Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2;Tsg and Cv2;Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.
BMP; Crossveinless-2; Chordin; Twisted Gastrulation; Tolloid; vertebra; morphogenetic field; cartilage; pattern formation
Dorsal-ventral patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional information gradient. The components involved include bone morphogenetic proteins (BMP/Dpp), a BMP antagonist (Chordin/Short gastrulation; Chd/Sog) and a secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. Here we describe Xenopus Twisted gastrulation (xTsg), another member of this signalling pathway. xTsg is expressed ventrally as part of the BMP-4 synexpression group and encodes a secreted BMP-binding protein that is a BMP signalling agonist. The data suggest a molecular mechanism by which xTsg dislodges latent BMPs bound to Chordin BMP-binding fragments generated by Xolloid cleavage, providing a permissive signal that allows high BMP signalling in the embryo. Drosophila Tsg also binds BMPs and is expressed dorsally, supporting the proposal that the dorsal-ventral axis was inverted in the course of animal evolution.
The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4−CD8− double-negative (DN) thymocytes and their differentiation to the CD4+CD8+ double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4+ or CD8+ single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.
BMP4; Twisted gastrulation; thymocyte development; chordin; morphogen
The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.
Twisted-gastrulation; BMP; Chordin; Tolloid; Crossveinless; TGFβ; Cell-cell signaling; Xenopus; Zebrafish; CR modules
Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: 1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; 2) CV2 complexes with Twisted gastrulation and BMP4; 3) CV2 is not a substrate for tolloid proteinases; 4) CV2 binds to purified Chordin protein with high affinity (KD in the 1 nM range); 5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; 6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.
The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.
Crossveinless-2; Chordin; Twisted gastrulation; Short gastrulation; BMP; Dpp; Tolloid; CR domain; von Willebrand factor type D domain; TIL domain; Extracellular matrix; Organogenesis; Somite; Sclerotome; Notochord; Neural crest; Dorsal root ganglion; Branchial arch; Cartilage; Vertebra; Intervertebral disc; Lung
The oral-aboral (OA) axis in the sea urchin is specified by the TGFβ family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In L. variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.
Chordin; BMP; p38 MAPK; Nodal; Neural; Axis
Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). We have analyzed the molecular mechanism by which Tsg regulates BMP signaling. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, as they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay revealed that Tsg has two distinct and sequential activities on BMP signaling. First, Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. Second, after cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated.
TGFβ; BMP; Chordin; Tolloid; Twisted gastrulation; Crossveinless; Xenopus
Twisted Gastrulation (Tsg) is a secreted molecule which regulates BMP signalling in the extracellular space as part of an evolutionarily conserved network of interacting proteins. In Xenopus, maternal xTsg mRNA can be found throughout the early embryo. After gastrulation, xTsg is expressed as part of the BMP4 synexpression group until late tadpole stages. Here we report the identification of a second Xenopus Tsg gene (xTsg-2). Xenopus Tsg-2 is highly homologous to xTsg. In particular, amino acid residues which have been shown to be required for the binding of xTsg to BMP and to Chordin are conserved. The expression of Xenopus Tsg-2 mRNA was restricted to late stages of embryonic development; it was detected at tadpole stages in lateral plate mesoderm, neural crest, branchial arches and head mesenchyme. In microinjection experiments, the activity of xTsg-2 mRNA was similar to that of xTsg. We conclude that two Tsg genes act in distinct temporal and spatial territories in the course of Xenopus embryonic development.
