Notch Signaling Target Genes Research Articles

Xenopus laevis ATF1, a novel target gene of Notch signaling, functions during gastrulation of Xenopus embryos

Xenopus laevis ATF1, a novel target gene of Notch signaling, functions during gastrulation of Xenopus embryos

WITHDRAWN: Xenopus laevis ATF1, a novel target gene of Notch signaling, functions during gastrulation of Xenopus embryos

WITHDRAWN: Xenopus laevis ATF1, a novel target gene of Notch signaling, functions during gastrulation of Xenopus embryos

XSu(H)2 is an essential factor for gene expression and morphogenesis of the Xenopus gastrula embryo

The CSL (CBF-1, Suppressor of Hairless, Lag-1) transcriptional factor is an important mediator of Notch signal transduction. It plays a key role in cell fate determination by cell-cell interaction. CSL functions as a transcriptional repressor before the activation of Notch signaling. However, once Notch signaling is activated, CSL is converted into a transcriptional activator. It remains unclear if CSL has any function during early development before neurogenesis, while transcriptional products exist from the maternal stage. Here, we analyzed the function of Xenopus Suppressor of Hairless (XSu(H)) using morpholino antisense oligonucleotides (MO), which interfere with the translation of transcripts. In Xenopus embryos, maternal transcripts of both XSu(H)1 and XSu(H)2 were ubiquitously observed until the blastula stage and thereafter only XSu(H)1 was zygotically transcribed. Knockdown experiments with MO demonstrated that XSu(H)2 depletion caused a decrease in the expression of the Xbrachyury, MyoD and JNK1 genes. Morphological and histological examinations indicated that XSu(H)2 depletion caused abnormal gastrulation, which resulted in severe defects of the notochord and somitic mesoderm. The effect of XSu(H)2-MO was completely rescued by co-injection of XSu(H)2 mRNAs, but not by XSu(H)1 mRNAs. XESR-1, a Notch signaling target gene, inhibited Xbrachyury expression. However, expression of the XESR-1 gene was not induced by depletion of XSu(H)2. Co-injection of the dominant-negative form of XESR-1 could not rescue the suppression of Xbrachyury expression in the XSu(H)2-depleted embryo. These results suggest that XSu(H)2 is involved in mesoderm formation and the cell movement of gastrula embryos in a different manner from the XESR-1-mediated Notch signaling pathway.

The Interaction of the Wnt and Notch Pathways Modulates NK vs. T Cell Commitment.

The Wnt and Notch signaling pathways have critical roles in cell fate decisions. However, the interaction of these pathways is poorly understood. Using highly purified Wnt3a and immobilized Notch ligand, Delta1ext-IgG, we investigated the mechanisms involved in Wnt and Notch signaling interactions during hematopoietic stem cell differentiation. When CD34+CD38- cord blood stem cells were cultured for 2 to 3 weeks with five growth factors (SCF 300ng/ml, Flt-3L 300ng/ml, TPO 100ng/ml, IL-6 100ng/ml, and IL-3 10ng/ml), most precursor cells lost CD34 expression and differentiated into mature cells, most of which were monocytes. However, as we previously reported, when cells were cultured with Delta1ext-IgG, we found an increased percentage of lymphoid progenitors (CD34+CD7+) and more mature lymphoid precursors (CD34-CD7+) in the cell population. The addition of purified Wnt3a (100ng/ml) alone without Delta1ext-IgG did not significantly change the percentage of CD7+ cells (1% vs. 3%). However, when both Wnt3a and Delta1ext-IgG were added, we saw an increased percentage of CD7+ cells (11% with Delta1ext-IgG alone to 27% with both) (Fig. 1A). Wnt3a also enhanced gene expression of CD3ε and preTα induced by Delta1ext-IgG. These results suggest that Wnt3a enhances the effect of Notch signaling on T-cell lineage development. To test the role of endogenous Wnt signaling, we added Dickkopf1, an inhibitor of Wnt signaling. When CD34+CD38- cells were cultured with Dickkopf1 (300ng/ml) alone for a 2 week, the percentage and number of CD56+ NK cells was unaffected. However, when cultured with Dickkopf1 and Delta1ext-IgG, the percentage and number of CD56+ NK precursor cells were increased (2% with Delta1ext-IgG alone vs. 17% with Dickkopf1 and Delta1ext-IgG; 0.2 ×105 vs. 1.5×105, p<0.01) (Fig. 1B). This result shows that decreased endogenous Wnt signaling enhances the generation of NK cells in the presence of Notch signaling. To address whether Wnt signaling affects Notch signaling by modulating protein levels, we assessed the amount of activated Notch1 intracellular domain with or without Wnt 3a by Western blot (Fig. 2A). After stimulation for 24 h, Wnt3a increased the amount of activated Notch1 intracellular domain induced by Delta1ext-IgG. Wnt3a also enhanced the expression of a primary target gene of Notch signaling, Hes1, determined by quantitative RT-PCR (Fig. 2B). In contrast, Dickkopf1 reduced Delta1ext-IgG-induced Hes1 expression. These results suggest that Wnt signaling directly modulates Notch signaling. Thus, these studies suggest that Wnt signaling is a key factor in cell fate determination at the point of NK/T cell commitment that is mediated via an interaction with Notch signaling. These studies also suggest that Wnt directly regulates Notch signaling by modulating protein turnover. [Display omitted]

Visualization of spatiotemporal activation of Notch signaling: Live monitoring and significance in neural development

Notch signaling plays various key roles in cell fate determination during CNS development in a context-dependent fashion. However, its precise physiological role and the localization of its target cells remain unclear. To address this issue, we developed a new reporter system for assessing the RBP-J-mediated activation of Notch signaling target genes in living cells and tissues using a fluorescent protein Venus. Our reporter system revealed that Notch signaling is selectively activated in neurosphere-initiating multipotent neural stem cells in vitro and in radial glia in the embryonic forebrain in vivo. Furthermore, the activation of Notch signaling occurs during gliogenesis and is required in the early stage of astroglial development. Consistent with these findings, the persistent activation of Notch signaling inhibits the differentiation of GFAP-positive astrocytes. Thus, the development of our RBP-J-dependent live reporter system, which is activated upon Notch activation, together with a stage-dependent gain-of-function analysis allowed us to gain further insight into the complexity of Notch signaling in mammalian CNS development.

Notch signaling in vascular development.

Notch signaling is an extremely conserved and widely used mechanism regulating cell fate in metazoans. Interaction of Notch receptors (Notch) with their ligands (Delta-like or Jagged) leads to cleavage of the Notch intracellular domain (NICD) that migrates into the nucleus. In the nucleus, NICD associates with a transcription factor, RBP-Jk. The NICD-RBP-Jk complex, in turn, upregulates expression of primary target genes of Notch signaling, such as hairy and enhancer of split (HES) and HES-related repressor protein (HERP) transcriptional repressors. Recent evidence has demonstrated that the Notch pathway is involved in multiple aspects of vascular development, including proliferation, migration, smooth muscle differentiation, angiogenic processes, and arterial-venous differentiation. In this brief review, we focus on ligands, receptors, and target genes of Notch signaling in the vascular system and discuss (1) tissue distribution; (2) gain- and loss-of-function studies; and (3) the role of Notch components in human diseases involving the vascular system.

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

We used the UAS/GAL4 two component system to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi we demonstrate that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi. Our results demonstrate that inducible RNAi is a powerful tool to study the role of essential genes throughout development.

Scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila.

The Notch pathway regulates the differentiation of many cell types throughout development of higher metazoa. Different cellular responses are elicited through specific activation of distinct Notch target genes. In the Drosophila wing, for example, the cut gene is activated by Notch signaling along the dorso-ventral boundary but, as we show here, not in other cell types. We identify additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. Our data support a model, whereby context specific involvement of distinct co-regulators modulates Notch target gene activation.

HERP, a new primary target of Notch regulated by ligand binding.

Notch signaling dictates cell fate and critically influences cell proliferation, differentiation, and apoptosis in metazoans. Ligand binding initiates the signal through regulated intramembrane proteolysis of a transmembrane Notch receptor which releases the signal-transducing Notch intracellular domain (NICD). The HES/E(spl) gene family is a primary target of Notch and thus far the only known Notch effector. A newly isolated HERP family, a HES-related basic helix-loop-helix protein family, has been proposed as a potential target of Notch, based on its induction following NICD overexpression. However, NICD is physiologically maintained at an extremely low level that typically escapes detection, and therefore, nonregulated overexpression of NICD-as in transient transfection-has the potential of generating cellular responses of little physiological relevance. Indeed, a constitutively active NICD indiscriminately up-regulates expression of both HERP1 and HERP2 mRNAs. However, physiological Notch stimulation through ligand binding results in the selective induction of HERP2 but not HERP1 mRNA and causes only marginal up-regulation of HES1 mRNA. Importantly, HERP2 is an immediate target gene of Notch signaling since HERP2 mRNA expression is induced even in the absence of de novo protein synthesis. HERP2 mRNA induction is accompanied by specific expression of HERP2 protein in the nucleus. Furthermore, using RBP-Jk-deficient cells, we show that an RBP-Jk protein, a transcription factor that directly activates HES/E(spl) transcription, also is essential for HERP2 mRNA expression and that expression of exogenous RBP-Jk is sufficient to rescue HERP2 mRNA expression. These data establish that HERP2 is a novel primary target gene of Notch that, together with HES, may effect diverse biological activities of Notch.