Mothers against dpp


The predicted MAD polypeptide lacks known protein motifs, but has strong sequence similarity to three polypeptides predicted from genomic sequence from the nematode C. elegans. The middle of each protein sequence contains a region of variable length that does not show extensive identity. This region is proline-rich in all four sequences and is also serine rich in MAD (Sekelsky, 1995).

Comparison of Medea with other Smad proteins indicates that Medea is most closely related to human Smad4, with an overall identity of 59%. The regions of greatest identity between Medea and Smad4 include the two MAD homology domains, MH1 and MH2, with 76 and 80 % identity, respectively. Both proteins share an insert in the MH2 domain of 30 and 34 amino acids, respectively. The linker regions between the MH1 and MH2 domains display little sequence conservation; however, like all Smad family members, this domain is rich in proline residues. Additional sequences within the linker region of Medea, including the presence of several unusual poly-glutamine repeats, account for the protein's larger size relative Smad4. Sequence comparison of Smad family members indicates that they can be divided into three groups: the Smad4 subgroup, which comprises Medea, Smad4 and the C. elegans sma4; a second group, which includes those Smads most closely related to Mad and a third group comprising the inhibitory Smads (for example, Drosophila Dad). Within the MH1 and MH2 domains there are amino acids conserved in most positively-acting Smad proteins and amino acids that are conserved only within a sub-group. Of note, a conserved SSXS motif present at the carboxy-terminus of all MAD subgroup proteins is absent in the Smad4 subgroup. Since serine residues in this motif are targets for type I receptor-mediated phosphorylation (see Drosophila Punt and Thickveins), the absence of these residues raises questions regarding the regulation and function of Medea in Dpp signaling (Wisotzkey, 1998 and Hudson, 1998).

The family of TGF-beta signaling molecules play inductive roles in various developmental contexts. One member of this family, Drosophila Decapentaplegic (Dpp) serves as a morphogen that patterns both the embryo and adult. Daughters against dpp (Dad), whose transcription is induced by Dpp shares, weak homology with Drosophila Mad (Mothers against dpp), a protein required for transduction of Dpp signals. Dad is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule, and in fact ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad blocks Dpp activity. Dpp target gene optomotor blind is absent in Dad-overexpressing cells. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by either protein alone. Dad can also antagonize the activity of a vertebrate homolog of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. It is concluded that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).

Interaction of Smads with DNA

Transcriptional regulation by TGFbeta signaling is mediated by the Smad family of transcription factors. It is generally accepted that Smads must interact with other transcription factors in order to bind to their targets. However, recently it has been shown that a complex of the Drosophila Smad proteins, Mad and Medea, binds with high affinity to silencer elements that repress brinker and bag of marbles in response to Dpp signaling. These silencers are bound by a heterotrimer containing two Mad subunits and one Medea subunit. The MH1 domains of all three subunits contribute directly to sequence-specific DNA contact, thus accounting for the exceptionally high stability of the Smad-silencer complex. The Medea MH1 domain binds to a canonical Smad box (GTCT), while the Mad MH1 domains bind to a GC-rich sequence resembling Mad binding sites previously identified in Dpp-responsive enhancer elements. The consensus for this sequence, GRCGNC, differs from that of the canonical Smad box, but it was found that Mad binding nonetheless required the same beta-hairpin amino acids that mediate base-specific contact with GTCT. Binding is also affected by alanine substitutions in Mad and Med at a subset of basic residues within and flanking helix 2, indicating a contribution to binding of the GRCGNC and GTCT sites. Slight alteration of the Dpp silencers causes them to activate transcription in response to Dpp signaling, indicating that the potential for Smad complexes to recognize specific targets need not be limited to repression (Gao, 2005).

This work demonstrates that Mad binds to GCrich sites -- originally defined as GCCGNCG and more recently in the context of silencers as GRCGNC -- by sequence-specific contact with two subunits. Sequence specific interaction with GC-rich sites has been demonstrated previously for Smad1 and for Mad, but the stoichiometry of these interactions was not documented. Finding that the 6 bp site is contacted by two Mad subunits raises new questions, since it is not clear how the two MH1 domains are arranged on the DNA, nor is it known why GNCG or GNCN (depending on orientation) is recognized rather than the GTCT site preferred by Smad3 and Smad4. Previous work involving Smad1- Smad3 chimeras identified Lys36 and Ser37 of Smad3 helix 2 vs. Asp35 and Ala36 in Smad1 as a key difference necessary for activation of a (CAGA)9-luc reporter. However, mutation of the corresponding residue in Mad (D49A) has little or no effect on binding to BrkS, and thus the structural determinants for the GRCGNC binding site preference of Mad (and by inference, Smad1) remain to be identified (Gao, 2005).

