Mesoderm Induction in Pleurodeles waltl: Distribution of Fibronectin and Chondroitin Sulfate in Induced Explants

In Pleurodeles , cell-matrix interactions play a major role in promoting active mesodermal cell migration during gastrulation. It was therefore important to determine whether the expression of define matrix molecules may be dependent on mesoderm induction. Results from induction experiments done with XTC cell line-conditioned medium show that mesoderm tissues induced in animal cap explants of Pleurodeles are identical to those from Xenopus . However, we also show that dorsally-induced explants in Pleurodeles elongate to a lesser degree than in Xenopus . This observation agrees well with the differences observed in the role of ECM in Pleurodeles and Xenopus gastrulation, respectively. Additional immunostaining studies demonstrate that the induction of mesodermal tissues is associated with the expression of chondroitin sulfate whereas fibronectin fibrils are already assembled in uninduced animal caps. These results suggest that mesoderm cell-matrix interactions in early amphibian embryo may be under the control of mesoderm induction.     

Notch signaling modulates the nuclear localization of carboxy-terminal-phosphorylated smad2 and controls the competence of ectodermal cells for activin A

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps [Coffman, C.R., Skoglund, P., Harris, W.A., Kintner, C.R., 1993. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73, 659-671], although the mechanism by which this modulation occurs remains unknown. Here, we show that Notch signaling also delays the onset of LMC in whole embryos, as it did in animal caps. To better understand this effect and the mechanism of LMC itself, we investigated at which step of activin signal transduction pathway the Notch signaling act to affect the timing of the LMC. In our system, ALK4 (activin type I receptor) maintained the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 could bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. We conclude that LMC was induced because C-terminal-phosphorylated smad2 lost its ability to bind to smad4, and consequently could not accumulate in the nucleus. Notch signal activation restored the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC.     

Mesoderm-independent regulation of gastrulation movements by the Src tyrosine kinase in Xenopus embryo

In vitro studies have demonstrated the involvement of Src kinases in several aspects of cell scattering, including cell dissociation and motility. We have therefore sought to explore their functions in the context of the whole organism. Loss-of-function microinjection studies indicate that the ubiquitous Src, Fyn, and Yes tyrosine kinases are specifically implicated in Xenopus gastrulation movements. Injection of mRNAs coding for dominant negative forms of the ubiquitous members of the Src family, namely Fyn, Src, and Yes, perturbs gastrulation movements, resulting in the inability to close the blastopore. Injection of mRNA coding for Csk, a natural inhibitor of Src kinase activity, produces the same phenotypic alterations. The ubiquitous Src kinases have redundant functions in gastrulation movements since overexpression of one member of the family can compensate for the inhibition of another. Interfering mutants of the Src family also inhibit activin-induced morphogenetic movements of animal cap explants isolated from injected embryos. In contrast, these mutants do not interfere with mesoderm induction, as inferred from the presence of mesoderm derivatives and from the expression of early mesodermal markers in injected embryos. In addition, Src kinase activity measured by an in vitro kinase assay is elevated in gastrulating embryos and in FGF- and activin-treated animal caps, confirming the implication of Src enzymatic activity during gastrulation. Altogether, our results demonstrate that Src kinases are essential components of the machinery that drives gastrulation movements independent of mesoderm induction and suggest that Src activity is primarily implicated in cellular movements that take place during the process of cell intercalation.     

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.     

A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.     

Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1.

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.     

Regulation of Xenopus gastrulation by ErbB signaling

During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Different signals are implicated in these processes. Our previous studies reveal that signals through ErbB receptor tyrosine kinases modulate Xenopus gastrulation, but the mechanisms employed are not understood. Here we report that ErbB signals control both C&E and head mesoderm migration. Inhibition of ErbB pathway blocks elongation of dorsal marginal zone explants and activin-treated animal caps without removing mesodermal gene expression. Bipolar cell shape and cell mixing in the dorsal region are impaired. Inhibition of ErbB signaling also interferes with migration of prechordal mesoderm on fibronectin. Cell-cell and cell-matrix interaction and cell spreading are reduced when ErbB signaling is blocked. Using antisense morpholino oligonucleotides, we show that ErbB4 is involved in Xenopus gastrulation morphogenesis, and it partially regulates cell movements through modulation of cell adhesion and membrane protrusions. Our results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to non-canonical Wnt and FGF signals, that controls gastrulation. We further demonstrate that regulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation.     

FRL-1, a member of the EGF-CFC family,is essential for neural differentiation in Xenopus early development

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

Molecular expression of Ly6k, a putative glycosylphosphatidyl-inositol-anchored membrane protein on the mouse testicular germ cells

Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.     

Endoderm is required for vascular endothelial tube formation, but not for angioblast specification

Angioblasts, the precursor cells that comprise the endothelial layer of blood vessels, arise from a purely mesodermal population. Individual angioblasts coalesce to form the primary vascular plexus through a process called vasculogenesis. A number of reports in the literature suggest that signals from the adjacent endoderm are necessary to induce angioblast specification within the mesoderm. We present evidence, using both embryological and molecular techniques, indicating that endoderm is not necessary for the induction of angioblasts. Xenopus embryos that had endoderm physically removed at the onset of gastrulation still express vascular markers. Furthermore, animal caps stimulated with bFGF form angioblasts in the absence of any detectable endodermal markers. These results show that endoderm is not required for the initial formation of angioblasts. While Xenopus embryos lacking endoderm contain aggregates of angioblasts, these angioblasts fail to assemble into endothelial tubes. Endothelial tube formation can be rescued, however, by implantation of endodermal tissue from sibling embryos. Based on these studies in Xenopus, and corroborating experiments using the quail embryo, we conclude that endoderm is not required for angioblast specification, but does play an essential role in the formation of vascular tubes.     

Direct activation of phospholipase C-gamma by fibroblast growth factor receptor is not required for mesoderm induction in Xenopus animal caps.

Members of the fibroblast growth factor (FGF) family induce mesoderm formation in explants of Xenopus embryonic ectoderm (animal caps). Recent studies have been directed at determining signaling pathways downstream of the FGF receptor that are important in mesoderm induction. We have recently shown that a point mutation in the FGF receptor changing tyrosine 766 to phenylalanine (Y/F mutation) abolishes phospholipase C-gamma (PLC-gamma) activation in mammalian cells. To explore the role of PLC-gamma activation in FGF-stimulated mesoderm induction, we constructed two chimeric receptors, each consisting of the extracellular portion of the platelet-derived growth factor beta receptor, with one having the transmembrane and intracellular portions of the wild-type FGF receptor 1 (PR-FR wt) and the other having the corresponding region of the Y/F766 mutant FGF receptor 1 (PR-FR Y/F766). When expressed in Xenopus oocytes, only PR-FR wt was able to mediate PLC gamma phosphorylation, inositol-1,4,5-trisphosphate accumulation, and calcium efflux in response to platelet-derived growth factor stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps as measured by cap elongation, muscle-specific actin mRNA induction, and skeletal muscle formation. These results demonstrate that PLC gamma activation by the FGF receptor is not required for FGF-stimulated mesoderm induction.     

HNF1(beta) is required for mesoderm induction in the Xenopus embryo

XHNF1(&bgr;) is a homeobox-containing gene initially expressed at the blastula stage in the vegetal part of the Xenopus embryo. We investigated its early role by functional ablation, through mRNA injection of an XHNF1(beta)/engrailed repressor fusion construct (XHNF1(beta)/EngR). Dorsal injections of XHNF1(beta)/EngR mRNA abolish dorsal mesoderm formation, leading to axial deficiencies; ventral injections disrupt ventral mesoderm formation without affecting axial development. XHNF1(beta)/EngR phenotypic effects specifically depend on the DNA-binding activity of its homeodomain and are fully rescued by coinjection of XHNF1(beta) mRNA. Vegetal injection of XHNF1(beta)/EngR mRNA blocks the mesoderm-inducing ability of vegetal explants. Both B-Vg1 and VegT maternal determinants trigger XHNF1(beta) expression in animal caps. XHNF1(beta)/EngR mRNA blocks B-Vg1-mediated, but not by eFGF-mediated, mesoderm induction in animals caps. However, wild-type XHNF1(beta) mRNA does not trigger Xbra expression in animal caps. We conclude that XHNF1(beta) function is essential, though not sufficient, for mesoderm induction in the Xenopus embryo.