The Snail transcription factor regulates diverse aspects of stem cell biology in organisms ranging from Drosophila to mammals. Here we have asked whether it regulates the biology of neural precursor cells (NPCs) in the forebrain of postnatal and adult mice, taking advantage of a mouse containing a floxed Snail allele (Snailfl/fl mice). We show that when Snail is inducibly ablated in the embryonic cortex, this has long-term consequences for cortical organization. In particular, when Snailfl/fl mice are crossed to Nestin-cre mice that express Cre recombinase in embryonic neural precursors, this causes inducible ablation of Snail expression throughout the postnatal cortex. This loss of Snail causes a decrease in proliferation of neonatal cortical neural precursors and mislocalization and misspecification of cortical neurons. Moreover, these precursor phenotypes persist into adulthood. Adult neural precursor cell proliferation is decreased in the forebrain subventricular zone and in the hippocampal dentate gyrus, and this is coincident with a decrease in the number of adult-born olfactory and hippocampal neurons. Thus, Snail is a key regulator of the numbers of neural precursors and newborn neurons throughout life.
Two Snail family genes, Snai1 and Snai2, encode E2 box-binding transcriptional repressors that are important for cartilage development during long bone formation in mice. We demonstrated previously that the Snai1 and Snai2 genes function redundantly, and compensate for each other’s loss during mouse chondrogenesis in vivo. A prediction from this genetic data is that the SNAI1 and SNAI2 proteins can bind to each other’s promoter to regulate gene expression. Here we demonstrate that expression of Snai1 and Snai2 RNA and protein is induced during chondrogenic differentiation of cultured mouse ATDC5 cells. Using chromatin immunoprecipitation assays, we then show that endogenous SNAI1 and SNAI2 proteins bind to a subset of E2 boxes in both their own and each other’s promoter in differentiating ATDC5 cells. Together with our previous genetic data, these results support the model that expression of the Snai1 and Snai2 genes is negatively regulated by their protein products occupying each other’s promoter during chondrogenesis, and help provide an explanation for the genetic redundancy observed in the mouse loss of function models.
Snail; Slug; transcriptional repressor; chondrogenesis; chromatin immunoprecipitation
Endochondral bone formation is a multistep process during which a cartilage primordium is replaced by mineralized bone. Several genes involved in cartilage and bone development have been identified as target genes for the Snail family of zinc finger transcriptional repressors, and a gain-of-function study has demonstrated that upregulation of Snai1 activity in mouse long bones caused a reduction in bone length. However, no in vivo loss-of-function studies have been performed to establish whether Snail family genes have an essential, physiological role during normal bone development. We demonstrate here that the Snai1 and Snai2 genes function redundantly during embryonic long bone development in mice. Deletion of the Snai2 gene, or limb bud-specific conditional deletion of the Snai1 gene, did not result in obvious defects in the skeleton. However, limb bud-specific Snai1 deletion on a Snai2 null genetic background resulted in substantial defects in the long bones of the limbs. Long bones of the Snai1/Snai2 double mutants exhibited defects in chondrocyte morphology and organization, inhibited trabecular bone formation and delayed ossification. Chondrocyte proliferation was markedly reduced, and transcript levels of genes encoding cell cycle regulators, such as p21Waf1/Cip1, were strikingly upregulated in the Snai1/Snai2 double mutants, suggesting that during chondrogenesis Snail family proteins act to control cell proliferation by mediating expression of cell cycle regulators. Snai2 transcript levels were increased in Snai1 mutant femurs, while Snai1 transcript levels were increased in Snai2 mutant femurs. In addition, in the mutant femurs the Snai1 and Snai2 genes compensated for each other's loss not only quantitatively, but also by expanding their expression into the other genes' normal expression domains. These results demonstrate that the Snai1 and Snai2 genes transcriptionally compensate temporally, spatially, and quantitatively for each other's loss, and demonstrate an essential role for Snail family genes during chondrogenesis in mice.