These findings contradict a previous report that Mad and Med bind to BrkS as a dimer. Although no documentation was provided for how this was determined experimentally, attempts to determine stoichiometry by means of antibody supershift experiments suggest a possible explanation for the discrepancy. It was found that epitope tags at the Mad amino terminus are only readily accessible to antibody on one of the two Mad subunits, possibly due to crowding by the adjacent DNA-bound MH1 domain of Med. This problem led to the use instead of protein fusions as a strategy for assessing stoichiometry (Gao, 2005).

Mad and Med appear to be the only factors that directly contact the bam and brk silencers. This conclusion is based on mutational analysis of the BrkS element which showed that mutations that disrupt silencing also disrupt binding of the Mad-Med complex. The only exceptions were mutation of GTCT to GTCG or to GGCG, and a 1-bp deletion between the Mad and Med sites, each of which allowed binding of the Mad- Med complex but disrupted recruitment of Shn (Gao, 2005).

Over-expression of activated Mad-Med complexes is sufficient to generate these gel-shift complexes and therefore it is unlikely that an unknown cofactor is required since it would presumably need to be expressed at high levels endogenously in both Drosophila and human cells. The demonstration that binding of the Mad-Med heterotrimer requires all three MH1 domains also weighs against cofactor involvement since it becomes difficult to explain how GRCGNC could be contacted by a cofactor in addition to two Mad MH1 domains (the Med MH1 contacting GTCT). Nonetheless, it is possible that a cofactor present in Drosophila and human extracts has gone undetected, although the evidence suggests it could not play a direct role in sequence-specific DNA contact (Gao, 2005).

The apparent ability of a Mad-Med complex to bind silencers without cofactors contrasts with the general reliance of Smads on DNA-binding cofactors for target specificity. brk could be considered a special case since it is negatively regulated by Dpp globally, while other Dpp targets require tissue-specific regulation. However, bam expression is specific to germline cells, and thus it is unexpected that DNA contact by Mad and Med would be sufficient for the regulatory specificity of the bam silencer. The existence of a similar Dpp-responsive silencer regulating gooseberry provides further evidence that these novel arrangements of Smad binding sites provide sufficient specificity for regulation in response to signaling. Nonetheless, tissue specificity might be augmented by cooperative interaction of DNA-bound corepressors with Smads bound to BrkS/BamSE-like sites (Gao, 2005).

The high affinity of Mad and Med for these silencers is explained by the trimeric stoichiometry and involvement of all MH1 domains in directly contacting DNA. The ability of a single Smad complex to engage all three MH1 domains in DNA contact has several implications. The most obvious is that Smad complexes may in some cases make a greater contribution to target recognition than was previously apparent. As in the case of brk, this provides a mechanism by which Dpp, BMP or even TGFbeta signaling might trigger a general response without the need for a tissue-specific cofactor. Such a response need not be limited to silencing since slight alterations in the silencers transform them into Dpp-responsive activating elements, possibly by allowing CBP to interact with the Mad-Med complex in the absence of Shn. Conserved sites exhibiting the BrkS/BamSE motif have been identified within BMP-response elements for the Id genes, which as a class are responsive to BMP signaling. Conversely, tissue-specificity might be conferred upon such tripartite Smad response elements by adjacent binding sites for other transcription factors (Gao, 2005).

A second implication is that Smad complexes may have greater flexibility in their ability to recognize binding sites than was previously apparent, particularly for moderate affinity sites to which only two MH1 domains make sequence-specific contact. The observed flexibility in spacing between the Mad and Med sites in BrkS and BamSE suggests that DNA binding by Med plus just one Mad MH1 domain might be able to occur in a variety of permutations. However, loss of binding when the Med site was reversed shows that the topology of Smad sites has strict limits. A moderate affinity site might also consist of just the GRCGNC Mad binding site without an adjacent Med binding site, as appears to be the case for many Dpp responsive enhancer elements. Such moderate affinity sites would activate transcription in response to Dpp signaling, but only when the necessary tissue-specific cofactor is present. Some Mad sites serve also as binding sites for the Brk protein, which opposes Dpp signaling by direct competition for Mad binding and by functioning as an active repressor. The third implication points to complexity in the response to BMP signaling. In vertebrates that possess three BMP responsive rSmads -- Smad1, Smad5 and Smad8 -- BMP signaling might trigger the formation of a variety of trimeric rSmad complexes with Smad4 (e.g., a Smad1-Smad5- Smad4 complex). There is the potential for six such combinations. If such mixed complexes do form, as has been shown for Smad2, Smad3 and Smad4 in activation of p15Ink4B, it will be important to determine whether differences exist among them in the range of binding sites that can be bound by means of two or three MH1 domains. The likelihood of differential cofactor interactions by Smad1, Smad5 and Smad8 adds an additional layer of complexity (Gao, 2005).

C. elegans Smads

Three C. elegans genes (sma-2, sma-3, and sma-4) have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a family of proteins, the dwarfins, that includes the MAD gene product, a participant in the decapentaplegic pathway in Drosophila. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. Highly conserved dwarfins from vertebrates have been isolated, indicating that these components are not idiosyncratic to invertebrates. All the described null mutations in Mad, sma2 and sma3 are missense or nonsense mutations that fall within a highly conserved, short portion of the C-terminal domain (Savage, 1996).

Signals from TGF-beta superfamily receptors are transduced to the nucleus by Smad proteins, which transcriptionally activate target genes. In C. elegans, defects in a TGF-beta-related pathway cause a reversible developmental arrest and metabolic shift at the dauer larval stage. Null mutations in daf-3 suppress mutations in genes encoding this TGF-beta signal, its receptors, and associated Smad signal transduction proteins. daf-3 encodes a Smad protein that is most closely related to mammalian DPC4, and is expressed throughout development in many of the tissues that are remodeled during dauer development. DAF-4, the type II TGF-beta receptor in this pathway, is also expressed in remodeled tissues. These data suggest that the DAF-7 signal from sensory neurons acts as a neuroendocrine signal throughout the body to directly regulate developmental and metabolic shifts in tissues that are remodeled during dauer formation. A full-length functional DAF-3/GFP fusion protein is predominantly cytoplasmic; this localization is independent of activity in the upstream TGF-beta-related pathway. However, this fusion protein is associated with chromosomes in mitotic cells, suggesting that DAF-3 binds DNA either directly or indirectly. DAF-3 transgenes also interfere with dauer formation, perhaps attributable to a dosage effect. A truncated DAF-3/GFP fusion protein that is predominantly nuclear interferes with dauer formation, implying a role for DAF-3 in the nucleus. These data suggest that DAF-7 signal transduction antagonizes or modifies DAF-3 Smad activity in the nucleus to induce reproductive development; when DAF-7 signals are disabled, unmodified DAF-3 Smad activity mediates dauer arrest and its associated metabolic shift. Therefore, daf-3 is unique in that it is antagonized, rather than activated, by a TGF-beta pathway (Patterson, 1997).

C. elegans dauer formation is controlled by multiple environmental factors. The chemosensory neuron ASI regulates dauer formation by secretion of DAF-7/TGF-beta, but the molecular targets of the DAF-7 ligand are incompletely defined and the cellular targets are unknown. A putative transducer of DAF-7 signaling called daf-14 has been characterized and cloned and found to encode a Smad protein. DAF-14 Smad has a highly unusual structure, completely lacking the N-terminal domain found in all other Smad proteins known to date. DAF-14 lacks the DNA binding domain. daf-14 genetically interacts with daf-8, which encodes another Smad, and the interaction suggests partial functional redundancy between these two Smad proteins. The cellular targets of DAF-7 signaling were examined by studying the sites of action of daf-14 and daf-4 (receptor type 2), the putative receptor for DAF-7. daf-14::gfp is expressed in multiple tissues that are remodeled during dauer formation. Cells that consistently express DAF-14::GFP include intestinal cells, lateral hypodermal cells, the anal sphincter muscle, phasmid sheath cells, neurons in the lateral ganglia, the excretory duct cell, and several small cells anterior to the nerve ring that were not identified. In the lateral ganglia, expression is prominent in only a subset of neurons. In particular, strongest expression is often seen in an unidentified interneuron pair in the vicinity of AVJ and RIV. Analysis of mosaics generated by free duplication loss and tissue-specific expression constructs indicate cell-nonautonomous function of daf-4, arguing against direct DAF-7 signaling to tissues throughout the animal. Instead, these experiments suggest the nervous system as a target of DAF-7 signaling and that the nervous system in turn regulates dauer formation by other tissues (Inoue, 2000).

Two models are considered for the regulation of dauer formation by the DAF-7 secreting cell, ASI. DAF-7 may influence the development of target tissues directly. Alternatively, dauer regulation by ASI may occur in more than one step, with DAF-7 involved only in signaling to an intermediate cell or cells, which in turn regulate the target tissues by a different mechanism. The experiments show that daf-4 type 2 receptor can function in the nervous system (or possibly the intestine) to prevent dauer formation in the hypodermis, the pharynx, and the intestine, arguing against the first model as an exclusive mechanism for the regulation of dauer formation. These results indicate that the target cells of DAF-7 signaling can in turn regulate dauer formation through a different as-yet-unknown mechanism (Inoue, 2000).

Zebrafish Smads and BMP signaling

The maternal effect dorsalization of zebrafish embryos from sbndtc24 heterozygous mothers is caused by a dominant negative mutation in Smad5 (Drosophila homolog: Mad), a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbndtc24 mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it has remained open to what extent Smad5 itself is required for dorsoventral patterning. The identification is reported of novel smad5 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. m169 mutant embryos were rescued 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 than bmp2b 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 reduced bmp7 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 (Kramer, 2002).