twisted gastrulation; BMP; chordin; tolloid; Xenopus
Bone morphogenetic protein (BMP) 1 is a vertebrate metalloproteinase of the astacin family. BMP1 plays a key role in regulating the formation of the extracellular matrix (ECM), particularly by processing the C-propeptide of fibrillar procollagens. BMP1 also promotes BMP signaling by releasing BMP signaling molecules from complexes with the BMP-antagonist chordin. As a result of BMP1′s dual role in both ECM formation and BMP signaling, we hypothesized that BMP1 could play a role in ovarian physiology. Using the sheep ovary as a model system, we showed that BMP1 was expressed in the ovary throughout early fetal stages to adulthood. Furthermore, in adult ovaries, BMP1 was expressed along with chordin, BMP4, and twisted gastrulation, which together form an extracellular regulatory complex for BMP signaling. Within ovine ovaries, immunohistochemical localization demonstrated that BMP1 was present in granulosa cells at all stages of follicular development, from primordial to large antral follicles, and that the levels of BMP1 were not affected by the final follicle selection mechanism. In cultured granulosa cells, BMP1 expression was not affected by gonadotropins, but BMP4 and activin A had opposing effects on the levels of BMP1 mRNA. BMP1 appeared to be secreted into the follicular fluid of antral follicles, where it is able to exert procollagen C-proteinase and chordinase activities. Interestingly, BMP1 activity in follicular fluid decreased with follicular growth.
Enzymatically active bone morphogenetic protein 1 (BMP1) is produced by granulosa cells and is present in the follicular fluid of ovine ovarian follicles.
BMP1; follicle; follicular development; granulosa cells; ovary
Bone Morphogenetic Proteins (BMPs) regulate dorsal/ventral (D/V) patterning across the animal kingdom; however, the biochemical properties of certain pathway components can vary according to species-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate BMP-binding proteins called Chordins constitutively, while the Drosophila Chordin ortholog, Short gastrulation (Sog), is only cleaved efficiently when bound to BMPs. We identified Sog characteristics responsible for making its cleavage dependent on BMP binding. “Chordin-like” variants that are processed independently of BMPs, changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change ultimately affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution appears to facilitate long-range ligand diffusion and formation of a robust morphogen gradient, enabling the bistable BMP signaling outputs required for early Drosophila patterning.
Chordin (Chd) is an abundant protein secreted by Spemann organizer tissue during gastrulation. Chd antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Recombinant Xenopus Chd binds to BMP-4 with high affinity (KD 3 × 10−10 M), binding specifically to BMPs but not to activin or TGF-β1. Chd protein is able to dorsalize mesoderm and to neuralize ectoderm in Xenopus gastrula explants at 1 nM. We propose that the noncell-autonomous effects of Spemann’s organizer on dorsoventral patterning are executed in part by diffusible signals that directly bind to and neutralize ventral BMPs during gastrulation.
Twisted gastrulation (TWSG1), an extracellular regulator of bone morphogenetic protein (BMP) signaling, is critical for embryonic brain development. Mice deficient in TWSG1 have abnormal forebrain development manifesting as holoprosencephaly. The expression and potential roles of TWSG1 in postnatal brain development are less well understood. We show that Twsg1 is expressed in the adult mouse brain in the choroid plexus (CP), hippocampus, and other regions, with the strongest expression observed in CP. TWSG1 was also detected in a human fetal brain at mid-gestation, with highest levels in the epithelium of CP. Bmp1, Bmp2, Bmp4-Bmp7 as well as BmprIA and BmprII, but not BmprIB, were expressed in CP. BMP antagonists Chordin (Chrd) and Noggin were not detected in CP, however Chrd-like 1 and brain-specific Chrd-like (Brorin) were expressed. Electrophysiological study of synaptic plasticity revealed normal paired-pulse facilitation and long-term potentiation in the CA1 region of hippocampus in Twsg1−/− mice. Among the homozygous mutants that survive beyond the first 2 weeks, the prevalence of hydrocephalus is 4.3%, compared to 1.5% in a wild type colony (p=0.0133) between 3 and 10 weeks of life. We detected a high level of BMP signaling in CP in wild type adult mice that was 17-fold higher than in the hippocampus (p=0.005). In contrast, TGFβ signaling was predominant in the hippocampus. Both BMP signaling and the expression of BMP downstream targets Msx1 and Msx2 were reduced in CP in Twsg1−/− mice. In summary, we show that Twsg1 is expressed in the adult mouse and human fetal CP. We also show that BMP is a branch of TGFβ superfamily that is dominant in CP. This presents an interesting avenue for future research in light of the novel roles of CP in neural progenitor differentiation and neuronal repair, especially since TWSG1 appears to be the main regulator of BMP present in CP.
