Stimulation Of Notch Signaling Research Articles

Moderate dose alcohol protects against serum amyloid protein A1-induced endothelial dysfunction via both notch-dependent and notch-independent pathways.

Arterial endothelium plays a critical role in maintaining vessel homeostasis and preventing atherosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol (EtOH) consumption is associated with reduced atherosclerosis and stimulates Notch signaling in endothelial cells. The aim of this study was to determine whether EtOH protects the endothelium against serum amyloid A1 (SAA1)-induced activation/injury, and to determine whether this protection is exclusively Notch-dependent. Human coronary artery endothelial cells (HCAEC) were stimulated or not with "pro-atherogenic" SAA1 (1μM) in the absence or presence of EtOH (25mM), the Notch ligand DLL4 (3μg/ml), or the Notch inhibitor DAPT (20μM). EtOH stimulated Notch signaling in HCAEC, as evidenced by increased expression of the Notch receptor and hrt target genes. Treatment with EtOH alone or stimulation of Notch signaling by DLL4 increased eNOS activity and enhanced HCAEC barrier function as assessed by trans-endothelial electrical resistance. Moreover, EtOH and DLL4 both inhibited SAA1-induced monolayer leakiness, cell adhesion molecule (ICAM, VCAM) expression, and monocyte adhesion. The effects of EtOH were Notch-dependent, as they were blocked with DAPT and by Notch receptor (N1, N4) knockdown. In contrast, EtOH's inhibition of SAA1-induced inflammatory cytokines (IL-6, IFN-γ) and reactive oxygen species (ROS) was Notch-independent, as these effects were unaffected by DAPT or by N1 and/or N4knockdown. EtOH at moderate levels protects against SAA1-induced endothelial activation via both Notch-dependent and Notch-independent mechanisms. EtOH's maintenance of endothelium in a nonactivated state would be expected to preserve vessel homeostasis and protect against atherosclerosis development.

Anabolic actions of Notch on mature bone

Notch controls skeletogenesis, but its role in the remodeling of adult bone remains conflicting. In mature mice, the skeleton can become osteopenic or osteosclerotic depending on the time point at which Notch is activated or inactivated. Using adult EGFP reporter mice, we find that Notch expression is localized to osteocytes embedded within bone matrix. Conditional activation of Notch signaling in osteocytes triggers profound bone formation, mainly due to increased mineralization, which rescues both age-associated and ovariectomy-induced bone loss and promotes bone healing following osteotomy. In parallel, mice rendered haploinsufficient in γ-secretase presenilin-1 (Psen1), which inhibits downstream Notch activation, display almost-absent terminal osteoblast differentiation. Consistent with this finding, pharmacologic or genetic disruption of Notch or its ligand Jagged1 inhibits mineralization. We suggest that stimulation of Notch signaling in osteocytes initiates a profound, therapeutically relevant, anabolic response.

Notch signaling promotes osteoclast maturation and resorptive activity.

The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors.

Abstract SY20-01: Role of Notch signaling in colon cancer invasion and metastasis