Hematopoiesis, the dynamic process of blood cell development, is regulated by the activity of the bone morphogenetic protein (BMP) signaling pathway and by many transcription factors. However, the molecules and mechanisms that regulate BMP/Smad signaling in hematopoiesis are largely unknown. This study shows that the Integrator complex, that associates with the C-terminal repeats of RNA polymerase II and mediates U1 and U2 snRNA 3' end processing, functions in zebrafish hematopoiesis by modulating Smad/BMP signaling. This study has identified several subunits of the Integrator complex in zebrafish. Antisense morpholino-mediated knockdown of the Integrator subunit 5 (Ints5) in zebrafish embryos affects U1 and U2 snRNA processing, leading to aberrant splicing of smad1 and smad5 RNA, and reduced expression of the hematopoietic genes stem cell leukemia (scl, also known as tal1) and gata1. Blood smears from ints5 morphant embryos show arrested red blood cell differentiation, similar to scl-deficient embryos. Interestingly, targeting other Integrator subunits also leads to defects in smad5 RNA splicing and arrested hematopoiesis, suggesting that the Ints proteins function as a complex to regulate the BMP pathway during hematopoiesis. This work establishes a link between the RNA processing machinery and the downstream effectors of BMP signaling, and reveals a new group of proteins that regulates the switch from primitive hematopoietic stem cell identity and blood cell differentiation by modulating Smad function (Tao, 2009).

Xenopus Smads and BMP signaling

Smads are central mediators of signal transduction for the TGFbeta superfamily. However, the precise functions of Smad-mediated signaling pathways in early development are unclear. A requirement for Smad2 signaling is demonstrated in dorsoanterior axis formation during Xenopus development. Using two point mutations of Smad2 previously identified in colorectal carcinomas, it has been shown that Smad2 ushers Smad4 to the nucleus to form a transcriptional activation complex with the nuclear DNA-binding protein FAST-1 and that the mutant proteins interact normally with FAST-1 but fail to recruit Smad4 into the nucleus. This mechanism of inhibition specifically restricts the dominant-negative activity of these mutants to the activin/Vg1 signaling pathway without inhibiting BMPs. Furthermore, expression of these mutants in Xenopus animal caps inhibits but does not abolish activin and Vg1 induction of mesoderm and in the embryo results in a truncated dorsoanterior axis. These studies define a mechanism through which mutations in Smad2 may block TGFbeta-dependent signaling and suggests a critical role for inductive signaling mediated by the Smad2 pathway in Xenopus organizer function (Hoodless, 1999).

There are two functionally related Xenopus Mad-related genes. The different Xmad proteins produce distinct biological responses. Xmad1 produces ventral mesoderm, apparently transducing a signal for BMP2 and BMP4, where Xmad2 (see Baboon) induces dorsal mesoderm like Vg1, Activin and Nodal. This suggests that individual Xmad proteins wait poised in the cytoplasm for instruction from a distinct subset of TGFß ligands and then convey specific information to the nucleus (Graff, 1996).

Bone morphogenetic protein 4 (BMP4), the vertebrate homolog of Dpp, controls the fundamental choice between neural and epidermal fates in the vertebrate ectoderm, under the control of antagonists secreted by the organizer region of the mesoderm. BMP4 can act as a morphogen, evoking distinct responses in Xenopus ectodermal cells at high and low concentrations, in a pattern consistent with the positions of the corresponding cell types in the embryo. Moreover, this complex cellular response to extracellular BMP4 concentration does not require subsequent cell-cell communication and is thus direct, as required of a classical morphogen. The same series of cell types--epidermis, cement gland and neural tissue--can be produced by progressively inhibiting endogenous BMP signaling with specific antagonists, including the organizer factor noggin. BMP4 suppresses the neural marker NCAM; high doses of BMP antagonists induce NCAM, and lower doses of antigonist induce cement gland. Expression of increasing doses of the signal transduction molecule Smad1 accurately reproduces the response to BMP4 protein. Since Smads have been shown to act in the nucleus, this finding implies a direct translation of extracellular morphogen concentration into transcription factor activity. It is proposed that a graded distribution of BMP activity controls the specification of several cell types in the gastrula ectoderm, and that this extracellular gradient acts by establishing an intracellular and then nuclear gradient of Smad activity (Wilson, 1997).

