Characterization of zebrafish smad1, smad2 and smad5: the amino-terminus of smad1 and smad5 is required for specific function in the embryo.

Members of the TGFbeta superfamily of signalling molecules play important roles in mesendoderm induction and dorsoventral patterning of the vertebrate embryo. We cloned three intracellular mediators of TGFbeta signalling, smad1, 2 and 5, from the zebrafish. The three smad genes are expressed ubiquitously at the onset of gastrulation. The pattern of expression becomes progressively restricted during somitogenesis suggesting that at later stages not only the distribution of the TGFbeta signal but also that of the intracellular smad signal transducer determine the regionally restricted effects of TGFbeta signalling. Forced expression of smad1 leads to an expansion of blood cells resembling the phenotype of moderately ventralized zebrafish mutants. In contrast to Smad1, neither Smad2 nor Smad5 caused a detectable effect when expressed as full-length molecules suggesting that these latter two Smads are more dependent on activation by the cognate TGFbeta ligands. N-terminal truncated Smad2 dorsalized embryos, in agreement with a role downstream of dorsalizing TGFbeta members such as Nodals. In contrast to the C-terminal MH2 domain of Smad2, the C-terminal region of Smad1 and Smad5 lead to pleiotropic effects in embryos giving rize to both dorsalized and ventralized characteristics in injected embryos. Analysis of truncated zebrafish Smad1 in Xenopus embryos supports the notion that the C-terminal domain of smad1 is both a hypomorph and antimorph which can act as activator or inhibitor depending on the region of expression in the embryo. These results indicate a specific function of the MH1 domain of Smad1 and 5 for activity of the molecules.     

The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo

Signaling by members of the TGFbeta superfamily is thought to be transduced by Smad proteins. Here, we describe a zebrafish mutant in smad5, designated somitabun (sbn). The dominant maternal and zygotic effect of the sbntc24 mutation is caused by a change in a single amino acid in the L3 loop of Smad5 protein which transforms Smad5 into an antimorphic version, inhibiting wild-type Smad5 and related Smad proteins. sbn mutant embryos are strongly dorsalized, similarly to mutants in Bmp2b, its putative upstream signal. Double mutant analyses and RNA injection experiments show that sbn and bmp2b interact and that sbn acts downstream of Bmp2b signaling to mediate Bmp2b autoregulation during early dorsoventral (D-V) pattern formation. Comparison of early marker gene expression patterns, chimera analyses and rescue experiments involving temporally controlled misexpression of bmp or smad in mutant embryos reveal three phases of D-V patterning: an early sbn- and bmp2b-independent phase when a coarse initial D-V pattern is set up, an intermediate sbn- and bmp2b-dependent phase during which the putative morphogenetic Bmp2/4 gradient is established, and a later sbn-independent phase during gastrulation when the Bmp2/4 gradient is interpreted and cell fates are specified.     

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.     

SnoN co-repressor binds and represses smad7 gene promoter

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.     

Peptidyl-prolyl cis-trans isomerase xFKBP1B induces ectopic secondary axis and is involved in eye formation during Xenopus embryogenesis

Although Xenopus FKBP1A (xFKBP1A) induces an ectopic dorsal axis in Xenopus embryos, involvement of xFKBP1B, a vertebrate paralogue of FKBP1A, in embryogenesis remains undetermined. Here, we demonstrate that xFKBP1B induces ectopic dorsal axis and involves in eye formation of Xenopus embryos. Injection of the xFKBP1B mRNA in ventral blastomeres of 4-cell stage Xenopus embryos induced a secondary axis and showed multiplier effect to that of xFKBP1A on this when xFKBP1A was co-injected. In addition, BMP4 and Smad1 mRNAs did not affect the ability of xFKBP1B to induce the ectopic secondary axis when either was co-injected with xFKBP1B in ventral blastomeres, whereas they downed out that of xFKBP1A, suggesting that xFKBP1A and xFKBP1B induce the ectopic secondary axis through affecting different pathways from each other. On the other hand, the injection of the FKBP1B mRNA in dorsal blastomeres showed eye malformation, and suppressed almost completely the expression of Rx1, Mitf, and Vax2 mRNAs. xFKBP1B was expressed in the dorsal side of the embryo including the eye during embryogenesis at least until stage 46. Injection of morpholino of the xFKBP1B mRNA in dorsal blastomeres induced additional retina or failed to close tapetum nigrum in the ventral side within the optic cap, whereas it did not affect the dorsal organ development. The injection of the morpholino reduced the expression of Xotx2 and Rx1 mRNAs in the eye. These observations suggest that xFKBP1B is a key factor that regulates the expression levels of the genes involved in eye formation during Xenopus embryogenesis.     

A role for BMP signalling in heart looping morphogenesis in Xenopus

The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.     

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.     

Kinesin-mediated transport of Smad2 is required for signaling in response to TGF-beta ligands

During vertebrate development, Activin/Nodal-related ligands signal through Smad2, leading to its activation and accumulation in the nucleus. Here, we demonstrate that Smad2 constantly shuttles between the cytoplasm and nucleus both in early Xenopus embryo explants and in living zebrafish embryos, providing a mechanism whereby the intracellular components of the pathway constantly monitor receptor activity. We have gone on to demonstrate that an intact microtubule network and kinesin ATPase activity are required for Smad2 phosphorylation and nuclear accumulation in response to Activin/Nodal in early vertebrate embryos and TGF-beta in mammalian cells. The kinesin involved is kinesin-1, and Smad2 interacts with the kinesin-1 light chain subunit. Interfering with kinesin activity in Xenopus and zebrafish embryos phenocopies loss of Nodal signaling. Our results reveal that kinesin-mediated transport of Smad2 along microtubules to the receptors is an essential step in ligand-induced Smad2 activation.     

Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.     

Smad2/3 activities are required for induction and patterning of the neuroectoderm in zebrafish

Smad2 and Smad3, two essential nuclear effectors of transforming growth factor (Tgf)-β signals, have been found to be implicated in mesoderm and endoderm development in vertebrate embryos. However, their roles in the induction and patterning of the neuroectoderm are not well established. In this study, we show that interference with Smad2/3 activities in zebrafish embryos, by injecting dnsmad3b mRNA encoding a dominant negative Smad3b mutant, inhibits the expression of the early neural markers sox2 and sox3 at the onset of gastrulation and results in reduction of the anterior neuroectodermal marker otx2 as well as the posterior neuroectodermal marker hoxb1b during late gastrulation, suggesting a role of Smad2/3 activities in neural induction. Conversely, excess Smad2/3 activities, caused by injecting smad3b mRNA, lead to an enhancement of sox2 and sox3 expression in the ventral domains but an inhibition of their expression in the dorsalmost region at early stages. Overexpression of smad3b also causes ventral expansion of the otx2 and hoxb1b expression domains accompanied with rostral shift of the hoxb1b domain at late gastrulation stages. Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. Knockdown of chordin partially inhibits effect of smad3b overexpression on neural induction, implying that Smad2/3 exert their effect on neural induction in part by regulating the expression of Bmp antagonists. Furthermore, down-regulation or up-regulation of Smad2/3 activities in MZoep mutant embryos, which lack the organizer and mesendodermal tissues due to deficiency of Nodal signaling, still affects induction and patterning of the neuroectoderm, suggesting that Smad2/3 activities are implicated in neural development in the absence of the organizer and mesendodermal tissues. We additionally demonstrate that Smad2/3 activities cooperate with Wnt and Fgf signals in neural development. Thus, Smad2/3 activities play important roles not only in mesendodermal development but also in neural development during early vertebrate embryogenesis.     

Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination

During vertebrate development, signaling by the TGFbeta ligand Nodal is critical for mesoderm formation, correct positioning of the anterior-posterior axis, normal anterior and midline patterning, and left-right asymmetric development of the heart and viscera. Stimulation of Alk4/EGF-CFC receptor complexes by Nodal activates Smad2/3, leading to left-sided expression of target genes that promote asymmetric placement of certain internal organs. We identified Ttrap as a novel Alk4- and Smad3-interacting protein that controls gastrulation movements and left-right axis determination in zebrafish. Morpholino-mediated Ttrap knockdown increases Smad3 activity, leading to ectopic expression of snail1a and apparent repression of e-cadherin, thereby perturbing cell movements during convergent extension, epiboly and node formation. Thus, although the role of Smad proteins in mediating Nodal signaling is well-documented, the functional characterization of Ttrap provides insight into a novel Smad partner that plays an essential role in the fine-tuning of this signal transduction cascade.