Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos

Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2^−/− or Chd^−/− mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2^−/− mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2^−/− embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2^−/−vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.     

Twisted gastrulation mutation suppresses skeletal defect phenotypes in Crossveinless 2 mutant mice

Bone morphogenetic protein (BMP) signaling controls various aspects of organogenesis, including skeletal development. We previously demonstrated that the pro-BMP function of Crossveinless 2 (Cv2) is required for axial and non-axial skeletal development in mice. Here, we showed that skeletal defects in the Cv2-null mutant were reversed by the additional deletion of Twisted gastrulation (Tsg). Whereas the Cv2(-/-) mutant lacks a substantial portion of the lumbar vertebral arches, Cv2(-/-);Tsg(-/-) mice have almost normal arches. Suppression of Cv2(-/-) phenotypes is also seen in the non-axial skeleton, including the ribs, humerus, skull, and laryngeal and tracheal cartilages. In contrast, the Tsg(-/-) phenotype in the head is not significantly affected by the Cv2 mutation. These findings demonstrate that Tsg mutation is epistatic to Cv2 mutation in the major skeletal phenotypes, suggesting that the pro-BMP activity of Cv2 is, at least in part, dependent on Tsg. We also present genetic evidence for the context-dependent functional relationship between Tsg and Cv2 during mouse development.     

Cv2, functioning as a pro-BMP factor via twisted gastrulation, is required for early development of nephron precursors

The fine-tuning of BMP signals is critical for many aspects of complex organogenesis. In this report, we show that the augmentation of BMP signaling by a BMP-binding secreted factor, Crossveinless2 (Cv2), is essential for the early embryonic development of mammalian nephrons. In the Cv2-null mouse, the number of cap condensates (clusters of nephron progenitors, which normally express Cv2) was decreased, and the condensate cells exhibited a reduced level of aggregation. In these Cv2^-/- condensates, the level of phosphorylated Smad1 (pSmad1) was substantially lowered. The loss of a Bmp7 allele in the Cv2^-/- mouse enhanced the cap condensate defects and further decreased the level of pSmad1 in this tissue. These observations indicated that Cv2 has a pro-BMP function in early nephrogenesis. Interestingly, the renal defects of the Cv2^-/- mutant were totally suppressed by a null mutation of Twisted gastrulation (Tsg), which encodes another BMP-binding factor, showing that Cv2 exerts its pro-BMP nephrogenic function Tsg-dependently. By using an embryonic kidney cell line, we presented experimental evidence showing that Cv2 enhances pro-BMP activity of Tsg. These findings revealed the molecular hierarchy between extracellular modifiers that orchestrate local BMP signal peaks in the organogenetic microenvironment.     

The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing

In the early Drosophila embryo, Bone morphogenetic protein (BMP) activity is positively and negatively regulated by the BMP-binding proteins Short gastrulation (Sog) and Twisted gastrulation (Tsg). We show here that a similar mechanism operates during crossvein formation, utilizing Sog and a new member of the tsg gene family, encoded by the crossveinless (cv) locus. The initial specification of crossvein fate in the Drosophila wing requires signaling mediated by Dpp and Gbb, two members of the BMP family. cv is required for the promotion of BMP signaling in the crossveins. Large sog clones disrupt posterior crossvein formation, suggesting that Sog and Cv act together in this context. We demonstrate that sog and cv can have both positive and negative effects on BMP signaling in the wing. Moreover, Cv is functionally equivalent to Tsg, since Tsg and Cv can substitute for each other's activity. We also confirm that Tsg and Cv have similar biochemical activities: Sog/Cv complex binds a Dpp/Gbb heterodimer with high affinity. Taken together, these studies suggest that Sog and Cv promote BMP signaling by transporting a BMP heterodimer from the longitudinal veins into the crossvein regions.     