A Xenopus homolog of human DPC4 is XSmad4. Smad4/DPC4 is the shared hetero-oligomerization partner for the other SMADs. XSmad4 has 89% identity to human DPC4 and only 40-46% similarity to human or Xenopus XSMad1 and XSMad2 (having undergone a name change acceptable to both C. elegans and Drosophila biologists, and formerly known as XMad1 and XMad2). XSmad4 transcripts are present in the maternal RNA pool and are ubiquitously expressed at least until the neural-groove stage. Smad4 proteins are important because they are dimerization partners of the other Smads. Human DPC4 transcripts were injected into the animal pole of two-cell Xenopus embryos. Human DPC4 alone can act as a ventral mesoderm inducer in the context of Xenopus animal cap explants, thus mimicking the effect of low concentrations of activin. A mutant form of DPC4, with a small C-terminal deletion, acts as a dominant negative form, failing to induce mesoderm in animal cap assays. Injection of the dominant negative DPC4 prevents brachyury expression in embryos, indicating the potential requirement for endogenous DPC4 in mesoderm induction. Mesoderm induction by XSmad2, an activin mediatior, is completely inhibited by coinjection of dominant negative DPC4. The action of XSmad1, a BMP mediator, is likewise inhibited by coinjection of dominant negative DPC4. DPC4 can physically interact with both XSmad1 and XSmad2. These complexes are formed when cells were stimulated with BMP4 for XSmad1 and activin or TGF-ß for XSmad2. The XSmads become phosphorylated upon stimulation. Thus each member of the TGF-ß family signals through its own SMad, requiring partnership with DPC4/SMad4 (Lagna, 1996).

Misexpression of Smad5 in the Xenopus embryo causes ventralization and induces ventral mesoderm. Moreover, Smad5 induces epidermis in dissociated ectoderm cells that would otherwise form neural tissue. Both of these activities require Smad4 (DPC4) activity, the promiscuous partner of the other Smads. It is proposed that Smad5 acts downstream of the BMP4 signaling pathway in Xenopus embryos and directs the formation of ventral mesoderm and epidermis (Suzuki, 1997).

During early embryogenesis of Xenopus, dorsoventral polarity of the mesoderm is established by dorsalizing and ventralizing agents, which are presumably mediated by the activity of an activin/BVg1-like protein and Bone Morphogenetic Proteins (BMP). Interestingly, these two TGF-beta subfamilies are found in overlapping regions during mesoderm patterning. This raises the question of how the presumptive mesodermal cells recognize the multiple TGF-beta signals and differentially interpret this information to assign a particular cell fate. The well characterized model of Xenopus mesoderm induction was exploited to determine the intracellular interactions between BMP-2/4 and activin/BVg1 signaling cascades. Using a constitutively active BMP-2/4 receptor that transduces BMP-2/4 signals in a ligand-independent fashion, it has been demonstrated that signals provided by activin/BVg1 and BMP modulate each other's activity; this crosstalk occurs through intracellular mechanisms. In assays using BMP-2/4 and activin/BVg1-specific reporters, it has been determined that the specificity of BMP-2/4 and activin/BVg1 signaling is mediated by Smad1 and Smad2, respectively. These Smads should be considered as the mediators of the intracellular antagonism between BMP-2/4 and activin/BVg1, possibly signaling through sequestration of a limited pool of Smad4. Consistent with such a mechanism, Smad4 interacts functionally with both Smad1 and -2 to potentiate their signaling activities; a dominant negative variant of Smad4 can inhibit both activin/BVg1 and BMP-2/4 mediated signaling. An activin/BVg1-dependent transcriptional complex contains both Smad2 and Smad4 and thereby provides a physical basis for the functional involvement of both Smads in TGF-beta-dependent transcriptional regulation. Thus, Smad4 plays a central role in synergistically activating activin/BVg1 and BMP-dependent transcription, and functions as an intracellular sensor for TGF-beta-related signals (Candia, 1997).

Bone morphogenetic protein (BMP) receptors signal by phosphorylating Smad1, which then associates with Smad4; this complex moves into the nucleus and activates transcription. A natural inhibitor of this process, Smad6, is a longer version of the previously reported JV15-1. In Xenopus embryos and in mammalian cells, Smad6 specifically blocks signaling by the BMP/Smad1 pathway. Smad6 inhibits BMP/Smad1 signaling without interfering with receptor-mediated phosphorylation of Smad1. Smad6 specifically competes with Smad4 for binding to receptor-activated Smad1, yielding an apparently inactive Smad1-Smad6 complex. Therefore, Smad6 selectively antagonizes BMP-activated Smad1 by acting as a Smad4 decoy. In Xenopus, Smad6 can induce cement gland and neural tissues in ectodermal explants in a cell-autonomous and dose-dependent manner, without inducing mesoderm. Smad6 also inhibits induction of a ventral mesoderm marker by a BMP receptor. Similar effects are observed with Dad, a Drosophila homolog of Smad6. Smad6 does interfere with formation of the primary axis, a process that requires signaling via the activin receptor. When directly challenged by Smad6 in a Xenopus animal cap assay, Smad1, but not Smad2, action is inhibited by Smad6. Smad6 inhibits BMP/Smad1 signaling selectively, without inhibiting Smad2 signaling in Xenopus embryos or TGFbeta and activin effects in mammalian cells. Smad6 may be an intracellular complement to the BMP inhibitory functions of Noggin, Chordin, and/or Follistatin and may play a key role in cell-autonomous determination of cell fate (Hata, 1998).

A new inhibitory Smad in Xenopus has been isolated and characterized. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-beta-like signals is mediated by receptor ALK-4 and Smad7 is shown to block signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway (Bhushan, 1998).

Bone morphogenetic proteins (BMPs) participate in the development of nearly all organs and tissues. BMP signaling is mediated by specific Smad proteins, Smad1 and/or Smad5, which undergo serine phosphorylation in response to BMP-receptor activation and are then translocated to the nucleus where they modulate transcription of target genes. A distantly related member of the Xenopus Smad family, Smad8, has been identified that lacks the C-terminal SSXS phosphorylation motif present in other Smads, and which appears to function in the BMP signaling pathway. During embryonic development, the spatial pattern of expression of Smad8 mirrors that of BMP-4. An intact BMP signaling pathway is required for its expression. Overexpression of Smad8 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to induction of a secondary axis on the ventral side of intact embryos and to direct neural induction in ectodermal explants. Smad8 can block BMP-4-mediated induction of ventral mesoderm-specific gene expression in ectodermal explants. However, overexpression of Smad8 within dorsal cells causes patterning defects that are distinct from those reported in BMP-4-deficient embryos, suggesting that Smad8 may interact with additional signaling pathways. Indeed, overexpression of Smad8 blocks expression of Xbra in whole animals, and partially blocks activin signaling in animal caps. Smad8 inhibits involution of mesodermal cells during gastrulation, a phenotype that is not observed following blockade of activin or BMPs in Xenopus. Together, these results are consistent with the hypothesis that Smad8 participates in a negative feedback loop in which BMP signaling induces the expression of Smad8, which then functions to negatively modulate the amplitude or duration of signaling downstream of BMPs and, possibly, downstream of other transforming growth factor-beta family ligands (Nakayama, 1998a).

Bone morphogenetic proteins (BMPs) transmit signals via the intracellular protein Smad1, which is phosphorylated by ligand bound receptors, translocates to the nucleus, and functions to activate BMP target genes. Recently, a subclass of Smad proteins has been shown to inhibit, rather than transduce, BMP signaling, either by binding to the intracellular domain of BMP receptors, thereby preventing phosphorylation-mediated activation of Smad1, or by binding directly to Smad1, thereby inhibiting its ability to activate gene transcription. A Xenopus Smad (Smad6) has been identified that is 52% identical to mammalian Smad6, an inhibitory Smad. The spatial pattern of expression of Smad6 changes dynamically during embryogenesis and is similar to that of BMP-4 at the tailbud stage. Overexpression of Smad6 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to dorsalization of mesoderm and neuralization of ectoderm. Xenopus Smad6 completely blocks the activity of exogenous BMP-4, and, unlike human Smad6, partially blocks the activity of activin, in a mesoderm induction assay. Smad6 protein accumulates at the membrane in some cells but is partially or completely restricted to nuclei of most overexpressing cells. Thus Smad6 functions as an intracellular antagonist of activin and BMP-4 signaling. The finding that Smad6 protein is partially or completely restricted to the nuclei of most overexpressing cells suggests that it may employ a novel or additional mechanism of action to antagonize TGF-beta family signaling other than that reported for other inhibitory Smads (Nakayama, 1998b).

The Spemann organizer induces neural tissue, dorsalizes mesoderm and generates a second dorsal axis. Smad10, is shown to have all three of these Spemann activities. The primary structure of Smad 10 is most closely related to the common-partner Smad, Smad4. Smad 10 also contains carboxyl-terminal serines that are sites of phosphorylation in the ligand-activated Smads. therefore, Smad10 may be a hybrid of these two classes of Smads and function in a new manner. Smad10 is expressed at the appropriate time to transduce Spemann signals endogenously. Like the organizer, Smad10 generates anterior and posterior neural tissues. Smad10 appears to function downstream of the Spemann organizer, consistent with a role in mediating organizer-derived signals. Interestingly, Smad10, unlike previously characterized mediators of Spemann activity, does not appear to block BMP signals. This finding, coupled with the functional activity and expression profile, suggests that Smad10 mediates Spemann action in a novel manner. Absence of BMP signaling is not the only condition for dorsal mesodermal induction. A TGFbeta signal transduced via a Smad is thought to be essential for induction of dorsal mesoderm. Although BMP4 signaling counteracts neural induction by BMP inhibitors, it does not reverse Smad10-mediated neural induction. It is plausible that Smad10 is the transducer of the neural induction signal and that the pattern of the induced neural tissue is then modified by factors such as the BMP inhibitors, FGF signals or Wnt signals. Discovery of a ligand that activates Smad10 will provide insights into this novel signaling cascade (LeSueur, 1999)

Signaling by members of the TGFbeta superfamily is thought to be transduced by Smad proteins. This paper describes 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, similar 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. A comparison among early marker gene expression patterns, chimera analyses and rescue experiments involving temporally controlled misexpression of bmp or smad in mutant embryos revealed 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 (Hild, 1999).

Bone morphogenetic protein-4 (BMP-4) induces epidermis and represses neural fate in Xenopus ectoderm. p42 Erk MAP kinase (MAPK) is implicated in the response to neural induction. The effects of BMP-4 on MAPK activity were examined in gastrula ectoderm. Expression of a dominant negative BMP-4 receptor results in a 4.5-fold elevation in MAPK activity in midgastrula ectoderm. MAPK activity is reduced in ectoderm expressing a constitutively active BMP-4 receptor, or ectoderm treated with BMP-4 protein in the presence or absence of cycloheximide. Overexpression of TAK1 (Drosophila homolog: TGF-ß activated kinase 1) leads to a reduction in MAPK activity in early gastrula ectoderm. The inhibitory effects of TAK1 can be reversed by 1 mM SB 203580, a p38 inhibitor. Treatment of isolated ectoderm with SB 203580 leads to expression of otx2, NCAM, and noggin. Western blot analyses indicate that the BMP-4 pathway does not activate JNKs in ectoderm. These findings indicate that BMP-4 inhibits ectodermal MAPK activity through a TAK1/p38-type pathway. MAPK has been shown to inactivate Smad1. Thus, these results suggest that BMP-4 and MAPK pathways are mutually antagonistic in Xenopus ectoderm, and that interactions between these pathways may govern the choice between epidermal and neural fate (Goswami, 2001).

Two signaling pathways are activated by signaling through the BMP-4 receptor complex. The first involves phosphorylation of the BMP-4 effector Smad1 at the C terminus; thus phosphorylated, Smad1 binds Smad4, translocates to the nucleus, and participates in transcriptional regulation. The second pathway is mediated by TGF-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. In mammalian cells, TAK1 has been shown to act via either Jun N-terminal kinases (JNKs), both members of the MAP kinase family. In C. elegans, TAK1 activates lit-1, a homolog of the distantly related MAP kinase family member Nemo-like kinase; this pathway culminates in the inhibitory phosphorylation of TCF-1/Lef-1 and the down-regulation of wnt/beta-catenin-inducible transcription. The TAK1/NLK pathway has also been shown to inhibit wnt/beta-catenin-dependent signaling in vertebrates. In Xenopus embryos, TAK1 has been implicated in the establishment of ventral mesoderm in response to BMP-4 (Goswami, 2001 and references therein).

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, an inner nuclear membrane protein, XMAN1, has been identified 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 (Osada, 2003).

Synexpression groups are genetic modules composed of genes that share both a complex expression pattern and the biological process in which they function. The regulation of BMP4 synexpression has been identied by studying the enhancers of bambi, smad7 and vent2 in Xenopus. A BMP4 synexpression promoter module has been identifed that is compact and that (1) requires direct BMP responsiveness through Smad and Smad-cofactor binding motifs; (2) may contain an evolutionary conserved BMP-responsive element, bre7 (TGGCGCC), which is crucial for expression of bambi and smad7 and is highly prognostic for novel BMP-responsive enhancers (BREs), and (3) requires a narrow window of BMP inducibility, because minor enhancement or reduction of BMP responsiveness abolishes synexpression. Furthermore, a bioinformatic model was used to predict in silico 13 novel BREs, and five found in the id1-4 genes were tested. The results highlight that in vivo analysis is required to reveal the physiological, spatio-temporal regulation of BMP-responsive genes (Karaulanov, 2004).

Neural differentiation is induced by inhibition of BMP signaling. Secreted inhibitors of BMP such as Chordin from the Spemann organizer contribute to the initial step of neural induction. Xenopus Smad-interacting protein-1 gene (XSIP1) is expressed in neuroectoderm from the early gastrula stage through to the neurula stage. XSIP1 is able to inhibit BMP signaling and overexpression of XSIP1 induces neural differentiation. To clarify the function of XSIP1 in neural differentiation, a loss-of-function study of XSIP1 was performed. Knockdown of XSIP1 inhibits SoxD expression and neural differentiation. These results indicate that XSIP1 is essential for neural induction. Furthermore, loss-of-function experiments show that SoxD is essential for XSIP1 transcription and for neural differentiation. However, inhibition of XSIP1 translation prevents neural differentiation induced by SoxD; thus, SoxD is not sufficient to mediate neural differentiation. Expression of XSIP1 is also required for inhibition of BMP signaling. Together, these results suggest that XSIP1 and SoxD interdependently function to maintain neural differentiation (Nitta, 2004).