Twsg1; chordin-like; hydrocephalus; cerebrospinal fluid; choroid plexus; BMP
This commentary is a concise discussion of the interactions between bone morphogenetic proteins (BMPs) and their binding proteins in bone and cartilage morphogenesis. BMPs are a family of growth and differentiation factors, and they act on mesenchymal cells to induce cartilage and bone differentiation in concentration-dependent thresholds. The BMP–BMP receptor binding leads to a cascade of signaling and transcription of BMP response genes. BMP binding proteins, noggin, chordin and DAN, act as antagonists and determine the bioavailability of BMPs for binding to cognate receptors to elicit the biological response. Noggin null mice with unrestricted action of BMPs exhibit defects in joint morphogenesis. BMPs and their binding proteins may reciprocally regulate the dynamic topography of joints, muscle, tendons and ligaments during morphogenesis of the skeleton. In addition, BMP actions may be potentiated by twisted gastrulation. BMPs and their binding proteins may play a critical role in regeneration of cartilage in osteoarthritis.
BMP; BMP receptors; extracellular matrix; Smads
BMP signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1–3], and its role in axial patterning apparently predates the birth of Bilateria [4–7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8–10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (Super-phylum Lophotrochozoa) featuring stereotyped developmental cell lineages; but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, while a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is up-regulated by BMP5-8 in dorsolateral territory, rather than ventral, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the ‘conserved’ BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.
Crystals of the complex of the first von Willebrand type C domain (VWC1) of crossveinless 2 (CV2) bound to bone morphogenetic protein 2 (BMP2) exist in two tetragonal crystal forms belonging to either space group P41212 or I41, with one complete BMP2 dimer and two CV2 VWC1 domains per asymmetric unit, and diffract to 2.6 Å resolution.
Crossveinless 2 (CV2) is a member of the chordin family, a protein superfamily that modulates the activity of bone morphogenetic proteins such as BMP2. The BMPs represent a large group of secreted proteins that control many steps during embryonal development and in tissue and organ homeostasis in the adult organism. The gene encoding the first von Willebrand type C domain (VWC1) of CV2 was cloned, expressed in Escherichia coli and purified to homogeneity. The binary complex of CV2 VWC1 and BMP2 was purified and subjected to crystallization. Crystals of SeMet-labelled proteins were obtained in two different forms belonging to the tetragonal space groups P41212 and I41, with unit-cell parameters a = b = 86.7, c = 139.2 Å and a = b = 83.7, c = 139.6 Å, respectively. Initial analysis suggests that a complete binary complex consisting of one BMP2 dimer bound to two CV2 VWC1 domains is present in the asymmetric unit.
TGF-β superfamily; chordin; BMP modulators; BMP inhibitors
Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.
Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl ┤ Xlr ┤ Chd ┤ BMP → P-Smad1 → Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10−8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.
The transforming growth factor β (TGF-β) superfamily, including the bone morphogenetic protein (BMP) and TGF-β/activin A subfamilies, is regulated by secreted proteins able to sequester or present ligands to receptors. KCP is a secreted, cysteine-rich (CR) protein with similarity to mouse Chordin and Xenopus laevis Kielin. KCP is an enhancer of BMP signaling in vertebrates and interacts with BMPs and the BMP type I receptor to promote receptor-ligand interactions. Mice homozygous for a KCP null allele are hypersensitive to developing renal interstitial fibrosis, a disease stimulated by TGF-β but inhibited by BMP7. In this report, the effects of KCP on TGF-β/activin A signaling are examined. In contrast to the enhancing effect on BMPs, KCP inhibits both activin A- and TGF-β1-mediated signaling through the Smad2/3 pathway. These inhibitory effects of KCP are mediated in a paracrine manner, suggesting that direct binding of KCP to TGF-β1 or activin A can block the interactions with prospective receptors. Consistent with this inhibitory effect, primary renal epithelial cells from KCP mutant cells are hypersensitive to TGF-β and exhibit increased apoptosis, dissociation of cadherin-based cell junctions, and expression of smooth muscle actin. Furthermore, KCP null animals show elevated levels of phosphorylated Smad2 after renal injury. The ability to enhance BMP signaling while suppressing TGF-β activation indicates a critical role for KCP in modulating the responses between these anti- and profibrotic cytokines in the initiation and progression of renal interstitial fibrosis.
