike in inhibition of Notch1 or Dll4, vessel density and branching in tumors developed in Notch4-deficient mice were unchanged. However, final tumor size was similar between tumors grown in wild type and Notch4 null hosts. Conclusion Our results suggest a novel role for Notch4 in the establishment of tumor colonies and vessel perfusion of transplanted mammary tumors.

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance (1, 2), while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation (3-6). Recently, stem cells have been shown to regulate themselves using an autocrine mechanism (7). The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells (1). Since then, an increasing body of work has focused on defining stem cell niches (1-6). Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium, basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells (8, 9). Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells (9, 10). Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly, this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation, lineage tracing and signalling pathway modulation, we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals, the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus, a parent stem/progenitor cell can serve as a functional daughter cell niche., To establish whether post-mitotic ciliated cells send a conventional feedback signal to regulate the replication of their parent stem and progenitor cells, we genetically ablated ciliated cells using FOXJ1-creER; LSL-DTA [...]

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer [...]

HSCs either self-renew or differentiate to give rise to multipotent cells whose progeny provide blood cell precursors. However, surprisingly little is known about the factors that regulate this choice of self-renewal versus differentiation. One candidate is the Notch signaling pathway, with ex vivo studies suggesting that Notch regulates HSC differentiation, although a functional role for Notch in HSC self-renewal in vivo remains controversial. Here, we have shown that Notch2, and not Notch1, inhibits myeloid differentiation and enhances generation of primitive [Sca-1.sup.+]c-[kit.sup.+] progenitors following in vitro culture of enriched HSCs with purified Notch ligands. In mice, Notch2 enhanced the rate of formation of short-term repopulating multipotential progenitor cells (MPPs) as well as long-term repopulating HSCs, while delaying myeloid differentiation in BM following injury. However, consistent with previous reports, once homeostasis was achieved, neither Notch1 nor Notch2 affected repopulating cell self-renewal. These data indicate a Notch2-dependent role in assuring orderly repopulation by HSCs, MPPs, myeloid cells, and lymphoid cells during BM regeneration., Introduction HSCs either self-renew or give rise to multipotent cells whose progeny provide precursors committed to the lymphoid and myeloid lineages. Recent studies have identified factors that maintain HSCs in [...]

Am J Physiol Lung Cell Mol Physiol 298: L45-L56, 2010. First published November 6, 2009; doi: 10.1152/ajplung.90550.2008.--Distal lung development occurs through coordinated induction of myofibroblasts, epithelial cells, and capillaries. Lunatic Fringe (Lfng) is a [[beta].sub.1-3] N-acetylglucosamine transferase that modifies Notch receptors to facilitate their activation by Delta-like (D111/4) ligands. Lfng is expressed in the distal lung during saccular development, and deletion of this gene impairs myofibroblast differentiation and alveogenesis in this context. A similar defect was observed in [Notch2.sup.[beta]geo/+] [Notch3.sup.[beta]-geo/[beta]-geo] compound mutant mice but not in [Notch2.sup.[beta]geo/+] or [Notch3.sup.[beta]-geo/[beta]-geo] single mutants. Finally, to directly test for the role of Notch signaling in myofibroblast differentiation in vivo, we used ROSA26-[rtTA.sup./+];[tetO-CRE.sup./+];RBPJ[[kappa].sup.flox/flox] inducible mutant mice to show that disruption of canonical Notch signaling during late embryonic development prevents induction of smooth muscle actin in mesenchymal cells of the distal lung. In sum, these results demonstrate that Lfng functions to enhance Notch signaling in myofibroblast precursor cells and thereby to coordinate differentiation and mobilization of myofibroblasts required for alveolar septation. alveolar development