Abstract While there have been substantial advances in our understanding of the mechanisms of cancer metastasis, efficient remedies for prevention and treatment of metastasis are still missing. The invasion-metastasis cascade consists of local invasion, intravasation, transport, extravasation, formation of micrometastases, and colonization (1,2). This sequence is completed only infrequently, causing metastatic inefficiency, and the least efficient of these steps appears to be colonization (3). Spread of metastatic cancer cells via blood circulation is responsible for the majority of distant metastases although they may travel also through the lymph ducts to nodes (4). In colorectal cancer, metastatic tropism to the liver and lungs can be explained largely by the organization of venous circulation of the intestines through portal vein to the liver, and further to the lungs through pulmonary artery. Among endogenous colon tumor models, the widely used Apc (Adenomatous polyposis coli) mutant mice form adenomas in the small intestine, with various multiplicities (3᠄300 per animal) depending on the mutational allele, although several adenomas are also found in the colon (5). Additional mutations introduced into Apc mutant mice can modify the tumor phenotype. For example, knocking out Smad4 gene in the TGF- family signaling converts the benign intestinal adenomas to very invasive adenocarcinomas (6). Even in such a model, the adenocarcinomas are only locally invasive, and neither intravasation nor distant metastasis is observed during the short life span of these mice. Accordingly, we have screened for candidate genes whose inactivation can stimulate metastasis of mouse colon cancer cells from the rectum to the liver, the commonest site of metastasis using an orthotopic transplantation model. We found such a metastasis suppressor of CRC; Aes that turned out to be an endogenous inhibitor of Notch signaling transcription that is carried out by a complex of Notch intracellular domain (NICD), Maml and Rbpj (7, 8). Upon introduction of a conditional Aes knockout mutation into adenomatous intestinal epithelium of Apc+/∆716 polyposis mice, their tumors become malignant due to Notch signaling activation, showing marked submucosal invasion and intravasation. Consistently, transendothelial migration (TEM) is increased significantly, when CRC cells are activated for Notch signaling and placed on an endothelial cell (EC) layer in culture. When human CRC cells that were inhibited for Aes expression were transplanted to the nude mouse rectum, the number of metastatic foci increased significantly. Thus, reduced level of Aes and stimulation of Notch signaling are implicated in the invasion and intravasation of CRC cells during metastasis. The molecular mechanism of Notch inhibition by Aes is through transcriptional repression by sequestering Rbpj, NICD and Maml1 to the nuclear matrix. The cellular mode of metastasis suppression includes inhibition of transendothelial migration (TEM) of tumor cells, which blocks intravasation. Heterotypic interaction between cancer epithelium and host stromal cells activates Notch signaling and promotes TEM of metastasizing cancer cells. Because TEM in metastasis is common to many solid tumors, this mechanism is important in various types of cancers. As a model for colon cancer invasion and intravasation, compound mutant mice for the Apc and Aes genes should be useful to evaluate new therapeutics including Notch signaling inhibitors against colon cancer metastasis. Some more recent advances will be presented and discussed regarding the molecular mechanism responsible for metastatic invasion downstream of Notch signaling activation.

Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells

Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment.

Abstract 518: Differential Effects of Alcohol and Its Primary Metabolite Acetaldehyde on Vascular Smooth Muscle Cell Notch Signaling and Growth

Moderate consumption of alcohol (ethanol/EtOH) is a negative risk factor for atherosclerosis while binge drinking and chronic alcohol abuse is associated with increased cardiovascular disease and mortality. As the balance between the levels of EtOH itself and its primary metabolite, acetaldehyde, is likely different in these two drinking groups, we assessed the hypothesis that differential effects of EtOH and acetaldehyde on vascular smooth muscle cell (SMC) Notch signaling and growth may be key in mediating the effects of various drinking patterns on cardiovascular disease. Human coronary artery smooth muscle cells (HCASMC) were treated (24 h) with either EtOH (25 mM) or acetaldehyde (10 or 25 μM). We confirmed our previously reported inhibitory effects of EtOH on Notch signaling and subsequently on the growth of HCASMC. In contrast, acetaldehyde treatment stimulated HCASMC proliferation (determined by cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling as determined by increased Notch target gene (HRT 1-3) mRNA levels. Interaction of a ligand (e.g., Dll4) with the Notch receptor initiates proteolytic cleavage by γ-secretase, resulting in the release of the active Notch-intracellular domain (NICD). A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Dll4-stimulated γ-secretase activity in a solubilized membrane fraction from HCASMC (similar to the effect of the control inhibitor, DAPT), but no effect of acetaldehyde treatment. Similarly, EtOH, but not acetaldehyde, affected the association and distribution of the γ-secretase catalytic subunit, presenilin-1, with lipid rafts as determined by dual fluorescent labeling and confocal microscopic visualization (Alexa 488-conjugated cholera toxin B subunit for lipid rafts, and Alexa 594-conjugated anti-presenilin-1 antibody). In conclusion, EtOH and acetaldehyde have opposite effects on SMC Notch signaling and growth. These differential effects of alcohol and its metabolite may be relevant in mediating the opposing effects of different drinking patterns (e.g., daily moderate vs weekend binge) on cardiovascular disease.