SNAIL; SLUG; PRRX1-CRE; FUNCTIONAL REDUNDANCY; ENDOCHONDRAL OSSIFICATION
The Snail1 transcriptional repressor plays a key role in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, in adult animals it is limited to a subset of mesenchymal cells where it has a largely unknown function. Using a mouse model with inducible depletion of Snail1, here we demonstrate that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-β1 which shows a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals causes a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiate to osteoblasts or adipocytes and, in contrast to controls, are resistant to the TGF-β1-induced differentiation block. These results demonstrate a new role for Snail1 in TGF-β response and MSC maintenance.
Snail1; mesenchymal stem cells; TGF-β; Akt
During mammalian palatogenesis, palatal shelves initially grow vertically from the medial sides of the paired maxillary processes flanking the developing tongue and subsequently elevate and fuse with each other above the tongue to form the intact secondary palate. Pathological palate-mandible or palate-tongue fusions have been reported in humans and other mammals, but the molecular and cellular mechanisms that prevent such aberrant adhesions during normal palate development are unknown. We previously reported that mice deficient in Jag2, which encodes a cell surface ligand for the Notch family receptors, have cleft palate associated with palate-tongue fusions. In this report, we show that Jag2 is expressed throughout the oral epithelium and is required for Notch1 activation during oral epithelial differentiation. We show that Notch1 is normally highly activated in the differentiating oral periderm cells covering the developing tongue and the lateral oral surfaces of the mandibular and maxillary processes during palate development. Oral periderm activation of Notch1 is significantly attenuated during palate development in the Jag2 mutants. Further molecular and ultrastructural analyses indicate that oral epithelial organization and periderm differentiation are disrupted in the Jag2 mutants. Moreover, we show that the Jag2 mutant tongue fused to wildtype palate shelves in recombinant explant cultures. These data indicate that Jag2-Notch1 signaling is spatiotemporally regulated in the oral epithelia during palate development to prevent premature palatal shelf adhesion to other oral tissues and to facilitate normal adhesion between the elevated palatal shelves.
epithelial differentiation; maxillary-mandibular fusion; periderm; palate development; palatal fusion; palate-tongue fusion; synechiae; Jag2; Notch1
The Snail gene family encodes DNA-binding zinc finger proteins that function as transcriptional repressors. While the Snai1 and Snai2 genes are required for normal development in mice, Snai3 mutant mice exhibit no obvious abnormalities. The Snai3 gene is expressed at high levels in skeletal muscle. However, we demonstrate by histological analysis that Snai3 null mutant mice exhibit normal skeletal muscle. During hindlimb muscle regeneration after cardiotoxin-mediated injury, the Snai3 null mice exhibited efficient regeneration. To determine whether the Snai3 gene functions redundantly with the Snai1 gene during skeletal muscle regeneration, we performed hindlimb muscle regeneration in mice with skeletal muscle-specific deletion of the Snai1 gene on a Snai3 null genetic background. These mice also exhibited efficient regeneration, demonstrating that there is no major role for the Snai1 and Snai3 genes in regulating skeletal muscle regeneration in mice.
Jagged2 preferentially signals through Notch3 to promote γδ T cell development.
In humans, high Notch activation promotes γδ T cell development, whereas lower levels promote αβ-lineage differentiation. How these different Notch signals are generated has remained unclear. We show that differential Notch receptor–ligand interactions mediate this process. Whereas Delta-like 4 supports both TCR-αβ and -γδ development, Jagged1 induces mainly αβ-lineage differentiation. In contrast, Jagged2-mediated Notch activation primarily results in γδ T cell development and represses αβ-lineage differentiation by inhibiting TCR-β formation. Consistently, TCR-αβ T cell development is rescued through transduction of a TCR-β transgene. Jagged2 induces the strongest Notch signal through interactions with both Notch1 and Notch3, whereas Delta-like 4 primarily binds Notch1. In agreement, Notch3 is a stronger Notch activator and only supports γδ T cell development, whereas Notch1 is a weaker activator supporting both TCR-αβ and -γδ development. Fetal thymus organ cultures in JAG2-deficient thymic lobes or with Notch3-blocking antibodies confirm the importance of Jagged2/Notch3 signaling in human TCR-γδ differentiation. Our findings reveal that differential Notch receptor–ligand interactions mediate human TCR-αβ and -γδ T cell differentiation and provide a mechanistic insight into the high Notch dependency of human γδ T cell development.