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.10.056 Byline: Steve Rodriguez (a), Heather M. Sickles (a), Chris DeLeonardis (a), Ana Alcaraz (a), Thomas Gridley (b), David M. Lin (a) Keywords: Notch; Glia; Olfactory; Sustentacular; Neurodegeneration; Hes1; Hey1; Six1; Notch2; GST Abstract: Notch receptors are expressed in neurons and glia in the adult nervous system, but why this expression persists is not well-understood. Here we examine the role of the Notch pathway in the postnatal mouse main olfactory system, and show evidence consistent with a model where Notch2 is required for maintaining sustentacular cell function. In the absence of Notch2, the laminar nature of these glial-like cells is disrupted. Hes1, Hey1, and Six1, which are downstream effectors of the Notch pathway, are down-regulated, and cytochrome P450 and Glutathione S-transferase (GST) expression by sustentacular cells is reduced. Functional levels of GST activity are also reduced. These disruptions are associated with increased olfactory sensory neuron degeneration. Surprisingly, expression of Notch3 is also down-regulated. This suggests the existence of a feedback loop where expression of Notch3 is initially independent of Notch2, but requires Notch2 for maintained expression. While the Notch pathway has previously been shown to be important for promoting gliogenesis during development, this is the first demonstration that the persistent expression of Notch receptors is required for maintaining glial function in adult. Author Affiliation: (a) Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA (b) The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA Article History: Received 17 May 2007; Revised 25 October 2007; Accepted 26 October 2007

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene ([Jag1.sup.del1]) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed [Jag1.sup.del1] heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in [Jag1.sup.del1] heterozygous mice from this same cross.

L5Jcs1 is a perinatal lethal mutation uncovered in a screen for ENU-induced mutations on mouse chromosome 5. L5Jcs1 homozygotes exhibit posterior-to-anterior transformations of the vertebral column midsection, similar to mice deficient for Hoxc8 and Hoxc9. Positional cloning efforts identified a mutation in a novel, evolutionarily conserved, and ubiquitously expressed gene dubbed Tapt1 (Transmembrane anterior posterior transformation 1). TAPT1 is predicted to contain several transmembrane domains, and part of the gene is orthologous to an unusual alternatively spliced human transcript encoding the cytomegalovirus gH receptor. We speculate that TAPT1 is a downstream effector of HOXC8 that may act by transducing or transmitting extracellular information required for axial skeletal patterning during development.

In Drosophila, mutations in the Twist gene interact with mutations in the Snail gene. We show that the mouse Twist1 mutation interacts with Snail and Snai2 mutations to enhance aberrant cranial suture fusion, demonstrating that genetic interactions between genes of the Twist and Snail families have been conserved during evolution.

Radial glia functions as neural progenitors and as scaffolding supporting neuronal migration during CNS development. A study identifies brain lipid-binding protein as the first predominantly CNS-specific Notch target gene and mediates some aspects of Notch signaling in radial glia.

The mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit happloinsufficient lethality because of defects in vascular remodeling is reported. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations.

Teeth develop as epithelial appendages, and their morphogenesis is regulated by epithelial--mesenchymal interactions and conserved signaling pathways common to many developmental processes. A key event during tooth morphogenesis is the transition from bud to cap stage when the epithelial bud is divided into specific compartments distinguished by morphology as well as gene expression patterns. The enamel knot, a signaling center, forms and regulates the shape and size of the tooth. Mesenchymal signals are necessary for epithelial patterning and for the formation and maintenance of the epithelial compartments. We studied the expression of Notch pathway molecules during the bud-to-cap stage transition of the developing mouse tooth. Lunatic fringe expression was restricted to the epithelium, where it formed a boundary flanking the enamel knot. The Lunatic fringe expression domains overlapped only partly with the expression of Notch1 and Notch2, which were coexpressed with Hes1. We examined the regulation of Lunatic fringe and Hes1 in cultured explants of dental epithelium. The expression of Lunatic fringe and Hes1 depended on mesenchymal signals and both were positively regulated by FGF-10. BMP-4 antagonized the stimulatory effect of FGF-10 on Lunatic fringe expression but had a synergistic effect with FGF-10 on Hes1 expression. Recombinant Lunatic fringe protein induced Hes1 expression in the dental epithelium, suggesting that Lunatic fringe can act also extracellularly. Lunatic fringe mutant mice did not reveal tooth abnormalities, and no changes were observed in the expression patterns of other Fringe genes. We conclude that Lunatic fringe may play a role in boundary formation of the enamel knot and that Notch-signaling in the dental epithelium is regulated by mesenchymal FGFs and BMP. Key Words: signaling center; enamel knot; tooth development; Lunatic fringe; Notch; Jagged; BMP; FGF; Hes1.