Neural induction constitutes the first step in the generation of the vertebrate nervous system from embryonic ectoderm. Work with Xenopus ectodermal explants has suggested that epidermis is induced by BMP signals, whereas neural fates arise by default following BMP inhibition. In amniotes and ascidians, however, BMP inhibition does not appear to be sufficient for neural fate acquisition, which is initiated by FGF signalling. The roles of the BMP and FGF pathways during neural induction in Xenopus have been reevaluated. Ectopic BMP activity converts the neural plate into epidermis, confirming that this pathway must be inhibited during neural induction in vivo. Conversely, inhibition of BMP, or of its intracellular effector SMAD1 in the non-neural ectoderm leads to epidermis suppression. In no instances, however, is BMP/SMAD1 inhibition sufficient to elicit neural induction in ventral ectoderm. By contrast, neural specification occurs when weak eFGF or low ras signalling are combined with BMP inhibition. Using all available antimorphic FGF receptors (FGFR), as well as the pharmacological FGFR inhibitor SU5402, it was demonstrated that pre-gastrula FGF signalling is required in the ectoderm for the emergence of neural fates. Finally, although the FGF pathway contributes to BMP inhibition, as in other model systems, it is also essential for neural induction in vivo and in animal caps in a manner that cannot be accounted for by simple BMP inhibition. Taken together, these results reveal that in contrast to predictions from the default model, BMP inhibition is required but not sufficient for neural induction in vivo. This work contributes to the emergence of a model whereby FGF functions as a conserved initiator of neural specification among chordates (Delaune, 2005).

Chicken MAD homologs

Determination of the left-right (L-R) axis implicates several genes, among which TGFbeta-related molecules such as Activin betaB, lefty1 and 2 and Nodal. Bmp4 and its signal transduction pathway partners BMPR IA and Smad1 are transiently expressed on the right side of Hensen's node, when L-R polarity is being established. Moreover, Smad1 is expressed asymmetrically in the nascent notochord. These observations suggest a role for a BMP4-dependent autocrine or paracrine mechanism during early L-R determination (Monsoro-Burq, 2000).

An antibody specific for the phosphorylated and activated form of Smad1 has been used to examine endogenous patterns of BMP signaling in chick embryos during early development. Complex spatial and temporal distributions of BMP signaling are found that elucidate how BMPs may function in multiple patterning events in the early chick embryo. In the pregastrula embryo, BMP signaling is initially ubiquitous and is extinguished in the epiblast at the onset of primitive streak formation. At the head process stage, BMP signaling is inactivated in prospective neural plate, while it is strongly activated at the neural plate border, a region which is populated by cells that will give rise to neural crest. During later development, a dynamic spatiotemporal activation of BMP signaling is found along the rostrocaudal axis, in the dorsal neural tube, in the notochord, and in the somites during their maturation process (Faure, 2002).

Models for where and when BMPs signal during development have in the past been based primarily on inferences from the pattern of expression of BMP ligands and their inhibitors. While this examination of phosphoSmad1 levels during chick development confirms that, in most instances, patterns of Smad1 activation are broadly consistent with the expression of ligands and inhibitors, two types of exceptions to this consistency emerge from this work. First, at several points in early chick embryogenesis, Smad1 activation is detected when the known BMP ligands are not expressed. This is observed in the notochord at stage 14 and in the somites as soon as stage 10. While a large number of vertebrate BMP ligands have been identified, their expression and role in embryogenesis have not been systematically examined. These observations indicate that the ligands studied to date are not sufficient to fully explain endogenous patterns of BMP signaling. Second, at several developmental stages in chick, the BMP antagonist Noggin is clearly localized to a region of Smad1 activation. This is observed in Hensen’s node at stage 5, in the dorsal neural tube at stage 10, and in the dorsomedial differentiated somite at stage 14. This is not consistent with the simple equation of Noggin expression with BMP antagonism. One explanation for the colocalization of BMP signal activation and Noggin expression is that Noggin is activated downstream of BMP signaling as part of a negative feedback loop. If so, however, this feedback loop is clearly restricted by additional mechanisms to only a small subset of the cell types in which BMP signaling is activated, since Noggin expression is not generally correlated with BMP signaling during development. The fact that Smad1 phosphorylation and Noggin expression can persist in the same set of cells also suggests that there are ligands present in these regions that are resistant to Noggin inhibition (Faure, 2002).

Based on recent data, a new view is emerging that vertebrate Dachshund (Dach) proteins are components of Six1/6 transcription factor-dependent signaling cascades. Although Drosophila data strongly suggest a tight link between Dpp signaling and the Dachshund gene, a functional relationship between vertebrate Dach and BMP signaling remains undemonstrated. Chick Dach1 is shown to interact with the Smad complex and the corepressor mouse Sin3a, thereby acting as a repressor of BMP-mediated transcriptional control. In the limb, this antagonistic action regulates the formation of the apical ectodermal ridge (AER) in both the mesenchyme and the AER itself, and also controls pattern formation along the proximodistal axis of the limb. These data introduce a new paradigm of BMP antagonism during limb development mediated by Dach1, which is now proven to function in different signaling cascades with distinct interacting partners (Kida, 2004).

Mammalian MAD homologs

continue: see Mothers against Dpp Evolutionary homologs part 2/3 | part 3/3

Mothers against dpp: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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