Notch-Nrf2 Axis: Regulation of Nrf2 Gene Expression and Cytoprotection by Notch Signaling

The Notch signaling pathway enables regulation and control of development, differentiation, and homeostasis through cell-cell communication. Our investigation shows that Notch signaling directly activates the Nrf2 stress adaptive response pathway through recruitment of the Notch intracellular domain (NICD) transcriptosome to a conserved Rbpjκ site in the promoter of Nrf2. Stimulation of Notch signaling through Notch ligand expression in cells and by overexpression of the NICD in Rosa(NICD/-)::AlbCre mice in vivo induces expression of Nrf2 and its target genes. Continuous and transient NICD expression in the liver produces a Notch-dependent cytoprotective response through direct transcriptional activation of Nrf2 signaling to rescue mice from acute acetaminophen toxicity. This response can be reversed upon genetic disruption of Nrf2. Morphological studies showed that the characteristic phenotype of high-density intrahepatic bile ducts and enlarged liver in Rosa(NICD/-)::AlbCre mice could be at least partially reversed after Nrf2 disruption. Furthermore, the liver and bile duct phenotypes could be recapitulated with constitutive activation of Nrf2 signaling in Keap1(F/F)::AlbCre mice. It appears that Notch-to-Nrf2 signaling is another important determinant in liver development and function and promotes cell-cell cytoprotective signaling responses.

Abstract P098: Inhibitory Role of Notch1 in Calcific Aortic Valve Disease Is Mediated by Sox9

Introduction: Aortic valve calcification is the most common form of valvular heart disease; however the mechanism(s) underlying calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. Objective: To investigate the molecular changes associated with the calcification of aortic valve that occurs with inhibition of Notch signaling. Methods and Results: The expression of Notch signaling pathway members was validated in the aortic valve cusps from adult mice, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1 signaling, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of many cartilage-specific genes that constitute the valve extracellular matrix (ECM). Analysis of these cartilage-specific genes demonstrated that several were transcriptional targets of Sox9, a master regulator of chondrogenesis, which has been previously shown to be essential for proper valve development and maintenance. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, utilizing transfection studies, addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. Conclusions: Loss of Notch signaling contributes to aortic valve calcification by a Sox9-dependent mechanism. Further elucidation of the Notch1-Sox9 molecular pathway and its role in the maintenance of the ECM will lead to an improved mechanistic understanding of aortic valve calcification and development of novel therapeutic strategies for CAVD.

Abstract 5319: The role of Notch and Delta-like 1 in antitumor immunity

Abstract Defects in T cell function are evident in tumor bearing hosts. One potential mechanism for these hematopoietic defects is disruption of Notch signaling which is essential for T- versus B-lineage commitment, and it is clear that Delta-like 1 (DLL1) and Delta-like 4 (DLL4) are critical in this process 1-4. Here we show that tumor-associated defects in T cell development are characterized by attenuated levels of DLL1 and DLL4 in the bone marrow (BM) microenvironment, and elevated circulating vascular endothelial growth factor (VEGF) is one causative factor. We also show that elective therapeutic stimulation of Notch signaling in animals using multivalent form of DLL1 significantly reduces tumor growth, rescues T cell development and enhances T cell-mediated immune responses and that over-expression of DLL1 alone in BM precursors also inhibits tumor growth and improves anti-tumor immunity. Thus, stimulation of DLL1-Notch signaling represents a new strategy to overcome tumor immune dysfunction. As neutralizing DLL4 has been proposed as an approach to inhibit tumor growth by regulating tumor angiogenesis, our data suggest that combining stimulation or blockade of specific context-dependent Notch signaling may optimally inhibit tumor growth via multiple distinct mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5319.

Notch signaling maintains proliferation and survival of the HL60 human promyelocytic leukemia cell line and promotes the phosphorylation of the Rb protein

The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a gamma-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein.

Notch Signaling Contributes to the Expression of Cardiac Markers in Human Circulating Progenitor Cells

It has been demonstrated that adult human circulating endothelial progenitor cells (EPCs) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPCs in a coculture system with neonatal cardiomyocyte. After coculture, Notch activation was transiently detected in EPCs, as determined by immunohistochemical detection of NICD (the intracellular cleavage fragment of Notch-1) and expression of human Notch target genes. Inhibition of gamma-secretase blocked Notch cleavage and NICD translocation. Furthermore, the expression of the cardiac marker protein alpha-sarcomeric actinin and troponin T was significantly suppressed by gamma-secretase inhibition or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because noncanonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and Wnt11. Wnt5a and Wnt11 expression in the human cells was induced by the coculture and was blocked by gamma-secretase inhibition. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted Wnt5a expression in EPCs. These data suggest that Notch is activated upon coculture of EPCs with neonatal rat cardiac myocytes. Gamma-secretase-dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of noncanonical Wnt proteins, which may act in a paracrine manner to further amplify cardiac differentiation.