Research has been conducted on the role of the Notch4 gene in the intercellular signaling mechanism. The generation of the Notch4-deficient mice by gene targeting is described.

The mouse Nkx3.1 (ital) homeobox gene has roles in prostate development and in cancer. This gene is the earliest known marker of prostate epithelium in embryogenesis and is expressed after that at all stages of prostate differentiation in vivo and in tissue recombinants. NKX3.1 (ital) may be a prostate-specific tumor suppressor gene. Loss a single allele may constitute a predisposition to carcinogenesis in the prostate.

The lunatic fringe (Lfng) gene is expressed in mice during somitogenesis, in a form that suggests a potential role for it in somite formation regulation. The role of the Lfng gene during embryogenesis, has been analyzed, and mice homozygous for a targeted mutation of the Lfng gene had defects in somite formation. Somites in the mutant embryos were of irregular size and shape, and anterior/posterior patterning was disturbed.

The Slug gene encodes a zinc finger protein, homologous to the product of the Drosophila Snail gene, that is implicated in the generation and migration of both mesoderm and neural crest cells in several vertebrate species. We describe here the cloning and genetic analysis of the mouse Slug (Slugh) gene. Slugh encodes a 269-amino-acid protein that shares 92% amino acid identity with the product of the chicken Slug gene. We have characterized Slugh gene expression during early mouse embryogenesis by whole mount in situ hybridization of Slugh mRNA and through detection of [Beta]-galactosidase expression from an in-frame [Slugh.sup.1acZ] allele generated through homologous recombination. Slugh expression is first detected in extraembryonic mesoderm and is later detected in many mesodermal subsets, although it is not detected in the primitive streak. In contrast to many other vertebrates, the mouse Slug gene is not expressed in premigratory neural crest cells but is expressed in migratory neural crest cells. Analysis of a targeted null mutation that deleted all Slugh coding sequences revealed that Slugh is not required for mesoderm formation or for neural crest generation, migration, or development in mice. These results indicate that neither the expression pattern nor the biological function of the Slug gene is conserved among all vertebrates. These data also raise interesting questions about the regulation of neural crest generation, which is one of the distinguishing characteristics of the vertebrate subphylum.

Jagged2 gene and Notch signaling, which is a intercellular signaling mechanism, play an important part in the limb, thymic and craniofacial development of mice. Mutant mice without a domain of the Jagged2 protein had fatal defects in craniofacial morphogenesis. The mice also had fusion in the limbs and modified thymic morphology.

The mechanism involved in the T cell lineage decision was studied using transgenic mice as host systems, supplemented with flow cytometry and polymerase chain reaction-restriction fragment length polymorphism. The results revealed that T cells that are heterozygous for a mutation of the Notch1 gene have a greater tendency to develop as gamma-delta T cells rather than as alpha-beta T cells. A constitutively activated form of Notch gene, on the other hand, stimulates the thymocytes to adopt the alpha-beta T cell fate. Notch, together with the newly formed T cell antigen receptor are thus identified as determinants of T cell lineage decision.

In vivo analyses to study the functions of receptor tyrosine kinases, Tie-1 and Tie-2, during vascular endothelial cell growth and differentiation indicate distinct, important roles for these structurally related receptors in the formation of blood vessels. Homologous recombination was used to make targeted mutations of these receptors' genes in mice. Tie-1 deficient mice were unable to structurally stabilize their vascular endothelial cells, resulting in localized hemorrhage and oedema. Tie-2 deficient mice had defective vasculogensis and low endothelial cell survival rates.

Histological analysis of the skeleton of mutant Hoxa-2 mice proves the existence of a branchial Hox code. The elements, usually derived from the second arch, are replaced by elements resembling more anterior structures such as Meckel's cartilage. Mice born with Hoxa-2 mutants consequently exhibit multiple cranial skeletal defects, accompanied by a cleft palate, and die within 24 hours of birth.