Bmp and nodal independently regulate lefty1 expression to maintain unilateral nodal activity during left-right axis specification in zebrafish

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.     

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.     

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.     

BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo

A model of left-right axis formation in the chick involves inhibition of bone morphogenetic proteins by the antagonist Car as a mechanism of upregulating Nodal in the left lateral plate mesoderm. By contrast, expression of CFC, a competence factor, which is absolutely required for Nodal signaling in the lateral plate mesoderm is dependent on a functional BMP signaling pathway. We have therefore investigated the relationship between BMP and Nodal in further detail. We implanted BMP2 and Noggin-expressing cells into the left lateral plate and paraxial mesoderm and observed a strong upregulation of Nodal and its target genes Pitx2 and Nkx3.2. In addition Cfc, the Nodal type II receptor ActrIIa and Snr were found to depend on BMP signaling for their expression. Comparison of the expression domains of Nodal, Bmp2, Car and Cfc revealed co-expression of Nodal, Cfc and Bmp2, while Car and Nodal only partially overlapped. Ectopic application of BMP2, Nodal, and Car as well as combinations of this signaling molecules to the right lateral plate mesoderm revealed that BMP2 and Car need to synergize in order to specify left identity. We propose a novel model of left-right axis formation, which involves BMP as a positive regulator of Nodal signaling in the chick embryo.     

Anteriorward shifting of asymmetric Xnr1 expression and contralateral communication in left-right specification in Xenopus

Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left-right (L-R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L-R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with recent findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains the midline barrier. Our findings suggest that there is long-range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.     

BMP signaling positively regulates Nodal expression during left right specification in the chick embryo

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.     

Relation of Nodal expression to the specification of the dorsal‐ventral axis and tissue patterning in the starfish Patiria pectinifera

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid‐blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left‐right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF‐beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.     

Lpp is involved in Wnt/PCP signaling and acts together with Scrib to mediate convergence and extension movements during zebrafish gastrulation

The zyxin-related LPP protein is localized at focal adhesions and cell–cell contacts and is involved in the regulation of smooth muscle cell migration. A known interaction partner of LPP in human is the tumor suppressor protein SCRIB. Knocking down scrib expression during zebrafish embryonic development results in defects of convergence and extension (C&E) movements, which occur during gastrulation and mediate elongation of the anterior–posterior body axis. Mediolateral cell polarization underlying C&E is regulated by a noncanonical Wnt signaling pathway constituting the vertebrate planar cell polarity (PCP) pathway. Here, we investigated the role of Lpp during early zebrafish development. We show that morpholino knockdown of lpp results in defects of C&E, phenocopying noncanonical Wnt signaling mutants. Time-lapse analysis associates the defective dorsal convergence movements with a reduced ability to migrate along straight paths. In addition, expression of Lpp is significantly reduced in Wnt11 morphants and in embryos overexpressing Wnt11 or a dominant-negative form of Rho kinase 2, which is a downstream effector of Wnt11, suggesting that Lpp expression is dependent on noncanonical Wnt signaling. Finally, we demonstrate that Lpp interacts with the PCP protein Scrib in zebrafish, and that Lpp and Scrib cooperate for the mediation of C&E.     

Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells

To understand the mechanisms by which thrombin induces vascular smooth muscle cell (VSMC) DNA synthesis and motility, we have studied the role of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling. Thrombin stimulated the phosphorylation of Akt and S6K1 in VSMC in a sustained manner. Blockade of PI3K-Akt-mTOR-S6K1 signaling by LY-294002, and rapamycin suppressed both thrombin-induced VSMC DNA synthesis and migration. Adenovirus-mediated expression of dominant-negative Akt also inhibited thrombin-induced VSMC DNA synthesis and migration. Furthermore, thrombin induced the expression of Fra-1 in a sustained PI3K-Akt-dependent and mTOR-independent manner in VSMC. Suppression of Fra-1 by its small interfering RNA attenuated both thrombin-induced VSMC DNA synthesis and migration. Thrombin also induced the expression of FGF-2 in a PI3K-Akt-Fra-1-dependent and mTOR-independent manner, and neutralizing anti-FGF-2 antibodies inhibited thrombin-stimulated VSMC DNA synthesis and motility. In addition, thrombin stimulated the tyrosine phosphorylation of EGF receptor (EGFR), and inhibition of its kinase activity significantly blocked Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility induced by thrombin in VSMC. Together these observations suggest that thrombin induces both VSMC DNA synthesis and motility via EGFR-dependent stimulation of PI3K/Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation.