XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos

Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.     

Cysteine-rich region of X-Serrate-1 is required for activation of Notch signaling in Xenopus primary neurogenesis

The Notch family genes encode single-pass transmembrane proteins which function in a variety of cell fate specifications in invertebrates and vertebrates. In Xenopus primary neurogenesis, the Notch ligands, X-Delta-1 and X-Serrate-1, mediate Notch signaling and regulate cell differentiation. In the present study, we examined the role of the Serrate-specific cysteine-rich (CR) region in the primary neurogenesis of Xenopus embryos. The ligand constructs containing the DSL (Delta/Serrate/Lag-2) domain in the extracellular region caused a reduction in primary neurons, whereas the DSL-deleted form of X-Delta-1 resulted in the overproduction of primary neurons. However, the DSL-deleted form of X-Serrate-1 or the construct containing only the CR region in the extracellular domain (SerCR) reduced the number of primary neurons. In contrast, the CR-deleted form of X-Serrate-1 (SerACR) lost activity as a Notch ligand, regardless of the presence of the DSL domain within the extracellular domain. Overexpression of X-Delta-1 and X-Serrate-1 strongly induced the expression of Xenopus ESR-1 (XESR-1), a gene related to Drosophila Enhancer of split. SerCR alone also moderately induced the expression of XESR-1, but the SerACR form did not induce this expression. Co-injection of X-Notch-1deltaICD, which deletes the intracellular domain (ICD), with SerCR suppressed a neurogenic phenotype, although co-injection of X-Su(H)1DBM with SerCR did not, indicating that SerCR affects primary neurogenesis through the Notch/Su(H) pathway. These results suggestthatthe CR region of Xenopus Serrate is required for the activation of Notch signaling and cell fate specification in primary neurogenesis.     

Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.     

The intracellular domain of X-Serrate-1 is cleaved and suppresses primary neurogenesis in Xenopus laevis

The Notch ligands, Delta/Serrate/Lag-2 (DSL) proteins, mediate the Notch signaling pathway in a numerous developmental processes in multicellular organisms. Although the ligands induce the activation of the Notch receptor, the intracellular domain-deleted forms of the ligands cause dominant-negative phenotypes, implying that the intracellular domain is necessary for the Notch signal transduction. Here we examined the role of the intracellular domain of Xenopus Serrate (XSICD) in Xenopus embryos. X-Serrate-1 has the putative nuclear localization sequence (NLS) in downstream of the transmembrane domain. Biochemical analysis revealed that XSICD fragments are cleaved from the C-terminus side of X-Serrate-1. Fluorescence microscopic analysis showed that the nuclear localization of XSICD occurs in the neuroectoderm of the embryo injected with the full-length X-Serrate-1/GFP. Overexpression of XSICD showed the inhibitory effect on primary neurogenesis. However, a point mutation in the NLSs of XSICD inhibited the nuclear localization of XSICD, which caused the induction of a neurogenic phenotype. The animal cap assay revealed that X-Serrate-1 suppresses primary neurogenesis in neuralized animal cap, but X-Delta-1 does not. Moreover, XSICD could not activate the expression of the canonical Notch target gene, XESR-1 in contrast to the case of full-length X-Serrate-1. These results suggest that the combination of XSICD-mediated intracellular signaling and the extracellular domain of Notch ligands-mediated activation of Notch receptor is involved in the primary neurogenesis. Moreover, we propose a bi-directional signaling pathway mediated by X-Serrate-1 in Notch signaling.     

Notch 1 overexpression inhibits osteoblastogenesis by suppressing Wnt/beta-catenin but not bone morphogenetic protein signaling

Notch proteins are transmembrane receptors that control cell-fate decisions. Upon ligand binding, Notch receptors undergo proteolytic cleavage leading to the release of their intracellular domain (NICD). Overexpression of NICD impairs osteoblastogenesis, but the mechanisms are not understood. We examined consequences of the constitutive activation of Notch 1 in ST-2 cells. Notch opposed the effects of bone morphogenetic protein (BMP)-2 and Wnt 3a on alkaline phosphatase activity (APA). BMP-2 induced the phosphorylation of Smad 1/5/8 and the transactivation of a BMP/Smad-responsive construct (12xSBE-Oc-pGL3), but the effect was not modified by Notch. BMP-2 had minimal effects on the phosphorylation of the mitogen-activated protein kinases ERK, p38, and JNK, in the absence or presence of NICD. Notch overexpression decreased the transactivating effect of Wnt 3a, cytoplasmic beta-catenin levels, and Wnt-dependent gene expression. Transfection of a mutant beta-catenin expression construct, or the use of a glycogen synthase kinase 3beta inhibitor to stabilize beta-catenin, partially blocked the inhibitory effect of NICD on Wnt signaling and on APA. HES-1 or Groucho1/TLE1 RNA interference enhanced basal and induced Wnt/beta-catenin signaling opposing NICD effects, but only HES-1 silencing enhanced Wnt 3a effects on APA. In conclusion, NICD overexpression prevents BMP-2 and Wnt biological effects by suppressing Wnt but not BMP signaling. HES-1 appears to mediate effects of Notch on osteoblastogenesis.     

Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart morphogenesis in mouse

Notch signaling is implicated in many developmental processes. In our current study, we have employed a transgenic strategy to investigate the role of Notch signaling during cardiac development in the mouse. Cre recombinase-mediated Notch1 (NICD1) activation in the mesodermal cell lineage leads to abnormal heart morphogenesis, which is characterized by deformities of the ventricles and atrioventricular (AV) canal. The major defects observed include impaired ventricular myocardial differentiation, the ectopic appearance of cell masses in the AV cushion, the right-shifted interventricular septum (IVS) and impaired myocardium of the AV canal. However, the fates of the endocardium and myocardium were not disrupted in NICD1-activated hearts. One of the Notch target genes, Hesr1, was found to be strongly induced in both the ventricle and the AV canal of NICD1-activated hearts. However, a knockout of the Hesr1 gene from NICD-activated hearts rescues only the abnormality of the AV myocardium. We searched for additional possible targets of NICD1 activation by GeneChip analysis and found that Wnt2, Bmp6, jagged 1 and Tnni2 are strongly upregulated in NICD1-activated hearts, and that the activation of these genes was also observed in the absence of Hesr1. Our present study thus indicates that the Notch1 signaling pathway plays a suppressive role both in AV myocardial differentiation and the maturation of the ventricular myocardium.     

A developmentally regulated, nervous system-specific gene in Xenopus encodes a putative RNA-binding protein.

A gene from Xenopus laevis that is expressed specifically in the nervous system beginning at the stage of neural plate formation has been isolated and several cDNAs have been sequenced. The sequence of the predicted protein contains two copies of a presumed RNA-binding domain, each of which includes two short conserved motifs characteristic for ribonucleoproteins (RNPs), called the RNP-1 and RNP-2 consensus sequences. We name this gene Xenopus nrp-1, for nervous system-specific RNP protein-1. Sequence comparisons suggest that the nrp-1 protein is a heterogeneous nuclear RNP protein, but it is clearly distinct from previously reported hnRNP proteins such as the A1, A2/B1, and C1 proteins. nrp-1 RNA undergoes an alternative splicing event giving rise to two predicted protein isoforms that differ from each other by seven amino acids. In situ hybridization to tadpole brain shows that the nrp-1 gene is expressed in the ventricular zone where cell proliferation takes place. The occurrence of an RNP protein with nervous system-limited expression suggests that it may be involved in the tissue-specific control of RNA processing.