XMam1, Xenopus Mastermind1, induces neural gene expression in a Notch-independent manner

Mastermind, which is a Notch signal component, is a nuclear protein and is thought to contribute to the transactivation of target genes. Previously we showed that XMam1, Xenopus Mastermind1, was essential in the transactivation of a Notch target gene, XESR-1, and was involved in primary neurogenesis. To examine the function of XMam1 during Xenopus early development in detail, XMam1-overexpressed embryos were analyzed. Overexpression of XMam1 ectopically caused the formation of a cell mass with pigmentation on the surface of embryos and expressed nrp-1. The nrp-1-positive cell mass was produced by XMam1 without expression of the Notch target gene, XESR-1, and not by the activation form of Notch, NICD. The ectopic expression of nrp-1 was not inhibited by co-injection of XMam1 with a molecule known to inhibit Notch signaling. The nrp-1 expression was also recognized in the animal cap injected with XMam1DeltaN, which lacks the basic domain necessary for interacting with NICD and Su(H). These results show that XMam1 has the ability to induce the cell fate into the neurogenic lineage in a Notch-independent manner.     

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.     

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.     

The role of BMP signaling in outgrowth and patterning of the Xenopus tail bud.

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

X-Serrate-1 is involved in primary neurogenesis in Xenopus laevis in a complementary manner with X-Delta-1

Notch, Delta and Serrate encode transmembrane proteins that function in cell fate specification in the Drosophila melanogaster embryo. Here we report gene expression patterns and functional characterization of a Xenopus Serrate homolog, X-Serrate-1. The isolated cDNA encoded a transmembrane protein with a Delta/Serrate/LAG-2 domain, 16 epidermal growth factor-like repeats and a cysteine-rich region. Expression of X-Serrate-1 was observed ubiquitously from unfertilized egg to tadpole, but an upregulation occurred in the tailbud stage embryo. Adult expression was found in eye, brain, kidney, heart, spleen and ovary. Whole-mount in situ hybridization revealed that the organ-related expression in eye, brain, heart and kidney occurred from an early stage of rudiment formation. Overexpression of X-Serrate-1 led to a reduction of primary neurons, whereas an intracellularly deleted form of X-Serrate-1 increased the number of primary neurons. Although the function of X-Serrate-1 in primary neurogenesis was quite similar to that of X-Delta-1, expression of X-Serrate-1 and X-Delta-1 did not affect each other. Co-injection experiments showed that wild-type X-Serrate-1 and X-Delta-1 suppressed overproduction of primary neurons induced by dominant-negative forms of X-Delta-1 and X-Serrate-1, respectively. These results suggest that X-Serrate-1 regulates the patterning of primary neurons in a complementary manner with X-Delta-1-mediated Notch signaling.     

Functional association of retinoic acid and hedgehog signaling in Xenopus primary neurogenesis.

Previous work has shown that the posteriorising agent retinoic acid can accelerate anterior neuronal differentiation in Xenopus laevis embryos (Papalopulu, N. and Kintner, C. (1996) Development 122, 3409-3418). To elucidate the role of retinoic acid in the primary neurogenesis cascade, we investigated whether retinoic acid treatment of whole embryos could change the spatial expression of a set of genes known to be involved in neurogenesis. We show that retinoic acid expands the N-tubulin, X-ngnr-1, X-MyT1, X-&Dgr;-1 and Gli3 domains and inhibits the expression of Zic2 and sonic hedgehog in the neural ectoderm, whereas a retinoid antagonist produces opposite changes. In contrast, sonic and banded hedgehog overexpression reduced the N-tubulin stripes, enlarged the neural plate at the expense of the neural crest, downregulated Gli3 and upregulated Zic2. Thus, retinoic acid and hedgehog signaling have opposite effects on the prepattern genes Gli3 and Zic2 and on other genes acting downstream in the neurogenesis cascade. In addition, retinoic acid cannot rescue the inhibitory effect of Notch(ICD), Zic2 or sonic hedgehog on primary neurogenesis. Our results suggest that retinoic acid acts very early, upstream of sonic hedgehog, and we propose a model for regulation of differentiation and proliferation in the neural plate, showing that retinoic acid might be activating primary neurogenesis by repressing sonic hedgehog expression.     