Embryos and developing organs have the remarkable ability of self-regenerating after experimental manipulations. In the Xenopus blastula half-embryos can regenerate the missing part, producing identical twins. Studies on the molecular nature of Spemann’s organizer have revealed that self-regulation results from the battle between two signaling centers under reciprocal transcriptional control. Long-range communication between the dorsal and ventral sides is mediated by the action of growth factor antagonists – such as the BMP antagonist Chordin – that regulate the flow of BMPs within the embryonic morphogenetic field. BMPs secreted by the dorsal Spemann organizer tissue are released by metalloproteinases of the Tolloid family, which cleave Chordin at a distance of where they were produced. The dorsal center secretes Chordin, Noggin, BMP2 and ADMP. The ventral center of the embryo secretes BMP4, BMP7, Sizzled, Crossveinless-2 and Tolloid-related. Crossveinless-2 binds Chordin/BMP complexes, facilitating their flow towards the ventral side, where BMPs are released by Tolloid allowing peak BMP signaling. Self-regulation occurs because transcription of ventral genes is induced by BMP while transcription of dorsal genes is repressed by BMP signals. This assures that for each action of Spemann’s organizer there is a reaction in the ventral side of the embryo. Because both dorsal and ventral centers express proteins of similar biochemical activities, they can compensate for each other. A novel biochemical pathway of extracellular growth factor signaling regulation has emerged from these studies in Xenopus. This remarkable dorsal-ventral positional information network has been conserved in evolution and is ancestral to all bilateral animals.
Morphogenetic fields; Embryonic induction; Dorsal-Ventral patterning; BMP; Chordin; Crossveinless-2; Tolloid; Sizzled; Hox genes; Urbilateria
The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.
We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b−/−) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.
Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.
The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites in biochemical experiments. Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein was unable to antagonize BMP activity. Furthermore, Xolloid digestion released biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicated that the in vivo function of Xolloid is to limit the extent of Spemann’s organizer field. We propose that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis:
Transforming growth factor β1 (TGFβ1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFβ-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)–like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFβ1 via cleavage of LAP by non–BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFβ1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFβ1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.
The morphogenetic field concept was proposed by experimental embryologists to account for the self-regulative behavior of embryos. Such fields have remained an abstract concept until the recent identification of their molecular components using a combination of genetics, biochemistry, and theoretical modeling. One of the best studied models of a morphogenetic field is the Dorsal-Ventral (D-V) patterning of the early frog embryo. This patterning system is regulated by the bone morphogenetic protein (BMP) signaling pathway and an intricate network of secreted protein antagonists. This biochemical pathway of interacting proteins functions in the extracellular space to generate a D-V gradient of BMP signaling, which is maintained during extensive morphogenetic movements of cell layers during gastrulation. The D-V field is divided into a dorsal and a ventral center, in regions of low and high BMP signaling respectively, under opposite transcriptional control by BMPs. The robustness of the patterning is assured at two different levels. First, in the extracellular space by secreted BMP antagonists that generate a directional flow of BMP ligands to the ventral side. The flow is driven by the regulated proteolysis of the Chordin inhibitor and by the presence of a molecular sink on the ventral side that concentrates BMP signals. The tolloid metalloproteinases and the Chordin-binding protein Crossveinless-2 (CV2) are key components of this ventral sink. Second, by transcriptional feedback at the cellular level: The dorsal and ventral signaling centers adjust their size and level of BMP signaling by transcriptional feedback. This allows cells on one side of a gastrula containing about 10,000 cells to communicate with cells in the opposite pole of the embryo.
A network of BMP ligands and antagonists regulates embryonic patterning in frogs. Proteolysis of inhibitors, a molecular sink, and transcriptional feedback loops ensure its robustness.
Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.
Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.
We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.
We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.