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.     

Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.     

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.     

The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell-cell adhesion

BACKGROUND: Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive. RESULTS: Via bead implantations, we show that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca(2+)/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction. CONCLUSIONS: We propose that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula.     

Essential role of Bmp7 (snailhouse) and its prodomain in dorsoventral patterning of the zebrafish embryo

Bone morphogenetic proteins (Bmps) are signaling molecules that have been implicated in a variety of inductive processes. We report here that zebrafish Bmp7 is disrupted in snailhouse (snh) mutants. The allele snh(st1) is a translocation deleting the bmp7 gene, while snh(ty68) displays a Val->Gly exhange in a conserved motif of the Bmp7 prodomain. The snh(ty68) mutation is temperature-sensitive, leading to severalfold reduced activity of mutant Bmp7 at 28 degrees C and non-detectable activity at 33 degrees C. This prodomain lesion affects secretion and/or stability of secreted mature Bmp7 after processing has occurred. Both snh(st1) and snh(ty68) mutant zebrafish embryos are strongly dorsalized, indicating that bmp7 is required for the specification of ventral cell fates during early dorsoventral patterning. At higher temperature, the phenotype of snh(ty68) mutant embryos is identical to that caused by the amorphic bmp2b mutation swirl swr(ta72) and similar to that caused by the smad5 mutation somitabun sbn(dtc24). mRNA injection studies and double mutant analyses indicate that Bmp2b and Bmp7 closely cooperate and that Bmp2b/Bmp7 signaling is transduced by Smad5 and antagonized by Chordino.     

Functional characterization and genetic mapping of alk8

The novel type I TGFβ family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.     

Modulation of BMP activity in dorsal-ventral pattern formation by the chordin and ogon antagonists

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.     

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 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.     

Zebrafish bmp4 functions during late gastrulation to specify ventroposterior cell fates

Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling.     

XMAN1, an inner nuclear membrane protein,antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.     

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.