The Snail gene family encodes zinc finger-containing transcriptional repressor proteins. Three members of the Snail gene family have been described in mammals, encoded by the Snai1, Snai2, and Snai3 genes. The function of the Snai1 and Snai2 genes have been studied extensively during both vertebrate embryogenesis and tumor progression and metastasis, and play critically important roles during these processes. However, little is known about the function of the Snai3 gene and protein. We describe here generation and analysis of Snai3 conditional and null mutant mice. We also generated an EYFP-tagged Snai3 null allele that accurately reflects endogenous Snai3 gene expression, with the highest levels of expression detected in thymus and skeletal muscle. Snai3 null mutant homozygous mice are viable and fertile, and exhibit no obvious phenotypic defects. These results demonstrate that Snai3 gene function is not essential for embryogenesis in mice.
Notch signaling is an evolutionarily-conserved, intercellular signaling mechanism that plays myriad roles during vascular development and physiology in vertebrates. These roles include the regulation of arteriovenous specification and differentiation in both endothelial cells and vascular smooth muscle cells, regulation of blood vessel sprouting and branching during normal and pathological angiogenesis, and the physiological responses of vascular smooth muscle cells. Defects in Notch signaling also cause inherited vascular diseases, such as the degenerative vascular disorder Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). This review summarizes recent studies that highlight the multiple roles the Notch signaling pathway plays during vascular development and physiology.
The Notch-regulated ankyrin repeat protein (Nrarp) is a component of a negative feedback system that attenuates Notch pathway-mediated signaling. In vertebrates, the timing and spacing of formation of the mesodermal somites is controlled by a molecular oscillator termed the segmentation clock. Somites are also patterned along the rostral-caudal axis of the embryo. Here we demonstrate that Nrarp-deficient embryos and mice exhibit genetic background-dependent defects of the axial skeleton. While progression of the segmentation clock occurred in Nrarp-deficient embryos, they exhibited altered rostrocaudal patterning of the somites. In Nrarp mutant embryos, the posterior somite compartment was expanded. These studies confirm an anticipated, but previously undocumented role for the Nrarp gene in vertebrate somite patterning, and provide an example of the strong influence that genetic background plays on the phenotypes exhibited by mutant mice.
Notch pathway; negative feedback; rostrocaudal somite patterning
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 activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell–mediated human disorders.
In the mouse ovary, oocytes initially develop in clusters termed germ-cell nests. Shortly after birth, these germ-cell nests break apart, and the oocytes individually become surrounded by somatic granulosa cells to form primordial follicles. Notch signaling plays essential roles during oogenesis in Drosophila, and recent studies have suggested that Notch signaling also plays an essential role during oogenesis and ovary development in mammals. However, no in vivo loss-of-function studies have been performed to establish whether Notch family receptors have an essential physiological role during normal ovarian development in mutant mice.
Female mice with conditional deletion of the Notch2 gene in somatic granulosa cells of the ovary exhibited reduced fertility, accompanied by the formation of multi-oocyte follicles, which became hemorrhagic by 7 weeks of age. Formation of multi-oocyte follicles resulted from defects in breakdown of the primordial germ-cell nests. The ovaries of the Notch2 conditional mutant mice had increased numbers of oocytes, but decreased numbers of primordial follicles. Oocyte numbers in the Notch2 conditional mutants were increased not by excess or extended cellular proliferation, but as a result of decreased oocyte apoptosis.
Our work demonstrates that Notch2-mediated signaling in the somatic-cell lineage of the mouse ovary regulates oocyte apoptosis non-cell autonomously, and is essential for regulating breakdown of germ-cell nests and formation of primordial follicles. This model provides a new resource for studying the developmental and physiological roles of Notch signaling during mammalian reproductive biology.
oogenesis; Notch signaling; apoptosis.
Cardiac valves originate from endocardial cushions (EC) formed by endothelial-to-mesenchymal transformation (EMT) during embryogenesis. The zinc-finger transcription factor Snai1 has previously been reported to be important for EMT during organogenesis, yet its role in early valve development has not been directly examined. In this study we show that Snai1 is highly expressed in endothelial, and newly transformed mesenchyme cells during EC development. Mice with targeted snai1 knockdown display hypocellular ECs at E10.5 associated with decreased expression of mesenchyme cell markers and downregulation of the matrix metalloproteinase (mmp) family member, mmp15. Snai1 overexpression studies in atrioventricular canal collagen I gel explants indicate that Snai1 is sufficient to promote mmp15 expression, cell transformation, and mesenchymal cell migration and invasion. However, treatment with the catalytically active form of MMP15 promotes cell motility, and not transformation. Further, we show that Snai1-mediated cell migration requires MMP activity, and caMMP15 treatment rescues attenuated migration defects observed in murine ECs following snai1 knockdown. Together, findings from this study reveal previously unappreciated mechanisms of Snai1 for the direct regulation of MMPs during EC development.
Snai1; MMP15; endocardial cushion; heart valve; endothelial-to-mesenchymal transformation
The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism that is required for embryonic development, cell fate specification, and stem cell maintenance. Discovered and studied initially in Drosophila melanogaster, the Notch pathway is conserved and functionally active throughout the animal kingdom. In this paper, we summarize the biochemical mechanisms of Notch signaling and describe its role in regulating one particular developmental pathway, oogenesis in Drosophila.
Notch signaling in the cardiovascular system is important during embryonic development, vascular repair of injury, and vascular pathology in humans. The vascular smooth muscle cell (VSMC) expresses multiple Notch receptors throughout its life cycle, and responds to Notch ligands as a regulatory mechanism of differentiation, recruitment to growing vessels, and maturation. The goal of this review is to provide an overview of the current understanding of the molecular basis for Notch regulation of VSMC phenotype. Further, we will explore Notch interaction with other signaling pathways important in VSMC.
Notch; vascular smooth muscle; signaling
The formation of mammalian secondary palate requires a series of developmental events such as growth, elevation and fusion. Despite recent advances in the field of palate development, the process of palate elevation remains poorly understood. The current consensus on palate elevation is that the distal end of the vertical palatal shelf corresponds to the medial edge of the elevated horizontal palatal shelf. We provide evidence suggesting that the prospective medial edge of the vertical palate is located toward the interior side (the side adjacent to the tongue), instead of the distal end, of the vertical palatal shelf and that the horizontal palatal axis is generated through palatal outgrowth from the side of the vertical palatal shelf rather than rotating the pre-existing vertical axis orthogonally. Since palate elevation represents a classical example of embryonic tissue re-orientation, our findings here may also shed light on the process of tissue re-orientation in general.
palate development; palate elevation; tissue re-orientation
The Notch signaling pathway is an evolutionarily-conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. The Jagged2 (Jag2) gene, which encodes a ligand for Notch pathway receptors, is required for craniofacial, limb and T cell development. Mice homozygous for a Jag2 null allele die at birth from cleft palate, precluding study of Jag2 function in postnatal and adult mice. We have generated a Jag2 conditional null allele by flanking the first two exons of the Jag2 gene with loxP sites. Cre-mediated deletion of the Jag2flox allele generates the Jag2del2 allele, which behaves genetically as a Jag2 null allele. This Jag2 conditional null allele will enable investigation of Jag2 function in a variety of tissue-specific contexts.
