Mesoderm Formation In Xenopus Embryos Research Articles

TMEPAI, a Transmembrane TGF-β-Inducible Protein, Sequesters Smad Proteins from Active Participation in TGF-β Signaling

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.

IRE1β is required for mesoderm formation in Xenopus embryos

IRE1 is an atypical serine/threonine kinase transmembrane protein with RNase activity. In the unfolded protein response (UPR), they function as proximal sensor of the unfolded proteins in the endoplasmic reticulum (ER). Upon activation by ER stress, IRE1 performs an unconventional cytoplasmic splicing of XBP1 pre-mRNA and thus allows the synthesis of active XBP1, which activates UPR target genes to restore the homeostasis of the ER. IRE1/XBP1 signaling is hence essential for UPR but its function during embryogenesis is yet unknown. The transcripts of the two isoforms of IRE1 in Xenopus, xIRE1α and xIRE1β are differentially expressed during embryogenesis. We found that xIRE1β is sufficient for cytoplasmic splicing of xXBP1 pre-mRNA. Although gain of xIRE1β function had no significant effect on Xenopus embryogenesis, overexpression of both, xIRE1β and xXBP1 pre-mRNA, inhibits activin A induced mesoderm formation, suggesting that an enhanced activity of the IRE1/XBP1 pathway represses mesoderm formation. Surprisingly, while loss of XBP1 function promotes mesoderm formation, the loss of IRE1β function led to a reduction of mesoderm formation, probably by action of IRE1 being different from the IRE1/XBP1 pathway. Therefore, both gain and loss of function studies demonstrate that IRE1 is required for mesoderm formation in Xenopus embryos.

TMEPAI, a transmembrane TGF-β-inducible protein, sequesters Smad proteins in TGF-β signaling

AbstractTransforming growth factor-[beta] (TGF-[beta]) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-[beta] signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-[beta] signaling, antagonizes TGF-[beta] signaling by interfering with TGF-[beta] type I receptor (T[beta]RI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif (SIM). TMEPAI competes with Smad anchor for receptor activation (SARA) for R-Smad binding, thereby sequestering R-Smads from T[beta]RI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-[beta]-induced PAI-1 production, whereas specific siRNA-mediated knockdown of TMEPAI expression potentiated TGF-[beta]-induced Smad2 phosphorylation and cellular responsiveness by TGF-[beta]. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Taken together, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-[beta] signaling.

The VT+ and VT− Isoforms of the Fibroblast Growth Factor Receptor Type 1 Are Differentially Expressed in the Presumptive Mesoderm of Xenopus Embryos and Differ in Their Ability to Mediate Mesoderm Formation

Previously, we cloned a variant form of the type 1 fibroblast growth factor receptor (FGFR1), FGFR-VT-, from Xenopus embryos (Gillespie, L. L., Chen, G., and Paterno, G. D. (1995) J. Biol. Chem. 270, 22758-22763). This isoform differed from the reported FGFR1 sequence (FGFR-VT+) by a 2-amino acid deletion, Val(423)-Thr(424), in the juxtamembrane region. This deletion arises from the use of an alternate 5' splice donor site, and the activity of the VT+ and VT- forms of the FGFR1 was regulated by phosphorylation at this site. We have now investigated the expression pattern and function of these two isoforms in mesoderm formation in Xenopus embryos. Cells within the marginal zone are induced to form mesoderm during blastula stages. RNase protection analysis of blastula stage embryos revealed that the VT+ isoform was expressed throughout the embryo but that the VT- isoform was expressed almost exclusively in the marginal zone. The ratio of VT+:VT- transcripts in the marginal zone indicated that the VT+ form was predominant throughout blastula stages except for a brief interval, coinciding with the start of zygotic transcription, when a dramatic increase in VT- expression levels was detected. This increase could be mimicked in part by treatment of animal cap explants with FGF-2. Overexpression of the VT+ isoform in Xenopus embryos resulted in development of tadpoles with severe reductions in trunk and tail structures, while embryos overexpressing the VT- isoform developed normally. A standard mesoderm induction assay revealed that a 10-fold higher concentration of FGF-2 was required to reach 50% induction in VT+-overexpressing animal cap explants compared with those overexpressing the VT- isoform. Furthermore, little or no expression of the panmesodermal marker Brachyury (Xbra) was detected in VT+-overexpressing embryos, while VT--overexpressing embryos showed normal staining. This demonstrates that VT+ overexpression had a negative effect on mesoderm formation in vivo. These data are consistent with a model in which mesoderm formation in vivo is regulated, at least in part, by the relative expression levels of the VT+ and VT- isoforms.

Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus.

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on effector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. We present evidence that dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-specific gene induction by eFGF in Xenopus animal cap explants. We also show that dominant-negative mutants of Grb2 and Nck can inhibit eFGF-induced Erk1 activation in Xenopus animal caps, and that targeting the first two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Furthermore, combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibited Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in different cells.

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos.

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.

Targeted over-expression of FGF in chick embryos induces formation of ectopic neural cells.

Fibroblast growth factors (FGFs) are known to be involved mainly in mesoderm formation in Xenopus embryos but their participation in other inductive mechanisms such as neural induction has not been clearly established and is now under study. Here, we provide evidence that targeted over-expression of members of this family of growth factors in the periphery of full-length primitive streak chick embryos produces the formation of ectopic neural cells that are able to differentiate into neurons. The supernumerary neural plate obtained derives from the epiblast layer of the blastoderm and show signs of neural differentiation 24 h after the application of FGF. We have used cell labeling and have examined the expression of mesodermal markers to ascertain how this expansion of the neural forming region of the epiblast takes place. We conclude that the new neural cells formed are originated in the region of the epiblast fated to be epithelia and that the induction of the ectopic neural tissue is not mediated by an increase, migration or new formation of axial mesoderm. This strongly suggests that FGF is acting directly on epiblast cells, changing their fate from epidermal ectoderm to neural ectoderm. Therefore, our results show that FGF can induce neural ectoderm when acting on still uncommitted cells and, therefore, it is a putative candidate for acting in normal neural induction during development.

Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

Heparan sulfate proteoglycans are required for mesoderm formation in Xenopus embryos.

Mesoderm forms in the vertebrate embryo as a result of inductive interactions involving secreted growth factors and cell surface molecules. Proteoglycans have recently been implicated in the control of cell adhesion, migration and growth factor responsiveness. We have found that removal of glycosaminoglycan chains of proteoglycans from Xenopus ectodermal explants by heparinase, but not by chondroitinase, results in inhibition of elongation and mesodermal differentiation in response to signaling factors: activin, FGF and Wnt. Heparinase treatment differentially affected expression of early general and region-specific mesodermal markers, suggesting that mesodermal cell fates become specified in the early embryo via at least two signaling pathways which differ in their requirements for heparan sulfate proteoglycans. Addition of soluble heparan sulfate restored activin-mediated induction of muscle-specific actin gene in heparinase-treated explants. Finally, heparinase inhibited autonomous morphogenetic movements and mesodermal, but not neural, differentiation in dorsal marginal zone explants, which normally give rise to mesoderm in the embryo. These results directly demonstrate that heparan sulfate proteoglycans participate in gastrulation and mesoderm formation in the early embryo.

Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue.

The Brachyrury (T) gene is required cell-autonomously for mesoderm formation in the posterior of the mouse embryo, and both is complementary DNA sequence and expression pattern closely resemble those of a Xenopus homologue (Xbra), suggesting that these genes have an evolutionarily conserved function in vertebrate development. Strong expression of Xbra messenger RNA is found in the ring of involuting mesoderm during Xenopus gastrulation, and the expression of Xbra is an immediate-early response of animal pole blastomeres to mesoderm-inducing factors. To assess the role of Xbra in mesoderm formation, we increased its domain of expression in the embryo by microinjection of Xbra transcripts into the animal pole of Xenopus embryos at the one-cell stage. We show that expression of Xbra by cells of the early embryo is sufficient to direct their development into differentiated mesodermal tissues. At the molecular level this response shows a sharp threshold of sensitivity to the dose of Xbra RNA delivered, and we suggest that Xbra may act as a genetic switch initiating posterior mesodermal specification during embryogenesis.

Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in xenopus embryos

Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.