Invertebrate Type I BMP receptors

DAF-1 is more similar to type I than to type II receptors in the TGFbeta family. DAF-1 has ten cysteine residues that are conserved in the extracellular domains of type I receptors for TGFbeta, activin, BMPs and DPP, but not in type II receptors. Furthermore, DAF-1 has a glycine-serine rich sequence (GS domain) that is characteristic of type I receptors. Phosphorylation of serines in the GS domain by an associated type II receptor is required for activation of the TGFbeta type I kinase. However, DAF-1 is the most divergent member of the type I receptor clade. Whereas the similarity between the kinase domains of type I receptors averages 50%, DAF-1 is only 25% similar. The daf-4 gene encodes a type II bone morphogenetic protein receptor in C. elegans that regulates dauer larva formation, body size and male tail patterning. The putative type I receptor partner for DAF-4 in regulating dauer larva formation is DAF-1. Genetic tests of the mechanism of activation of these receptors show that DAF-1 can signal in the absence of DAF-4 kinase activity. A daf-1 mutation enhances dauer formation in a daf-4 null background, whereas overexpression of daf-1 partially rescues a daf-4 mutant. DAF-1 alone cannot fully compensate for the loss of DAF-4 activity, indicating that nondauer development normally results from the activities of both receptors. DAF-1 signaling in the absence of a type II kinase is unique in the type I receptor family. The activity may be an evolutionary remnant, owing to daf-1's origin near the type I/type II divergence, or it may be an innovation that evolved in nematodes. daf-1 and daf-4 promoters both mediated expression of green fluorescent protein in the nervous system, indicating that a DAF-1/ DAF-4 receptor complex may activate a neuronal signaling pathway. Signaling from a strong DAF-1/DAF-4 receptor complex or a weaker DAF-1 receptor alone may provide larvae with more precise control of the dauer/nondauer decision in a range of environmental conditions (Gunther, 2000).

In C. elegans, the TGFbeta-like type II receptor daf-4 is required for two distinct signaling pathways. In association with the type I receptor daf-1, it functions in the dauer pathway. In addition, daf-4 is also required for body size determination and male tail patterning, roles that do not require daf-1. In an effort to determine how two different signals are transmitted through daf-4, other potential signaling partners for DAF-4 were sought. A novel type I receptor has been cloned and characterized and it is shown to be encoded by sma-6. daf-1 and sma-6 are more closely related to each other than they are to the two Drosophila genes thick veins and saxophone. Mutations in sma-6 generate the reduced body size (Sma) and abnormal male tail (Mab) phenotypes identical to those observed in C. elegans daf-4 and sma-2, sma-3, sma-4 mutants, indicating that these genes function in a common signaling pathway. However, mutations in sma-6, sma-2, sma-3, or sma-4 do not produce constitutive dauers, which demonstrates that the unique biological functions of daf-4 are mediated by distinct type I receptors functioning in parallel pathways. It is proposed that the C. elegans model for TGFbeta-like signaling, in which distinct type I receptors determine specificity, may be a general mechanism for achieving specificity in other organisms. These findings distinguish between the manner in which signaling specificity is achieved in TGFbeta-like pathways and receptor tyrosine-kinase (RTK) pathways (Krishna, 1999).

There are several transforming growth factor-beta (TGF-beta) pathways in the nematode Caenorhabditis elegans. One of these pathways regulates body length and is composed of the ligand DBL-1 (Dpp BMP-like), serine/threonine protein kinase receptors SMA-6 and DAF-4, and cytoplasmic signaling components SMA-2, SMA-3, and SMA-4. To further examine the molecular mechanisms of body-length regulation in the nematode by the TGF-beta pathway, the regional requirement for the type-I receptor SMA-6 was examined. Using a SMA-6::GFP (green fluorescent protein) reporter gene, sma-6 was found to be highly expressed in the hypodermis, unlike the type-II receptor DAF-4, which is reported to be ubiquitously expressed. The ability of SMA-6 expression in different regions of the C. elegans body to rescue the sma-6 phenotype (small) was examined and it was found that hypodermal expression of SMA-6 is necessary and sufficient for the growth and maintenance of body length. GATA sequences in the sma-6 promoter contribute to the hypodermal expression of sma-6 (Yoshida, 2002).

The hypodermis is the most important target tissue for regulation of body length by region-specific expression of a functional SMA-6 fusion protein and by the rescue of the Sma phenotype of the sma-6 mutant. The hypodermis is a group of cells underlying the cuticle, most of which are multinucleated syncytia. In particular, hyp-7 is the largest hypodermal cell and covers a large part of the body. A high level of sma-6 expression is observed in hyp-7, which suggests that hyp-7 contributes largely to the regulation of body length. Hypodermal cells also define the change of shape from an oval to a worm shape during embryogenesis. A target gene has been identified that is normally suppressed by DBL-1 signaling and is also expressed in the hypodermis. Overexpression of this gene in the hypodermis but not in the intestine shortens body length. Taken together, the results indicate that the hypodermis is a key tissue involved in determining body length (Yoshida, 2002).

C. elegans SMA-10 regulates BMP receptor trafficking

The C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6 (see Drosophila Thickveins), part of the TGFβ family, is recycled through the retromer complex while the type II receptor, DAF-4 (see Drosophila Punt) is recycled in a retromer-independent, ARF-6 dependent manner. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein, has been reported. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. This study shows that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, multivesicular bodies (MVB), and the late endosome with a reduction in signaling strength. Trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling (Gleason, 2017).

Components of the dorsal-ventral pathway also contribute to anterior-posterior patterning in honeybee embryos (Apis mellifera)

A key early step in embryogenesis is the establishment of the major body axes; the dorsal-ventral (DV) and anterior-posterior (AP) axes. Determination of these axes in some insects requires the function of different sets of signalling pathways for each axis. Patterning across the DV axis requires interaction between the Toll and Dpp/TGF-beta pathways, whereas patterning across the AP axis requires gradients of Bicoid/Orthodenticle proteins and the actions of a hierarchy of gene transcription factors. This study examined the expression and function of Toll and Dpp signalling during honeybee embryogenesis to assess to the role of these genes in DV patterning. Pathway components that are required for dorsal specification in Drosophila are expressed in an AP-restricted pattern in the honeybee embryo, including Dpp and its receptor Tkv. Components of the Toll pathway are expressed in a more conserved pattern along the ventral axis of the embryo. Late-stage embryos from RNA interference (RNAi) knockdown of Toll and Dpp pathways had both DV and AP patterning defects, confirmed by staining with Am-sna, Am-zen, Am-eve, and Am-twi at earlier stages. Two orthologues of dorsal were observed in the honeybee genome, with one being expressed during embryogenesis and having a minor role in axis patterning, as determined by RNAi and the other expressed during oogenesis. This study has found that early acting pathways (Toll and Dpp) are involved not only in DV patterning but also AP patterning in honeybee embryogenesis. Changes to the expression patterns and function of these genes may reflect evolutionary changes in the placement of the extra-embryonic membranes during embryogenesis with respect to the AP and DV axes (Wilson, 2014).

Cloning and expression patterns of Type I BMP receptors

Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, a cDNA from NIH 3T3 cells has been cloned that encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the C. elegans BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. It is concluded that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs (Koenig, 1994).

Bone morphogenetic proteins (BMPs) are multifunctional proteins that can induce cartilage and bone growth in vivo. Members of the TGF beta superfamily exert their biological effects via heteromeric serine/threonine kinase complexes of type I and type II receptors. Six different type I receptors have been identified and termed activin receptor-like kinase-1 (ALK-1) to -6. ALK-5 is a TGF beta type I receptor; ALK-2 and ALK-4 are activin type I receptors, and ALK-3 and ALK-6 are type I receptors for osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7) and BMP-4. The mouse homolog of ALK-3 is highly conserved between mouse and man. ALK-3 messenger RNA (mRNA) is ubiquitously expressed in various adult mouse tissues, whereas ALK-6 mRNA is only found in brain and lung. The distribution of ALK-3 and ALK-6 mRNA in the postimplantation mouse embryo [6.5-15.5 days postcoitum (pc)] was studied by in situ hybridization. ALK-3 is nearly ubiquitously expressed throughout these stages of development, but is notably absent in the liver. In contrast, ALK-6 shows a more restricted expression pattern. ALK-6 mRNA is absent in early postimplantation embryos, is detected first in 9.5 days pc embryos, and persists until 15.5 days pc. In midgestation embryos, ALK-6 transcripts are detected in mesenchymal precartilage condensations, premuscle masses, blood vessels, central nervous system, parts of the developing ear and eye, and epithelium. The expression in sites of developing cartilage and bone supports the idea that ALK-3 and -6 are receptors for BMPs in vivo. In addition, the expression of these genes in many soft tissues suggests broader functions for BMPs in embryogenesis (Dewulf, 1995).