Abstract 689: Notch Signaling Stimulates Expression Of Wnts And Contributes To Cardiac Markers Gene Expression In Human Progenitor Cell

It has been demonstrated that adult human circulating endothelial progenitor cells (EPC) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a prominent role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPC in a co-culture system with neonatal rat cardiomyocyte (CM). EPC expressed the receptors Notch-1 and Notch-2, whereas CM expressed high level of Notch ligand Jagged-1. Therefore, we hypothesized that CM may activate Notch signaling within EPC. Indeed, after co-culture, Notch activation was detected in EPC by immunohistochemical detection of the intracellular cleavage fragment of Notch-1 (NICD), whereas NICD was only rarely detected in EPC before co-culture. Western blot analysis confirmed Notch cleavage after co-culture. RT-PCR directed against the human specific sequences of the Notch target genes Hey2 and Hes1 demonstrated a transient activation of the transcriptional activity of Notch in human EPC after co-culture with CM. Inhibition of γ-secretase significantly blocked Notch cleavages and NICD translocations. Furthermore, the expression of the cardiac marker protein γ-sarcomeric actinin and troponin T was significantly suppressed by γ-secretase inhibition (55.8 ± 8.1% and 54.0 ± 10.5% of control, respectively) or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because non-canonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and. Wnt5a and Wnt11 expressions in the human cells was induced by the co-culture and was blocked by γ-secretase inhibitor. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted NICD cleavage and Wnt5a expression in EPC. These data suggested that Notch is transiently activated upon co-culture of EPC with neonatal rat CM. γ-Secretase-dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of non-canonical Wnt proteins, which may act in an paracrine manner to further amplify cardiac differentiation.

Rbm15 Modulates Notch-Induced Transcriptional Activation and Affects Myeloid Differentiation

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJkappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJkappa.

Rbm15 Affects Notch Signaling and Myelopoiesis.

Acute Megakaryoblastic Leukemia (AMKL) presents with one of two different genotypic abnormalities; either Down Syndrome (DS) accompanied with GATA-1 mutations, or a non-DS translocation t(1;22)(p13;q13). In AMKL associated with DS, there is trisomy 21 and various partial loss of function mutations in GATA-1. In non-DS AMKL, the translocation t(1;22)(p13;q13) encodes a fusion protein, RBM15-MKL, comprised of the transcriptional co-factors RBM15 (chromosome 1) and MKL (chromosome 22). In order to elucidate the role of the RBM15-MKL fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL. Herein we demonstrate a role for Rbm15 in myeloid differentiation.Previous work in the lab showed that Rbm15 is expressed at highest levels in hematopoietic stem cells, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Therefore, we hypothesized that Rbm15 plays a role in myeloid differentiation. Indeed, we demonstrate that shRNA-mediated knockdown of Rbm15 enhanced the differentiation of 32DWT18 myeloid precursor cells. Recent studies have shown the Rbm15 homolog, Sharp, interacts with RBPJk, a critical transcription factor in the Notch signaling pathway; and since Notch signaling is essential for hematopoiesis, we hypothesized that Rbm15 may also modulate Notch signaling. We demonstrate that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 activity in non-hematopoietic cell lines, but stimulates this activity in hematopoietic cell lines including 32DWT18 and HEL (human erythroleukemia). In addition, we show that the N-terminus of Rbm15 co-immunoprecipitates with RBPJk and has a dominant negative effect by impairing the activation ability of HES1 promoter activity by full length-Rbm15.Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated, at least in part, by stimulation of Notch signaling via interaction with RBPJk. Consistent with a potential mechanistic role of Rbm15 and Notch in myelopoiesis, we have shown that shRNA-mediated knockdown of Rbm15 in 32DWT18 cells promotes myeloid differentiation, suggesting that the RBM15 component of the RBM15-MKL fusion protein may act by blocking differentiation. Currently, we are examining the effect of RBM15 overexpression and knockdown in primary mouse bone marrow cells using in vitro and in vivo assays.

The Snail repressor positions Notch signaling in the Drosophila embryo.

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. We have investigated the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, Notch(IC), in transgenic embryos using the eve stripe2 enhancer. The Notch(IC) stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. We suggest that these dual activities of Snail, repression of Notch target genes and stimulation of Notch signaling, help define precise lines of sim expression within the neurogenic ectoderm.