The assembly of POSH-JNK regulates Xenopus anterior neural development

POSH (Plenty of SH3s) has distinct roles as a scaffold for specific c-Jun N-terminal kinase (JNK) signaling modules and as an E3 ubiquitin ligase. The physiological function of POSH remains unclear, however, and its possible involvement in developmental processes motivated the present study wherein the Xenopus orthologue of POSH (xPOSH) was examined for its potential role during Xenopus early embryogenesis. Loss-of-function analysis using morpholino oligonucleotides demonstrated that POSH was essential for Xenopus anterior neural development, although not Spemann organizer formation and early neurogenesis, through the formation of an active JNK signaling complex. Moreover, POSH-mediated JNK pathway was essential for apoptosis in anterior neural tissues. Finally, the present findings demonstrate that RING (Really Interesting New Gene) domain-mediated E3 ubiquitin ligase activity of POSH was not involved in POSH-mediated JNK pathway in vivo. Together, these data suggest that the active JNK signaling complex formed of POSH and the JNK module is essential for the expression of anterior neural genes and apoptosis in Xenopus anterior development.     

Antagonistic role of XESR1 and XESR5 in mesoderm formation in Xenopus laevis

The notch signaling pathway is widely conserved from vertebrates to invertebrates and mediates the specification of numerous cell fates during developmental processes. In the Xenopus gastrula embryo, Xdelta1, one of the Notch ligands, is expressed in the prospective mesoderm prior to Xbra expression. Here, we examined the role of Notch signaling in mesoderm formation. Embryos injected with Xdelta1 morpholino oligo DNA showed a severe gastrulation defect and suppression of Xbra expression, which were completely rescued by co-injection with the active form of Notch. In order to fully understand the role of Notch signaling, we examined the expression of the Notch target genes XESR1 and XESR5. RT-PCR and whole-mount in situ hybridization analyses showed that XESR5 was highly expressed in the marginal zone of the early gastrula embryo, whereas expression of XESR1 was not detected. Animal cap assays indicated that expression of XESR5 was not induced by Notch signaling but by nodal signaling. To clarify the role of XESR5 in the gastrula embryo, a dominant negative form of XESR5 was injected into the prospective mesoderm. The truncated form of XESR5 induced the ectopic expression of XESR1, which caused a decrease in Xbra expression and defective gastrulation. In contrast, the truncated form of XESR1 caused an upregulation of XESR5 resulting in an increase in Xbra expression. The antagonistic effect of XESR1 and XESR5 suggests a dual regulation in which XESR5 produces a competent area for mesoderm formation by suppressing the gene expression of XESR1, while XESR1 sharpens the boundary of Xbra expression.     

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.     

Xenopus Tetraspanin-1 regulates gastrulation movements and neural differentiation in the early Xenopus embryo

The tetraspanin family of four-pass transmembrane proteins has been implicated in fundamental biological processes, including cell adhesion, migration, and proliferation. Tetraspanins interact with various transmembrane proteins, establishing a network of large multimolecular complexes that allows specific lateral secondary interactions. Here we report the identification and functional characterization of Xenopus Tetraspanin-1 (xTspan-1). At gastrula and neurula, xTspan-1 is expressed in the dorsal ectoderm and neural plate, respectively, and in the hatching gland, cement gland, and posterior neural tube at tailbud stages. The expression of xTspan-1 in the early embryo is negatively regulated by bone morphogenetic protein (BMP) and stimulated by Notch signals. Microinjection of xTspan-1 mRNA interfered with gastrulation movements and reduced ectodermal cell adhesion in a cadherin-dependent manner. Morpholino knock-down of endogenous xTspan-1 protein revealed a requirement of xTspan-1 for gastrulation movements and primary neurogenesis. Our data suggest that xTspan-1 could act as a molecular link between BMP signalling and the regulation of cellular interactions that are required for gastrulation movements and neural differentiation in the early Xenopus embryo.