A rat homolog cDNA was cloned of mouse and human type I receptors for BMP-2 and BMP-4. Tissue distribution of the receptor mRNA was studied by in situ hybridization using rats at embryonic days 9, 13, 15, and 18 as well as 1- and 5-day-old postnatal rats. In the rats at embryonic days 9, 13, and 15, the receptor mRNA is diffusely expressed over the embryonic bodies. At embryonic day 18, the receptor mRNA expression is high in the hair and whisker follicles, tooth bud, cartilage, bone, digestive organs, lung, kidney, heart, and meninges. The receptor mRNA is expressed over a much wider area than those of the ligands in many organs. In the lung and digestive organs, the receptor mRNA is diffusely expressed; it is most highly expressed in the bronchial epithelium of lungs and muscle layer of digestive organs. In both of these tissues mRNA expression of the ligands is undetectable. The receptor mRNA is highly expressed in the meninges, although neither of the ligands is expressed in or near this region. These results suggest that this receptor participates in both mesoderm formation in early embryogenesis and differentiation of mesodermal cells during maturation of organs, and further suggest the presence of another factor(s) that binds the type I receptor (Ikeda, 1996).

Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. A transmembrane protein, BAMBI, has been cloned that is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. BAMBI is co-expressed with the ventralizing morphogen BMP4 during Xenopus embryogenesis and it requires BMP signaling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signaling. Evidence is provided that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signaling by a regulatory mechanism involving the interaction of signaling receptors with a pseudoreceptor (Onichtchouk, 1999).

Vertebrate Type I BMP receptors: Interactions with Type II receptors and ligand

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-beta-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. A cDNA has been isolated encoding a transmembrane serine/threonine kinase from human skin fibroblasts, a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhances affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. A subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection of only BRK-2 + BRK-3; this suggests that the different heteromeric BMP receptor complexes have different signaling potentials (Nohno, 1995).

Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors: type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors, BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB), and a type II receptor, BMP receptor type II (BMPR-II). In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). The binding and signaling properties of GDF-5 through type I and type II receptors has also been studied. GDF-5 induces alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. 125I-GDF-5 binds to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. Analysis using COS-1 cells transfected with the receptor cDNAs reveals that GDF-5 binds to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells are transfected with type II receptor cDNAs, GDF-5 binds to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 binds to different sets of type I receptors, but the binding is most efficient to BMPR-IB, as compared with the other type I receptors. A transcriptional activation signal is efficiently transduced by BMPR-IB in the presence of either BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II (Nishitoh, 1996).

The BMP type II receptor (BMPR-II), a missing component of this BMP receptor system in vertebrates, has been cloned. BMPR-II is a transmembrane serine/threonine kinase that binds BMP-2 and BMP-7 in association with multiple type I receptors, including BMPR-IA/Brk1, BMPR-IB, and ActR-I, which is also an activin type I receptor. Cloning of BMPR-II results from the strong interaction of its cytoplasmic domain with diverse transforming growth factor beta family type I receptor cytoplasmic domains in a yeast two-hybrid system. In mammalian cells, however, the interaction of BMPR-II is restricted to BMP type I receptors and is ligand dependent. BMPR-II binds BMP-2 and -7 on its own, but binding is enhanced by coexpression of type I BMP receptors. BMP-2 and BMP-7 can induce a transcriptional response when added to cells coexpressing ActR-I and BMPR-II but not to cells expressing either receptor alone. The kinase activity of both receptors is essential for signaling. Thus, despite their ability to bind to type I and II receptors separately, BMPs appear to require the cooperation of these two receptors for optimal binding and for signal transduction. The combinatorial nature of these receptors and their capacity to crosstalk with the activin receptor system may underlie the multifunctional nature of their ligands (Liu, 1995).

Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-ß superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yield variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behave as antagonists, partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-ß superfamily (Kirsch, 2000).

The identification and characterization of two distinct binding epitopes in human BMP-2 as well as the detection of antagonistic BMP-2 variants, provides new insights into the primary steps and mechanism of BMP receptor activation. Receptor-binding epitopes have not been described before for any of the closely related members of the TGF-ß superfamily that signal via type I and type II receptor serine-kinases. All TGF-ß-like proteins are dimers, usually homodimers, where the monomers have been compared with an open hand, with the central alpha-helix (alpha3) being the wrist or heel and the two aligned two-stranded ß-sheets representing the four fingers, with loop 1 and loop 2 at the tips of each pair of fingers. The N-terminal segment exists at the position of the thumb. Consequently, the epitope 1 assembled around the central alpha-helix is called in the following the 'wrist epitope' and epitope 2 located at the back of the hand near the outer finger segments is called the 'knuckle epitope' (Kirsch, 2000 and references therein).

The wrist epitope has dimensions of ~20 x 25-30 Å (500-600 Å2). This large area would be compatible with the function as a high-affinity interaction site. The knuckle epitope seems to be smaller with dimensions of 10 x 20-25 Å (200-250 Å2) in accordance with the lower affinity of the interaction with BMPR-II at this site. The wrist epitope is highly discontinuous and it comprises different elements of both monomers. In heterodimers, e.g. of BMP-4 and BMP-7 or of the inhibin/activin-type factors, this has the interesting consequence that the two symmetry-related epitopes are no longer equivalent and may therefore exhibit different receptor-binding properties. In the knuckle epitope, the binding residues are provided by one monomer only and are located in sheets ß3, ß4, ß7 and ß8 and possibly also in ß9 (E109)(Kirsch, 2000 and references therein).

The juxtaposed knuckle and wrist epitopes are only 10-15 Å apart. The distances between the two type I (~40 Å) or the two type II chains (~55 Å), that possibly are assembled at BMP-2, are much larger. This appears to be especially relevant for the receptor serine-kinases. Their small ectodomain is connected to the membrane-spanning segment by a short linker of <12 residues. In the receptor complex with BMP-2, this short distance between epitopes 1 and 2 might facilitate the interaction of the type I and type II receptor serine-kinases (Kirsch, 2000).

The occurrence of the BMP-2 antagonists detected in this study is most likely to be a consequence of an ordered sequential binding mechanism operating during receptor activation. The antagonist blocks the high-affinity type I receptor chain via its intact wrist epitope, and the disrupted knuckle epitope prevents the subsequent interaction with the low-affinity type II chain(s). The similarly low IC50 of the antagonists as well as their efficient competition with BMP-2 for receptor binding could indicate that it is predominantly the type I chain(s) that adjust(s) the binding affinity of BMP-2 for the whole receptor complex. Interestingly, the velocity of complex formation and dissociation with BMPR-IA is equally critical, as revealed by the complete loss of biological activity of the respective double mutants. An intriguing finding is the dramatic loss of biological activity in variants of the knuckle epitope, considering that binding to the ectodomains of the type II receptor chain(s) is reduced only 10- to 15-fold. It is possible that the simultaneous binding of two type II chains is necessary for an efficient receptor activation, and therefore a decrease in binding affinity becomes aggravated (Kirsch, 2000 and references therein).

Phosphorylation of Smad1 at the conserved carboxyl terminal SVS sequence activates BMP signaling. Reported in this study is the crystal structure of the Smad1 MH2 domain in a conformation that reveals the structural effects of phosphorylation and a molecular mechanism for activation. Within a trimeric subunit assembly, the SVS sequence docks near two putative phosphoserine binding pockets of the neighboring molecule, in a position ready to interact upon phosphorylation. The MH2 domain undergoes concerted conformational changes upon activation, which signal Smad1 dissociation from the receptor kinase for subsequent heteromeric assembly with Smad4. Biochemical and modeling studies reveal unique favorable interactions within the Smad1/Smad4 heteromeric interface, providing a structural basis for their association in signaling (Qin, 2001).

Ligand binding to specific transmembrane receptor kinases induces receptor oligomerization and phosphorylation of the receptor-specific Smad protein (R-Smad) in the cytoplasm. The conserved signaling mechanism is the formation of a heteromeric complex between the phosphorylated R-Smad and the common mediator Smad4. The heteromeric complex enters the nucleus to regulate transcription of target genes. The R-Smads and Smad4 share a common domain configuration consisting of a conserved N-terminal DNA binding domain (MH1 domain) and a C-terminal MH2 domain separated by a variable linker region. The MH2 domain of an R-Smad, but not Smad4, has been shown to homo-oligomerize. Phosphorylation-triggered heteromeric assembly between Smad4 and R-Smad is mediated by the C-terminal MH2 domain. The sites of phosphorylation have been mapped to the last two serine residues within the conserved C-terminal SSXS sequence of the R-Smads. However, the role of phosphorylation in subunit assembly as well as the stoichiometry of the heteromeric complex remains controversial. Phosphorylation has been proposed to contribute directly to subunit assembly by bridging the MH2 domain interaction. Another model, however, suggests that phosphorylation uncouples the intramolecular inhibitory activity of the MH1 domain on the MH2 domain, allowing the MH2 domains to associate constitutively. The work described here suggests that the phosphorylated C-terminal tail of Smad1 functions as a subunit assembly switch by forming specific contacts with the phosphoserine binding pockets of the neighboring molecule. Furthermore, the MH2 domain undergoes concerted conformational changes upon trimerization, which may serve as a signaling switch. The phosphorylation-triggered Smad1-Smad4 complex is a trimer containing two Smad1 and one Smad4 subunits. Conservation of the amino acids involved in subunit assembly demonstrates a unifying structural mechanism of phosphorylation-triggered heteromeric assembly between R-Smads and Smad4 (Qin, 2001).

The current model suggests two mechanisms controlling specific phosphorylation of the R-Smads by the receptor kinase complexes. (1) A loop structure in the receptor kinase domain, referred to as the L45 loop, specifically interacts with the L3 loop of an R-Smad protein. (2) The receptor-associated recruiting molecule, such as SARA in the TGF-ß/activin pathway, can recruit specific R-Smad protein to the receptor kinase for phosphorylation. Although these mechanisms warrant specific phosphorylation of the R-Smads, how the recruited R-Smads release after phosphorylation is unknown. The current work provides insight into how the R-Smad disengages from the receptor kinase and the recruiting molecule after phosphorylation. The structure of the trimeric Smad1 reveals that the L3 loop and the three-helix bundle structure undergo concerted conformational changes upon activation, which may signal Smad1 dissociation from the receptor kinase complex. At the basal state, in which Smad1 is monomeric, the L3 loop of Smad1 interacts with the L45 loop of the receptor kinase. Upon activation and formation of a trimer, the L3 loop of Smad1 undergoes conformation change and interacts with the phosphorylated C-terminal tail of another Smad1 molecule. Thus, by employing distinct conformations to interact with mutually exclusive signaling partners, the L3 loop can serve as a switch for R-Smad dissociation from the receptor. Consistent with this model, the interaction between the receptor kinase complex and R-Smad is stronger when either the C-terminal phosphorylation sites of the R-Smad are mutated or when the kinase activity is rendered inactive by the catalytic site mutation. In addition, the trimerization-induced tilting of the three-helix bundle structure toward the subunit interface could also serve as a conformational switch to direct Smad1 dissociation from the receptor complex. In the analogous TGF-ß/activin pathway, SARA recruits and stabilizes the monomeric form of Smad2/3. Structural comparison between the trimeric Smad1 structure and the Smad2/SARA 1:1 complex structure reveals that SARA inhibits Smad2 trimerization by restricting the three-helix bundle movement, which is an essential mechanism of Smad protein trimerization. It is suggested that phosphorylation energetically favors trimerization, and that formation of trimers is sterically incompatible with SARA association. Similar mechanisms may exist for Smad1 through other receptor-associated molecules (Qin, 2001).

Dullard promotes degradation and dephosphorylation of BMP receptors and is required for neural induction

Bone morphogenetic proteins (BMPs) regulate multiple biological processes, including cellular proliferation, adhesion, differentiation, and early development. In Xenopus development, inhibition of the BMP pathway is essential for neural induction. dullard, a gene involved in neural development, functions as a negative regulator of BMP signaling. Dullard promotes the ubiquitin-mediated proteosomal degradation of BMP receptors (BMPRs). Dullard preferentially complexes with the BMP type II receptor (BMPRII) and partially colocalizes with the caveolin-1-positive compartment, suggesting that Dullard promotes BMPR degradation via the lipid raft-caveolar pathway. Dullard also associates with BMP type I receptors and represses the BMP-dependent phosphorylation of the BMP type I receptor. The phosphatase activity of Dullard is essential for the degradation of BMP receptors and neural induction in Xenopus. Together, these observations suggest that Dullard is an essential inhibitor of BMP receptor activation during Xenopus neuralization (Satow, 2006).

A BLAST domain search identified the catalytic phosphatase motif ψψψDXDX(T/V)ψψ (ψ: a hydrophobic amino acid residue) in Dullard. This motif is found in several serine/threonine phosphatases, including FCP1, a RNA polymerase II CTD phosphatase. In order to test whether Dullard functions as a phosphatase, mutants of dullard, D67E and D69E, were made in which the first or second essential aspartate residue, respectively, within the catalytic motif was changed to a glutamate, as described in FCP1 (Satow, 2006).

In vitro phosphatase assays with immunoprecipitated myc-tagged Dullard using p-nitrophenyl phosphate as a substrate revealed that Dullard has significant phosphatase activity. In contrast, the D67E and D69E mutants retained only weak phosphatase activity compared to the wild-type control. Animal cap assays also revealed that the phosphatase-inactive mutants had no BMP-antagonizing activity when coexpressed with BMP4. Furthermore, coinjection of mutant mRNA with dullard reversed the expression of BMP-responsive genes that were suppressed by dullard alone, indicating that D67E and D69E function in a dominant-negative manner. These results clearly show that the phosphatase activity of Dullard is responsible for its inhibitory effects on BMP signaling (Satow, 2006).

Vertebrate Type I BMP receptors: Effects of mutation

Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1, Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclear ß-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles (Andl, 2004).

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. Bmpr1a has been ablated specifically in the neural crest, beginning at the five-somite stage. Most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium (Stottmann, 2004).

The strategy for assessing the role of Bmpr1a in NCC development relies on the use of a conditional allele of Bmpr1a in concert with an established Wnt1-Cre transgene to drive recombination specifically in NCCs. To visualize Cre activity in NCCs, Wnt1-Cre mice were crossed to a Cre recombination reporter line, R26R. Recombination by Cre at the R26R locus brings a lacZ transgene under the irreversible control of a ubiquitous promoter. Embryos carrying R26R express lacZ in all cells that express active Cre recombinase, and in the descendents of those cells (Stottmann, 2004).

Recombination at the dorsal neural folds of the midbrain/hindbrain junction was visible by 4 s (the 4 somite stage), reflecting a domain of Wnt1 expression required for midbrain development. The first distinct NCCs were seen along the dorsal neural folds of the hindbrain at the 5 s stage; these are more numerous by 6 s. Recombination extended into the rostral spinal cord by 8 s. Transverse sections of Wnt-1Cre; R26R embryos at ~10 s (E8.5) revealed migration from the neural tube to the pharyngeal arches and other target tissues. Recombination then extended caudally along the dorsal neural tube to include the entire neural crest and its descendents. Wnt1-Cre activity in the neural crest at 5 s coincides with the time at which the first NCCs have been detected by cell labeling studies. Thus, Wnt1-Cre allows recombination of Bmpr1a in the neural crest from very early stages of its development (Stottmann, 2004).

Although Wnt1-Cre expression has been associated exclusively with the neural crest and its derivatives, the epicardium of Wnt1-Cre; R26R embryos contained a small proportion of recombined cells. This indicates that they are derived from a Cre-expressing lineage. Although such cells were not detected in every specimen (probably for technical reasons), control experiments indicate that the labeling was not a staining artifact. These labeled cells later populated the same tissues as epicardial derivatives (coronary arteries and scattered cells in the ventricular wall). Because these epicardial cells clearly underwent epithelial-mesenchymal transition (EMT) to clonally populate these structures, it is possible that this population represents a fraction of the epicardium that is competent to undergo EMT rather than remaining an obligate part of the epicardial mesothelium (Stottmann, 2004).

A Wnt1-Cre lineage of cells was observed in the septum transversum adjacent to the ventricles at E9.5. This is the location of the proepicardial organ, the source of epicardial cells. It is therefore suspected that the labeled cells of the epicardium came from this tissue, though it is also possible that they have migrated in from a known neural crest domain elsewhere. It is uncertain whether these labeled cells are of neural crest origin, or if they represent a hitherto undetected independent expression domain of Wnt1-Cre. However, the same distribution of labeled cells was found when PO-Cre was used as an independent neural crest lineage marker. Moreover, using the sensitive assay of RT-PCR, Cre expression was never seen in non-dorsal tissues dissected from Wnt1-Cre embryos from E9.5-10.5 (during which time the epicardium forms). These considerations led the authors to suspect that the labeled epicardial cells were of neural crest origin, though they probably arrived via the proepicardial organ (Stottmann, 2004).

Although an epicardial NCC lineage has not been otherwise observed in mice or chicks, recent evidence suggests NCCs contribute to ventricular myocardium in zebrafish. In mice and chickens with NCC defects, myocardial defects are seen before NCCs are known to populate the outflow tract, suggesting that NCCs produce factor(s) involved in myocardial development. Moreover, Pax3 expression in NCCs rescues the reduced myocardial tissue phenotype of Pax3-null mutants. It is suggested that lesions in an early population of NCC derivatives, migrating into the epicardium and/or outflow tract, account for previously observed myocardial defects associated with neural crest perturbations. Overall, this work demonstrates that BMPRIA is dispensable for most aspects of early neural crest development, and identifies a novel, crucial role for BMPRIA signal transduction in extra-cardiac cells during the development of the outflow tract and ventricular myocardium of the heart (Stottmann, 2004).

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, Bmpr1a, which encodes the BMP receptor type IA (BMPR1A), was disrupted in an HF cell-specific manner, using the Cre/loxP system. It was found that the differentiation of inner root sheath, but not outer root sheath, is severely impaired in mutant mice. The number of HFs is reduced in the dermis and subcutaneous tissue, and cycling epithelial cells are reduced in mutant mice HFs. These results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin (Yuhki, 2004).

During spinal cord development, distinct classes of interneurons arise at stereotypical locations along the dorsoventral axis. This study demonstrates that signaling through bone morphogenetic protein (BMP) type 1 receptors is required for the formation of two populations of commissural neurons, DI1 and DI2, that arise within the dorsal neural tube. A double knockout of both BMP type 1 receptors, Bmpr1a and Bmpr1b, has been generated in the neural tube. These double knockout mice demonstrate a complete loss of D1 progenitor cells, as evidenced by loss of Math1 expression, and the subsequent failure to form differentiated DI1 interneurons. Furthermore, the DI2 interneuron population is profoundly reduced. The loss of these populations of cells results in a dorsal shift of the dorsal cell populations, DI3 and DI4. Other dorsal interneuron populations, DI5 and DI6, and ventral neurons appear unaffected by the loss of BMP signaling. The Bmpr double knockout animals demonstrate a reduction in the expression of Wnt and Id family members, suggesting that BMP signaling regulates expression of these factors in spinal cord development. These results provide genetic evidence that BMP signaling is crucial for the development of dorsal neuronal cell types (Wine-Lee, 2004).

Cleft lip with or without cleft palate (CL/P) is genetically distinct from isolated cleft secondary palate (CP). Mutations in the Bmp target gene Msx1 in families with both forms of orofacial clefting has implicated Bmp signaling in both pathways. To dissect the function of Bmp signaling in orofacial clefting, the type 1 Bmp receptor Bmpr1a was conditionally inactivated in the facial primordia using the Nestin cre transgenic line. Nestin cre; Bmpr1a mutants had completely penetrant, bilateral CL/P with arrested tooth formation. The cleft secondary palate of Nestin cre; Bmpr1a mutant embryos is associated with diminished cell proliferation in maxillary process mesenchyme and defective anterior posterior patterning. By contrast, elevated apoptosis is observed in the fusing region of the Nestin cre; Bmpr1a mutant medial nasal process. Moreover, conditional inactivation of the Bmp4 gene using the Nestin cre transgenic line results in isolated cleft lip. The data uncover a Bmp4-Bmpr1a genetic pathway that functions in lip fusion, and reveal that Bmp signaling has distinct roles in lip and palate fusion (Liu, 2005).

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, a Cre-loxP strategy was used to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina (Murali, 2005).

In vertebrate limbs that lack webbing, the embryonic interdigit region is removed by programmed cell death (PCD). Established models suggest that bone morphogenetic proteins (BMPs) directly trigger such PCD, although no direct genetic evidence exists for this. Alternatively, BMPs might indirectly affect PCD by regulating fibroblast growth factors (FGFs), which act as cell survival factors. The mouse BMP receptor gene Bmpr1a was inactivated specifically in the limb bud apical ectodermal ridge (AER), a source of FGF activity. Early inactivation completely prevents AER formation. However, inactivation after limb bud initiation causes an upregulation of two AER-FGFs, Fgf4 and Fgf8, and a loss of interdigital PCD leading to webbed limbs. To determine whether excess FGF signaling inhibits interdigit PCD in these Bmpr1a mutant limbs, double and triple AER-specific inactivations of Bmpr1a, Fgf4 and Fgf8 were performed. Webbing persists in AER-specific inactivations of Bmpr1a and Fgf8 owing to elevated Fgf4 expression. Inactivation of Bmpr1a, Fgf8 and one copy of Fgf4 eliminates webbing. It is concluded that during normal embryogenesis, BMP signaling to the AER indirectly regulates interdigit PCD by regulating AER-FGFs, which act as survival factors for the interdigit mesenchyme (Pajni-Underwood, 2007).

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. This study provides in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. Transgenic zebrafish were used that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. It was found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated (Shin, 2007).

Holoprosencephaly (HPE) is a devastating forebrain abnormality with a range of morphological defects characterized by loss of midline tissue. In the telencephalon, the embryonic precursor of the cerebral hemispheres, specialized cell types form a midline that separates the hemispheres. In the present study, deletion of the BMP receptor genes, Bmpr1b and Bmpr1a, in the mouse telencephalon results in a loss of all dorsal midline cell types without affecting the specification of cortical and ventral precursors. In the holoprosencephalic Shh-/- mutant, by contrast, ventral patterning is disrupted, whereas the dorsal midline initially forms. This suggests that two separate developmental mechanisms can underlie the ontogeny of HPE. The Bmpr1a;Bmpr1b mutant provides a model for a subclass of HPE in humans: midline inter-hemispheric HPE (Fernandes, 2007).

The olfactory sensory epithelium and the respiratory epithelium are derived from the olfactory placode. However, the molecular mechanisms regulating the differential specification of the sensory and the respiratory epithelium have remained undefined. To address this issue, first, Msx1/2 and Id3 were identified as markers for respiratory epithelial cells, by performing quail chick transplantation studies. Next, chick explant and intact chick embryo assays of sensory/respiratory epithelial cell differentiation were established and two mice mutants deleted of Bmpr1a;Bmpr1b or Fgfr1;Fgfr2 in the olfactory placode were analyzed. In this study, evidence is provided that in both chick and mouse, Bmp signals promote respiratory epithelial character, whereas Fgf signals are required for the generation of sensory epithelial cells. Moreover, olfactory placodal cells can switch between sensory and respiratory epithelial cell fates in response to Fgf and Bmp activity, respectively. These results provide evidence that Fgf activity suppresses and restricts the ability of Bmp signals to induce respiratory cell fate in the nasal epithelium. In addition, in both chick and mouse the lack of Bmp or Fgf activity results in disturbed placodal invagination; however, the fate of cells in the remaining olfactory epithelium is independent of morphological movements related to invagination. In summary, a conserved mechanism in amniotes is presented in which Bmp and Fgf signals act in an opposing manner to regulate the respiratory versus sensory epithelial cell fate decision (Maier, 2010).

Vertebrate Type I BMP receptors: Signal transduction

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta superfamily, inhibits the terminal differentiation of C2C12 myoblasts and changes their differentiation pathway into cells expressing osteoblast phenotypes, such as alkaline phosphatase (ALP) activity and osteocalcin production. Two type I receptors for BMP-2 (BMPR-IA and BMPR-IB) have been cloned, but the role of the respective receptors in signal transduction is not clear. The signal transduction of BMP-2 was studied in C2C12 cells using constitutively activated mutant BMPR-IA and BMPR-IB. In a Northern blot analysis, C2C12 cells express BMPR-IA and BMPR-II mRNAs at detectable levels (but not BMPR-IB mRNA). When mutated BMPR-IA and BMPR-IB are transiently transfected into C2C12 cells, both BMPR-IA and BMPR-IB similarly induce ALP activity in the absence of BMP-2. Subclonal cell lines of C2C12 cells were established by stably transfecting mutated BMPR-IB. When the mutated BMPR-IB-transfected cells are cultured in medium with low serum (differentiation medium) without BMP-2, the cells differentiate into ALP-positive mononuclear cells, and not into myosin heavy chain-positive myotubes. These mutated BMPR-IB-transfected cells express ALP activity and osteocalcin mRNA in a time-dependent manner, but not muscle creatine kinase or myogenin mRNAs. These results indicate that the mutated BMP-2 type I receptors can constitutively transduce BMP-2 signals in the absence of the ligand in C2C12 cells (Akiyama, 1997).

The type I TGFß receptor (TßR-I) is activated by phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the kinase domain. The molecular mechanism of receptor activation has been studied using a homogeneously tetraphosphorylated form of TßR-I, prepared using protein semisynthesis. Phosphorylation of the GS region dramatically enhances the specificity of TßR-I for the critical C-terminal serines of Smad2. In addition, tetraphosphorylated TßR-I is bound specifically by Smad2 in a phosphorylation-dependent manner and is no longer recognized by the inhibitory protein FKBP12. Thus, phosphorylation activates TßR-I by switching the GS region from a binding site for an inhibitor into a binding surface for substrate. These observations suggest that phosphoserine/phosphothreonine-dependent localization is a key feature of the TßR-I/Smad activation process (Huse, 2001).

Signal transduction by the TGF-beta family involves sets of receptor serine/threonine kinases, Smad proteins that act as receptor substrates (see Drosophila Mothers against Dpp), and Smad-associated transcription factors that target specific genes. Discrete structural elements have been identified that dictate the selective interactions between receptors and Smads and between Smads and transcription factors in the TGF-beta and BMP pathways. Of particular interest is a nine-amino-acid segment in the receptor kinase domain, known as the L45 loop. Replacement of all but the L45 loop in the kinase domain of TbetaR-I with the corresponding regions from ALK2 yields a construct that still mediates TGF-beta responses. As predicted from the conserved structure of protein kinases, the L45 loop links beta-strands 4 and 5, and is not part of the catalytic center. The L45 loop differs between type I receptors of different signaling specificity, such as the TGF-beta receptors and the BMP receptors, but is highly conserved between receptors of similar signaling specificity such as TbetaR-I and the activin receptor ActR-IB, or the BMP receptors from human (BMPR-IA and BMPR-IB) and Drosophila (Thick veins). A cluster of four residues in the L45 loop of the type I receptor kinase domain, and a matching set of two residues in the L3 loop of the Smad carboxy-terminal domain establish the specificity of receptor-Smad interactions. The L3 loop of Smads has drawn attention as a target of inactivating mutations in Drosophila and Caenorhabditis elegans Smad family members. As inferred from the effect of similar mutations in vertebrate Smads, the L3 loop participates in different interactions that are essential for signaling. In Smad4 (see Drosophila Medea) the L3 loop is required for interaction with activated receptor regulated Smads (R-Smads), whereas in R-Smads the L3 loop is required for interaction with the receptors and, furthermore, it specifies these interactions. The present results show that matching combinations of L45 loops and L3 loops determine the specificity of the receptor-Smad interaction. Exchanging the subtype-specific residues in either the L45 loop or the L3 loop causes a switch in the specificity of this interaction, with an attendant switch in the signaling specificity of the pathway. As evidence of a functional match between a receptor L45 loop and an R-Smad L3 loop, the switch in the signaling specificity of a TGF-beta receptor construct containing the BMP receptor L45 loop can be reversed by a Smad2 construct containing the matching L3 loop sequence from Smad1. A cluster of residues in the highly exposed alpha-helix 2 of the Smad carboxy-terminal domain specifies the interaction with the DNA-binding factor Fast1 and, as a result, the gene responses mediated by the pathway. By establishing specific interactions, these determinants keep the TGF-beta and BMP pathways segregated from each other (D. Chen, 1998).

TGF-ß coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-ß receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482-491 in the kinase domain of the type I receptor is required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, and Drosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non-down-regulating) abolishes TGF-ß-dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-ß receptor complex (Garamszegi, 2001).

No canonical motifs or significant sequence conservation with protein kinases other than type I family members was found within the sequence encompassing amino acids 482-492; however, an extremely high degree of conservation was noted within the activin-like kinase family, including identity at 10/11 and 9/11 residues with the Drosophila Thickveins and Saxophone type I receptors. As such, the BOX region seems to be a sequence uniquely restricted to regulating type I TGF-ßR activity. In that regard, studies have shown that the decreased type I receptor activity of Saxophone (relative to Thickveins) in Drosophila can be mapped to the nonconserved proline within the BOX. Thus, the functional activity of the BOX seems to be evolutionarily conserved throughout the type I TGF-ßR family (Garamszegi, 2001).

The BOX region was shown to be necessary for receptor down-regulation, PAI-1 and fibronectin protein expression, transcriptional activation, Smad2 phosphorylation, and growth inhibitory responses from both chimeric and endogenous TGF-ß receptors. Moreover, the finding that similar effects are observed on Smad4-dependent and -independent signaling, as well as receptor endocytic activity in mesenchymal cells, indicates that mutation of the BOX region interferes with an early event(s) in receptor activation. As such, the role of the BOX region in TGF-ßR complex formation and type I receptor phosphorylation was determined. Although the native type I receptor BOX-ANA mutant is capable of forming a heteromeric complex with the type II TGF-ßR to a similar extent as the wild-type type I receptor, the associated type II receptor is unable to transphosphorylate and activate the mutant receptor in vivo (Garamszegi, 2001).

Computer modeling indicates that although the BOX region (amino acids 482-491) is significantly distal (33.7 Å, on average) to the regulatory juxtamembrane GS domain (amino acids 176-205), it is exposed on the same surface as Gly-261 and Gly-322, two residues required for activation of type I receptor subunits. Because Gly-261 and Gly-322 can be transcomplemented by inactive type I receptors containing a mutation in the ATP binding site, whether the BOX motif would respond similarly was determined. As expected for an activation domain, cotransfection of the Box 3 mutant with a kinase-impaired type I receptor restores TGF-ßR signaling. Because type I receptor kinase activity is required for TGF-ß signaling, and the only receptor capable of providing this function harbors the Box 3 mutation, this shows that the BOX mutation does not directly impair the receptor kinase. Moreover, because the endocytic requirement for type I receptor phosphorylation differs between epithelial and fibroblast cells, this provided an ideal opportunity to assess whether the observed effects on down-regulation are a specific reflection of an absence of type I receptor phosphorylation or caused by a general misfolding of the receptor. When the Box 3 mutant receptor is expressed in epithelial cells, the receptor complex down-regulates to a similar extent as wild-type. Thus, not only does a Box 3 mutant receptor have a functional kinase, but the mutation is capable of being recognized by the endocytic machinery (Garamszegi, 2001).

These results suggest that type I receptor activation involves the coordinated action of multiple regulatory domains. Furthermore, although the BOX is within the type I receptor kinase domain (amino acids 207-498), the absence of receptor activity cannot be explained simply by disrupting this region. For instance, (1) analogous mutations 5' or 3' to the BOX have no apparent effect on either receptor endocytosis or signaling; (2) the absence of type I receptor kinase activity, per se, has no effect on TGF-ßR down-regulation in fibroblasts; (3) modeling of energy-minimized Box 3 substitution mutants shows only a minor structural perturbation with a 0.63 Å overall shift in the backbone; (4) truncation after amino acid 216 (i.e., missing essentially the entire kinase domain) generates a type I receptor that down-regulates similar to wild-type, and (5) cotransfection with a kinase-impaired type I receptor generates an active signaling complex. As such, the manner in which mutations within this motif block type I receptor activation is not apparent from the structure. These observations indicate that the sequence between amino acids 482 and 491 in the type I receptor provides a critical function regulating GS domain phosphorylation and subsequent activation of the TGF-ß receptor complex (Garamszegi, 2001).

BMP7 and activin are members of the transforming growth factor beta superfamily. Endogenous activin and BMP7 signaling pathways are characterized in P19 embryonic carcinoma cells. BMP7 and activin bind to the same type II receptors, ActRII and IIB, but recruit distinct type I receptors into heteromeric receptor complexes. The major BMP7 type I receptor observed is ALK2, while activin binds exclusively to ALK4 (ActRIB). BMP7 and activin elicit distinct biological responses and activate different Smad pathways. BMP7 stimulates phosphorylation of endogenous Smad1 and 5 and the formation of complexes with Smad4, and induces the promoter for the homeobox gene, Tlx2. In contrast, activin induces phosphorylation of Smad2, association with Smad4, and induction of the activin response element from the Xenopus Mix.2 gene. Biochemical analysis reveals that constitutively active ALK2 associates with and phosphorylates Smad1 on the COOH-terminal SSXS motif, and also regulates Smad5 and Smad8 phosphorylation. Activated ALK2 also induces the Tlx2 promoter in the absence of BMP7. ALK1 (TSRI), an orphan receptor that is closely related to ALK2 also mediates Smad1 signaling. Thus, ALK1 and ALK2 induce Smad1-dependent pathways. ALK2 functions to mediate BMP7 but not activin signaling (Macias-Silva, 1998).

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Evidence is provided that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, Xenopus Tob2 has been isolated and has been shown to cooperate with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. These results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions (Yoshida, 2003).

Vertebrate Type I BMP receptors: Protein degradation

The Rho-associated serine/threonine kinases Rock1 and Rock2 play important roles in cell contraction, adhesion, migration, proliferation and apoptosis. Mammalian Rock2 acts as a negative regulator of the TGFbeta signaling pathway. Mechanistically, Rock2 binds to and accelerates the lysosomal degradation of TGFbeta type I receptors internalized from the cell surface in mammalian cells. The inhibitory effect of Rock2 on TGFbeta signaling requires its kinase activity. In zebrafish embryos, injection of rock2a mRNA attenuates the expression of mesodermal markers during late blastulation and blocks the induction of mesoderm by ectopic Nodal signals. By contrast, overexpression of a dominant negative form of zebrafish rock2a has an opposite effect on mesoderm induction, suggesting that Rock2 proteins are endogenous inhibitors for mesoderm induction. Thus, these data have unraveled previously unidentified functions of Rock2, in controlling TGFbeta signaling as well as in regulating embryonic patterning (Zhang, 2009).

Vertebrate Type I BMP receptors: Developmental roles

It has been an intriguing problem to solve: do the polypeptide growth factors belonging to the transforming growth factor-beta (TGF-beta) superfamily function as direct and long-range signaling molecules in pattern formation of the early embryo? In this study, the mechanism of signal propagation of bone morphogenetic protein (BMP) was determined in the ectodermal patterning of zebrafish embryos, in which BMP functions as an epidermal inducer and a neural inhibitor. To estimate the effective range of zbmp-2, whole-mount in situ hybridization analysis was performed. The zbmp-2-expressing domain and the neuroectoderm, marked by otx-2 expression, are complementary, suggesting that BMP has a short-range effect in vivo. Moreover, mosaic experiments using a constitutively active form of a zebrafish BMP type I receptor (CA-BRIA) demonstrate that the cell-fate conversion, revealed by ectopic expression of gata-3 and repression of otx-2, occurs in a cell-autonomous manner, denying the involvement of the relay mechanism. zbmp-2 is induced cell autonomously within the transplanted cells in the host ectoderm, suggesting that BMP cannot influence even the neighboring cells. This result is consistent with the observation that there is no gap between the expression domains of zbmp-2 and otx-2. Taken together, it is proposed that, in ectodermal patterning, BMP exerts a direct and cell-autonomous effect on the fates of uncommitted ectodermal cells, making them become epidermis (Nikaido, 1999).

To examine the role of BMP signaling during limb pattern formation, chicken cDNAs encoding were isolate encoding type I (BRK-1 and BRK-2) and type II (BRK-3) receptors for bone morphogenetic proteins. BRK-2 and BRK-3, which constitute dual-affinity signaling receptor complexes for BMPs, are co-expressed in condensing precartilaginous cells, while BRK-1 is weakly expressed in the limb mesenchyme. BRK-3 is also expressed in the apical ectodermal ridge and interdigital limb mesenchyme. BRK-2 is intensely expressed in the posterior-distal region of the limb bud. During digit duplication by implanting Sonic hedgehog-producing cells, BRK-2 expression is induced anteriorly in the new digit forming region as observed for BMP-2 and BMP-7 expression in the limb bud. Dominant-negative effects on BMP signaling were obtained by overexpressing kinase domain-deficient forms of the receptors. Chondrogenesis of limb mesenchymal cells is markedly inhibited by dominant-negative BRK-2 and BRK-3, but not by BRK-1. Although the bone pattern was not disturbed by expressing individual dominant-negative BRK independently, preferential distal and posterior limb truncations resulted from co-expressing the dominant-negative forms of BRK-2 and BRK-3 in the whole limb bud, thus providing evidence that BMPs are essential morphogenetic signals for limb bone patterning (Kawakami, 1996).

Members of the family of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior-posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB) were generated. The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appear normal, the transgenic hindlimbs exhibit several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs are also found. The bifurcation in the transgenic hindlimb occurs early in the limb development (E10.5) and is associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appears unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression is found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. Gremlin is a member of the DAN family of BMP antagonists, highly conserved through evolution, and able to bind and block BMP2, BMP4 and BMP7. The observation that Hoxd-12 and Hoxd-13 expression patterns are extended anteriorly provides a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior-posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression (Zhang, 2000).

Bmpr, also known as ALK-3 and Brk-1, encodes a type I transforming growth factor-beta (TGF-beta) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development Bmpr-mutant mice were generated. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects are first detected at E7.0 and are smaller than normal. Morphological and molecular examination demonstrates that no mesoderm forms formed in the mutant embryos. The growth characteristics of homozygous mutant blastocysts cultured in vitro are indistinguishable from those of controls; however, embryonic ectoderm (epiblast) cell proliferation is reduced in all homozygous mutants at E6.5 before morphological abnormalities become prominent. Teratomas arising from E7.0 mutant embryos contain derivatives from all three germ layers but are smaller and give rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the differentiation of a subset of mesodermal cell types (Mishina, 1995).

Previous work has demonstrated that the bone morphogenetic proteins (BMP)-2, BMP-4, and BMP-7 can promote the development of tyrosine hydroxylase (TH)-positive and catecholamine-positive cells in quail trunk neural crest cultures. It has been shown that mRNA for the type I bone morphogenetic protein receptor IA (BMPR-IA) is present in neural crest cells grown in the absence or presence of BMP-4. A replication-competent avian retrovirus was used to express a constitutively active form of BMPR-IA in neural crest cells in culture. Cultures grown in the absence of BMP-4 and infected with retrovirus containing a construct encoding this activated BMPR-IA develop five times more TH-immunoreactive and catecholamine-positive cells than uninfected control cultures or cultures infected with virus bearing the wild-type BMPR-IA cDNA. The number of TH-positive cells that develop is dependent on the concentration of virus bearing the activated receptor cDNA used in the experiments. Most TH-positive cells that develop also contain viral p19 protein. Total cell number is not affected by infection with the virus containing the activated receptor construct. The effect of the activated receptor is phenotype-specific since infection with the virus bearing the activated receptor cDNA alters neither the number nor morphology of microtubule-associated protein (MAP)2-immunoreactive cells, which are distinct from the TH-positive cell population. These findings are consistent with the observation that MAP2-positive cells are not affected by the presence of BMP-4. Taken together, these results suggest that activity of BMPR-IA is an important element in promoting the development of the adrenergic phenotype in neural crest cultures (Varley, 1998).

Cumulative evidence indicates that osteoblasts and adipocytes share a common mesenchymal precursor and that bone morphogenetic proteins (BMPs) can induce both osteoblast and adipocyte differentiation of this precursor. In the present study, the roles of BMP receptors were investigated in differentiation along these separate lineages using a well-characterized clonal cell line, 2T3, derived from the mouse calvariae. BMP-2 induces 2T3 cells to differentiate into mature osteoblasts or adipocytes depending on culture conditions. To test the specific roles of the type IA and IB BMP receptor components, truncated and constitutively active type IA and IB BMP receptor cDNAs were stably expressed in these cells. Overexpression of truncated type IB BMP receptor (trBMPR-IB) in 2T3 cells completely blocks BMP-2-induced osteoblast differentiation and mineralized bone matrix formation. Expression of trBMPR-IB also blocks mRNA expression of the osteoblast specific transcription factor, Osf2/Cbfa1, and the osteoblast differentiation-related genes, alkaline phosphatase (ALP) and osteocalcin (OC). BMP-2-induced ALP activity can be rescued by transfection of wild-type (wt) BMPR-IB into 2T3 clones containing trBMPR-IB. Expression of a constitutively active BMPR-IB (caBMPR-IB) induces formation of mineralized bone matrix by 2T3 cells without addition of BMP-2. In contrast, overexpression of trBMPR-IA blocked adipocyte differentiation and expression of caBMPR-IA induces adipocyte formation in 2T3 cells. Expression of the adipocyte differentiation-related genes, adipsin and PPARgamma, correlates with the distinct phenotypic changes found after overexpression of the appropriate mutant receptors. These results demonstrate that type IB and IA BMP receptors transmit different signals to bone-derived mesenchymal progenitors and play critical roles in both the specification and differentiation of osteoblasts and adipocytes (Y. G.Chen, 1998).

A mouse insertional mutant was used to delineate gene activities that shape the distal limb skeleton. A recessive mutation that results in brachydactyly was found in a lineage of transgenic mice. Sequences flanking the transgene insertion site were cloned, mapped to chromosome 3, and used to identify the brachydactyly gene as the type [IB bone morphogenetic protein receptor, BmprIB (ALK6)]. Expression analyses in wild-type mice have revealed two major classes of BmprIB transcripts. Rather than representing unique coding RNAs generated by alternative splicing of a single pro-mRNA transcribed from one promoter, the distinct isoforms reflect evolution of two BmprIB promoters: one located distally, driving expression in the developing limb skeleton, and one situated proximally, initiating transcription in neural epithelium. The distal promoter is deleted in the insertional mutant, resulting in a regulatory allele (BmprIBTg) lacking cis-sequences necessary for limb BmprIB expression. Mutants fail to generate digit cartilage, indicating that BMPRIB is the physiologic transducer for the formation of digit cartilage from the skeletal blastema. Expansion of BmprIB expression into the limb through acquisition of these distal cis-regulatory sequences appears, therefore, to be an important genetic component driving morphological diversity in distal extremities. GDF5 is a BMP-related signal, which is also required for proper digit formation. Analyses incorporating both Gdf5 and BmprIBTg alleles has revealed that BMPRIB regulates chondrogenesis and segmentation through both GDF5-dependent and -independent processes, and that, reciprocally, GDF5 acts through both IB and other type I receptors. Together, these findings provide in vivo support for the concept of combinatorial BMP signaling, in which distinct outcomes result both from a single receptor being triggered by different ligands and from a single ligand binding to different receptors (Baur, 2000).

Specificity of BMP action is thought to be achieved in part by differential affinities of distinct ligands for one of four type I receptors: TSR-I (ALK1), ACTRI (ALK2), BMPRIA (ALK3) and BMPRIB (ALK6). BMPRIA and BMPRIB are more closely related to each other than to other type I BMP receptors, raising the possibility of functional redundancy. This possibility is supported by the fact that, although BmprIB has a more limited distribution and is not detected until about E9.5, it is coexpressed with BmprIA in many tissues of the developing mouse embryo. Moreover, BMPRIA, BMPRIB and ActRI activate the same downstream signaling components, Smad1 and Smad5. Mice carrying a targeted disruption of BmprIB were generated by homologous recombination in embryonic stem cells. BmprIB -/ - mice are viable and, in spite of the widespread expression of BMPRIB throughout the developing skeleton, exhibit defects that are largely restricted to the appendicular skeleton. BMPRIB is required for chondrogenesis in the limb. Using molecular markers, it is shown that the initial formation of the digital rays occurs normally in null mutants, but proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region are markedly reduced. These results suggest that BMPRIB-mediated signaling is required for cell proliferation after commitment to the chondrogenic lineage. Analyses of BmprIB and Gdf5 single mutants (GDF-5 is a member of the BMP family that has been implicated in several skeletogenic events including the induction and growth of the appendicular cartilages, the determination of joint forming regions, and the establishment of tendons) as well as BmprIB;Gdf5 double mutants suggests that GDF5 is a ligand for BMPRIB in vivo (Yi, 2000).

The possibility that GDF5 acts through BMPRIB in vivo has been investigated. BMP7 and GDF5 bind and activate BMPRIB in vitro and are expressed in the developing skeletal system. GDF5 binds to multiple receptors, but activates BMPRIB most efficiently in vitro. Gdf5 and BmprIB are expressed in adjacent tissues during limb development, suggesting that GDF5 may act in a paracrine manner as a ligand for BMPRIB in vivo. BmprIB;Bmp7 double mutants were constructed in order to examine whether BMPRIB has overlapping functions with other type I BMP receptors. BmprIB;Bmp7 double mutants exhibit severe appendicular skeletal defects, suggesting that BMPRIB and BMP7 act in distinct, but overlapping pathways. These results also demonstrate that in the absence of BMPRIB, BMP7 plays an essential role in appendicular skeletal development. Therefore, rather than having a unique role, BMPRIB has broadly overlapping functions with other BMP receptors during skeletal development (Yi, 2000).

Bone morphogenetic proteins (BMPs) have diverse and sometimes paradoxical effects during embryonic development. To determine the mechanisms underlying BMP actions, the expression and function of two BMP receptors, BMPR-IA and BMPR-IB, were analyzed in neural precursor cells in vitro and in vivo. Neural precursor cells always express Bmpr-1a, but Bmpr-1b is not expressed until embryonic day 9 and is restricted to the dorsal neural tube surrounding the source of BMP ligands. BMPR-IA activation induces (and Sonic hedgehog prevents) expression of Bmpr-1b along with dorsal identity genes in precursor cells and promotes their proliferation. When BMPR-IB is activated, it limits precursor cell numbers by causing mitotic arrest. This results in apoptosis in early gestation embryos and terminal differentiation in mid-gestation embryos. Thus, BMP actions are first inducing (through BMPR-IA) and then terminating (through BMPR-IB), based on the accumulation of BMPR-IB relative to BMPR-IA. A feed-forward mechanism is described to explain how the sequential actions of these receptors control the production and fate of dorsal precursor cells from neural stem cells. It is suggested that an early, unpatterned precursor responds to BMPs through the actions of BMPR-IA, which is expressed ubiquitously among precursor cells from gastrulation onward. Activation of BMPR-IA induces the expression of dorsal identity genes, including Bmpr-1b, and promotes the proliferation of this dorsalized cell. Since cell surface levels of accumulating BMPR-IB exceed those of BMPR-IA, BMPs then cause a termination response of cell death or cell differentiation. The in vitro results indicate that a single BMP ligand can drive this feed-forward, self-limiting process and suggest that the dose of BMPs determines the rate at which it occurs (Panchision, 2001).

Signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. A conditional knockout of the gene encoding BMPR-IA (Bmpr) was generated that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER is disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants results in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development (Ahn, 2001).

It is hypothesized that BMP signaling pathway mediates the initial induction of DV pattern in the presumptive limb ectoderm before limb bud formation. Bmp4 and Bmp7 are expressed in the lateral mesoderm and the overlying ectoderm just prior to the induction of limb bud formation, and therefore are correctly positioned in both time and space to induce ventral limb ectoderm. Furthermore, BMPR-IA signaling is required for the induction of En1, and subsequently the specification of ventral limb identity. Finally, the dorsalizing effect of somites, is hypothesized to be mediated by the expression of the BMP antagonist, noggin, in the myotomal compartment of the somite. Noggin expression in the somites could explain the ability of somites to dorsalize the ectoderm in transplantation experiments because of their ability to inhibit BMP signals from the lateral mesoderm. Noggin expressed in the somites could neutralize the effects of BMPs in ectoderm that is in close apposition to the somites. Alternatively, the somitic mesoderm could induce the expression of an unidentified BMP antagonist in dorsal ectoderm, which suppresses the BMP signaling within the dorsal ectoderm itself. Finally, the mesoderm-derived inductive signal would have to be downregulated as the lateral mesoderm loses its ability to induce the overlying ectoderm and as the dorsally fated ectoderm moves over the limb mesenchyme. The expression of Bmp4 and Bmp7 are, in fact, rapidly downregulated in most of the lateral mesenchyme as the limb bud is formed, although Bmp4 expression is maintained in the distal limb where BMP signaling is required for AER formation (Ahn, 2001).

The data demonstrate a crucial role for BMPR-IA signaling in the formation of the AER. It is conceivable that the AER defects are secondary to DV patterning defects. However, this seems unlikely because the penetrance of the DV patterning defect is complete, whereas the AER defect is quite variable. This difference in penetrance argues that AER formation and DV patterning are two independent processes. This argument is further bolstered by analyses of the eudiplopodia chick mutant that suggest that AER formation is not strictly dependent on establishment of a DV border at the distal tip of the limb. Classical studies have shown that lateral mesoderm induces the AER. As Bmp4 and Bmp7 are expressed in lateral mesoderm, they are candidates for the initial inductive event required for AER formation (Ahn, 2001).

In summary, the conditional knockout of the gene for the most widely expressed type I BMP receptor, BMPR-IA, results in limb malformations that are due to the disruption of AER formation and loss of DV patterning. This is the first demonstration that BMPR-IA signaling is essential for these early events in limb morphogenesis (Ahn, 2001).

Through the action of its membrane-bound type I receptors, TGF-ß elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of the signaling responses induced by TGF-ß are mediated by Smad proteins, but certain evidence has suggested that TGF-ß can also signal independently of Smads. In mouse mammary epithelial (NMuMG) cells, which respond to TGF-ß treatment in multiple ways, TGF-ß-induced activation of p38 MAP kinase is required for TGF-ß-induced apoptosis, epithelial-to-mesenchymal transition (EMT), but not growth arrest. Using a mutant type I receptor which is incapable of activating Smads but still retains the kinase activity, it was found that activation of p38 is independent of Smads. This mutant receptor is sufficient to activate p38 and cause NMuMG cells to undergo apoptosis. However, it is not sufficient to induce EMT. These results indicate that TGF-ß receptor signals through multiple intracellular pathways and provide first-hand biochemical evidence for the existence of Smad-independent TGF-ß receptor signaling (Yu, 2002).

BMPs have been proposed to pattern the medial-lateral axis of the telencephalon in a concentration-dependent manner, thus helping to subdivide the embryonic telencephalon into distinct forebrain regions. Using a CRE/loxP genetic approach, this hypothesis was tested by disrupting the Bmpr1alpha gene in the telencephalon. In mutants, BMP signaling is compromised throughout the dorsal telencephalon, but only the most dorsalmedial derivative, the choroid plexus, fails to be specified or differentiate. Choroid plexus precursors remain proliferative and do not adopt the fate of their lateral telencephalic neighbors. These results demonstrate that BMP signaling is required for the formation of the most dorsal telencephalic derivative, the choroid plexus, and that BMP signaling plays an essential role in locally patterning the dorsal midline. The data fail to support a more global, concentration-dependent role in specifying telencephalic cell fates (Hébert, 2002).

Bmp7 or Bmp4 genes are critical for early eye development. On the basis of the asymmetry in the dorsal-ventral expression patterns of several members of this family, it has been proposed that these molecules are critical for some aspect of dorsal-ventral patterning in the eye; however, it has been difficult to test this hypothesis because of the early requirement for BMPs in eye development. Therefore the effects of loss of one of the BMP receptors, BmprIb, on the development of the eye was studied by using targeted deletion. BmprIb is expressed exclusively in the ventral retina during embryonic development and is required for normal ventral ganglion cell axon targeting to the optic nerve head. In mice with a targeted deletion of the BmprIb gene, many axons arising from the ventrally located ganglion cells fail to enter the optic nerve head, and instead, make abrupt turns in this region. A second phenotype in these mice is a significantly elevated inner retinal apoptosis during a distinct phase of postnatal development, at the end of neurogenesis. These results therefore show two distinct requirements for BmprIb in mammalian retinal development (Liu, 2002).

By conditional gene ablation in mice, ß-catenin, an essential downstream effector of canonical Wnt signaling, was found to be a key regulator of formation of the apical ectodermal ridge (AER) and of the dorsal-ventral axis of the limbs. By generation of compound mutants, ß-catenin was also shown to act downstream of the BMP receptor IA in AER induction, but upstream or parallel to the BMP receptor in dorsal-ventral patterning. Thus, AER formation and dorsal-ventral patterning of limbs are tightly controlled by an intricate interplay between Wnt/ß-catenin and BMP receptor signaling (Soshnikova, 2003).

The Wnt/ß-catenin and TGFß/BMP-signaling pathways coordinately govern many developmental processes. During limb development, Wnt and BMP signals control the formation of the AER and participate in the establishment of the dorsal-ventral axis. The interactions between the two signaling systems in the limb were, however, not understood, and the epistatic relationship between BMP and Wnt signals remained unclear. This study has analyzed the interactions between Wnt/ß-catenin and BMP receptor signaling during limb development using conditional mutagenesis, which allows introduction of loss-of-function and gain-of-function mutations of ß-catenin, the central and essential mediator of canonical Wnt signaling. In addition, compound mutant mice were generated that carry both a gain-of-function mutation in ß-catenin and loss-of-function mutations in Bmp receptor IA. Analysis of these compound Brn4Cre;DeltaN-ß-catenin: BmpRIAflox/flox mutant mice clearly demonstrates that ß-catenin acts downstream of the BMP receptor IA in AER induction. ß-Catenin-mediated signals do, however, control Bmp4 expression in the ectoderm, and are thus responsible for the formation of a positive feedback loop. In contrast, the data suggest that ß-catenin acts upstream of or in parallel to the BMP receptor IA during dorsal-ventral patterning. These intricate interactions between the Wnt/ß-catenin and BMP-signaling pathways provide the molecular basis that connects the development of proximal-distal and dorsal-ventral axes in the limb, and might thus ensure a tight spatial-temporal control of signaling responses (Soshnikova, 2003).

Deletion of various bone morphogenetic proteins (BMPs) and their downstream Smads in mice have clearly shown that BMP signaling is essential for the formation of primordial germ cells (PGCs). However, the molecular mechanism through which this takes place is still unclear. BMP4 produced in the extraembryonic ectoderm signals through ALK2, a type I BMP receptor, in the visceral endoderm (VE) to induce formation of PGCs from the epiblast. Embryonic day 5.5-6.0 (E5.5-E6.0) embryos cultured on fibronectin formed PGCs in the presence of VE, but not in its absence. Alk2-deficient embryos completely lack PGCs and the heterozygotes have reduced numbers, resembling Bmp4-deficient phenotypes. Expression of constitutively active ALK2 in the VE, but not in the epiblast, is sufficient to rescue the PGC phenotype in Bmp4-deficient embryos. In addition, it was shown that the requirement for the VE at E5.5-E6.0 can be replaced by culturing embryos stripped of VE on STO (SIM mouse embryo-derived thioguanine and ouabain resistant feeder) cells, indicating that STO cells provide or transduce signals necessary for PGC formation that are normally transmitted by the VE. A model is proposed in which direct signaling to proximal epiblast is supplemented by an obligatory indirect BMP-dependent signal via the VE (de Sousa Lopes, 2004).

Although BMPs produced by the extraembryonic tissues are required for PGC formation, there is no evidence that BMPs signal directly or exclusively to the proximal epiblast cells to induce several of them to become PGCs. This study shows that there is an absolute requirement for ALK2 in the VE at E5.5-E6.0 for PGC formation to take place: absence of VE in explants (cultured embryos) or ALK2 in embryos results in the complete absence of PGCs, mimicking the Bmp4 mutant phenotype, whereas PGC formation in explants from Bmp4 mutant embryos can be rescued by constitutively active ALK2, but only in the presence of VE. Therefore, it is suggested that BMP4 signals through ALK2 in the VE to induce formation of PGCs from the epiblast. Furthermore, it is demonstrated that the reason for inconsistent reports in the past on whether or not VE is required for PGC formation is the consequence of different substrata used for explant cultures: STO feeder cells have the ability to induce PGCs to form in embryos stripped of VE at E5.5-E6.0, but in similar experiments using fibronectin substrata, no PGCs form, demonstrating an absolute requirement for VE at this stage (de Sousa Lopes, 2004).

Bone morphogenetic protein (BMP) signaling pathways are essential regulators of chondrogenesis. However, the roles of these pathways in vivo are not well understood. Limb-culture studies have provided a number of essential insights, including the demonstration that BMP pathways are required for chondrocyte proliferation and differentiation. However, limb-culture studies have yielded contradictory results; some studies indicate that BMPs exert stimulatory effects on differentiation, whereas others support inhibitory effects. Therefore, this study characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1aCKO) and Bmpr1aCKO;Bmpr1b+/- (Bmpr1aCKO;1b+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. They also demonstrate that the balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. These studies indicate that BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1. These results provide a genetic in vivo demonstration that the progression of chondrocytes through the growth plate is controlled by antagonistic BMP and FGF signaling pathways (Yoon, 2006).

BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions

The finding that morphogens, signalling molecules that specify cell identity, also act as axon guidance molecules has raised the possibility that the mechanisms that establish neural cell fate are also used to assemble neuronal circuits. It remains unresolved, however, how cells differentially transduce the cell fate specification and guidance activities of morphogens. To address this question, this study examined the mechanism by which the Bone morphogenetic proteins (BMPs) guide commissural axons in the developing spinal cord. In contrast to studies that have suggested that morphogens direct axon guidance decisions using non-canonical signal transduction factors, the results indicate that canonical components of the BMP signalling pathway, the type I BMP receptors (BMPRs), are both necessary and sufficient to specify the fate of commissural neurons and guide their axonal projections. However, whereas the induction of cell fate is a shared property of both type I BMPRs, axon guidance is chiefly mediated by only one of the type I BMPRs, BMPRIB. Taken together, these results indicate that the diverse activities of BMP morphogens can be accounted for by the differential use of distinct components of the canonical BMPR complex (Yamauchi, 2008).

BMP signaling regulates sympathetic nervous system development through Smad4-dependent and -independent pathways

Induction of the sympathetic nervous system (SNS) from its neural crest (NC) precursors is dependent on BMP signaling from the dorsal aorta. To determine the roles of BMP signaling and the pathways involved in SNS development, components of the BMP pathways were conditionally knocked out. To determine if BMP signaling is a cell-autonomous requirement of SNS development, the Alk3 (BMP receptor IA) was deleted in the NC lineage. The loss of Alk3 does not prevent NC cell migration, but the cells die immediately after reaching the dorsal aorta. The paired homeodomain factor Phox2b, known to be essential for survival of SNS precursors, is downregulated, suggesting that Phox2b is a target of BMP signaling. To determine if Alk3 signals through the canonical BMP pathway, Smad4 was deleted in the NC lineage. Loss of Smad4 does not affect neurogenesis and ganglia formation; however, proliferation and noradrenergic differentiation are reduced. Analysis of transcription factors regulating SNS development shows that the basic helix-loop-helix factor Ascl1 is downregulated by loss of Smad4 and that Ascl1 regulates SNS proliferation but not noradrenergic differentiation. To determine if the BMP-activated Tak1 (Map3k7) pathway plays a role in SNS development, Tak1 was deleted in the NC lineage. Tak1 was shown not to be involved in SNS development. Taken together, these results suggest multiple roles for BMP signaling during SNS development. The Smad4-independent pathway acts through the activation of Phox2b to regulate survival of SNS precursors, whereas the Smad4-dependent pathway controls noradrenergic differentiation and regulates proliferation by maintaining Ascl1 expression (Morikawa, 2009).

Type I TGF-beta and activin receptors

Continued: Evolutionary Homologs part 2/2

thickveins: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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