Notch signaling; conditional null allele; Cre-loxP; gene targeting
Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling, increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell-specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms.
angiogenesis; arteriovenous malformation; EphrinB2; EphB4; Notch signaling pathway; vascular morphogenesis
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+c-kit+ 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.
Neuronal migration is a fundamental component of brain development whose failure is associated with various neurological and psychiatric disorders. Reelin is essential for the stereotypical inside-out sequential lamination of the neocortex, but the molecular mechanisms of its action still remain unclear. Here we show that regulation of Notch activity plays an important part in Reelin signal-dependent neuronal migration. We found that Reelin-deficient mice have reduced levels of the cleaved form of Notch intracellular domain (Notch ICD) and that loss of Notch signaling in migrating neurons results in migration and morphology defects. Further, overexpression of Notch ICD mitigates the laminar and morphological abnormalities of migrating neurons in Reeler. Finally, our in vitro biochemical studies show that Reelin signaling inhibits Notch ICD degradation via Dab1. Together, our results indicate that neuronal migration in the developing cerebral cortex requires a Reelin-Notch interaction.
migration; cerebral cortex; Reelin; Notch
Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMC) may contribute to neointimal formation following vascular injury.
Methods and Results
We performed carotid artery ligation in wild-type (WT), control (smCre-Tg), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared to WT or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation following carotid artery ligation. However, neointimal formation was similar between WT and N3-/- mice. Indeed, SMC derived from explanted aortas of either N1+/- or smN1+/- mice showed decreased chemotaxis and proliferation, and increased apoptosis compared to control or N3-/- mice. This correlated with decreased staining of PCNA positive cells and increased staining of cleaved Caspase-3 in the intima of N1+/- or smN1+/- mice. In SMC derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increase SMC proliferation or migration.
These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation following vascular injury through CHF1/Hey2 and suggest that therapies, which target Notch1/CHF1/Hey2 in SMC, may be beneficial in preventing vascular proliferative diseases.
smooth muscle cells; proliferation; vascular injury; signal transduction; arteriosclerosis
Members of the Snail gene family, which encode zinc finger proteins that function as transcriptional repressors, play essential roles during embryonic development in vertebrates. Mouse embryos with conditional deletion of the Snail1 (Snai1) gene in the epiblast, but not in most extraembryonic membranes, exhibit defects in left-right asymmetry specification and migration of mesoderm cells through the posterior primitive streak. Here we describe phenotypic defects that result in death of the mutant embryos by 9.5 days of gestation.
Endothelial cells differentiated in epiblast-specific Snai1-deficient embryos, but formation of an interconnected vascular network was abnormal. To determine whether the observed vascular defects were dependent on disruption of blood flow, we analyzed vascular remodeling in cultured allantois explants from the mutant embryos. Similar vascular defects were observed in the mutant allantois explants.
These studies demonstrate that lethality in the Snai1-conditional mutant embryos is caused by multiple defects in the cardiovascular system.
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.
Notch; glia; olfactory; sustentacular; neurodegeneration; Hes1; Hey1; Six1; Notch2; GST
Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1+/- mice, but not Notch4-/- mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, γ-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of γ-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of γ-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates γ-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs.
angiogenesis; endothelium; ischemia; vasculature
CD4+ T helper cells differentiate into Th1 or Th2 effector lineages, which orchestrate immunity to different types of microbes. Both Th1 and Th2 differentiation can be induced by Notch, but what dictates which of these programs is activated in response to Notch is not known. The requirement for Notch in T helper differentiation is controversial. Using T cell specific gene ablation of the Notch effector RBP-J or the Notch1 and 2 receptors, we show here that Notch is required on CD4+ T cells for physiological Th2 responses to parasite antigens. We find that Gata3 is necessary for Notch induced Th2 differentiation and identify an upstream gata3 promoter as a direct target for Notch signaling. Moreover, we observed that absence of Gata3 turns Notch from a Th2 inducer into a powerful inducer of Th1 differentiation. Therefore, Gata3 is a critical element determining inductive Th2 differentiation and limiting Th1 differentiation by Notch.