Dpp/BMP transport mechanism is required for wing venation in the sawfly Athalia rosae

The pattern of wing venation varies considerably among different groups of insects and has been used as a means of species-specific identification. However, little is known about how wing venation is established and diversified among insects. The decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling pathway plays a critical role in wing vein formation during the pupal stages in Drosophila melanogaster. A key mechanism is BMP transport from the longitudinal veins (LVs) to the posterior crossvein (PCV) by the BMP-binding proteins, short gastrulation (Sog) and twisted gastrulation2/crossveinless (Tsg2/Cv). To investigate whether the BMP transport mechanism is utilized to specify insect wing vein patterns in other than Drosophila, we used the sawfly Athalia rosae as a model, which has distinct venation patterns in the fore- and hindwings. Here, we show that Ar-dpp is ubiquitously expressed in both the fore- and hindwings, but is required for localized BMP signaling that reflects distinct wing vein patterns between the fore- and hindwings. By isolating Ar-tsg/cv in the sawfly, we found that Ar-Tsg/Cv is also required for BMP signaling in wing vein formation and retains the ability to transport Dpp. These data suggest that the BMP transport system is widely used to redistribute Dpp to specify wing venation and may be a basal mechanism underlying diversified wing vein patterns among insects.     

Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.     

crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling

The Twisted gastrulation (Tsg) proteins are modulators of bone morphogenetic protein (BMP) activity in both vertebrates and insects. We find that the crossveinless (cv) gene of Drosophila encodes a new tsg-like gene. Genetic experiments show that cv, similarly to tsg, interacts with short gastrulation (sog) to modulate BMP signalling. Despite this common property, Cv shows a different BMP ligand specificity as compared with Tsg, and its expression is limited to the developing wing. These findings and the presence of two types of Tsg-like protein in several insects suggest that Cv represents a subgroup of the Tsg-like BMP-modulating proteins.     

Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.     

Hedgehog-Gli Activators Direct Osteo-chondrogenic Function of Bone Morphogenetic Protein toward Osteogenesis in the Perichondrium

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1^−/− fetuses, and the phenotype was more severe in Gli1^−/−;Gli2^−/− newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.     

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.     

The Drosophila LEM-domain protein MAN1 antagonizes BMP signaling at the neuromuscular junction and the wing crossveins

BMP signaling responses are refined by distinct secreted and intracellular antagonists in different cellular and temporal contexts. Here, we show that the nuclear LEM-domain protein MAN1 is a tissue-specific antagonist of BMP signaling in Drosophila. MAN1 contains two potential Mad-binding sites. We generated MAN1^ΔC mutants, harbouring a MAN1 protein that lacks part of the C-terminus including the RNA recognition motif, a putative Mad-binding domain. MAN1^ΔC mutants show wing crossvein (CV) patterning defects but no detectable alterations in nuclear morphology. MAN1^ΔC pupal wings display expanded phospho-Mad (pMad) accumulation and ectopic expression of the BMP-responsive gene crossveinless-2 (cv-2) indicating that MAN1 restricts BMP signaling. Conversely, MAN1 overexpression in wing imaginal discs inhibited crossvein development and BMP signaling responses. MAN1 is expressed at high levels in pupal wing veins and can be activated in intervein regions by ectopic BMP signaling. The specific upregulation of MAN1 in pupal wing veins may thus represent a negative feedback circuit that limits BMP signaling during CV formation. MAN1^ΔC flies also show reduced locomotor activity, and electrophysiology recordings in MAN1^ΔC larvae uncover a new presynaptic role of MAN1 at the neuromuscular junction (NMJ). Genetic interaction experiments suggest that MAN1 is a BMP signaling antagonist both at the NMJ and during CV formation.     

von Willebrand factor type C domain-containing proteins regulate bone morphogenetic protein signaling through different recognition mechanisms

Bone morphogenetic protein (BMP) function is regulated in the extracellular space by many modulator proteins, including those containing a von Willebrand factor type C (VWC) domain. The function of the VWC domain-containing proteins in development and diseases has been extensively studied. The structural basis, however, for the mechanism by which BMP is regulated by these proteins is still poorly understood. By analyzing chordin, CHL2 (chordin-like 2), and CV2 (crossveinless 2) as well as their individual VWC domains, we show that the VWC domain is a versatile binding module that in its multiple forms and environments can expose a variety of binding specificities. Three of four, two of three, and one of five VWCs from chordin, CHL2, and CV2, respectively, can bind BMPs. Using an array of BMP-2 mutant proteins, it can be demonstrated that the binding-competent VWC domains all use a specific subset of BMP-2 binding determinants that overlap with the binding site for the type II receptors (knuckle epitope) or for the type I receptors (wrist epitope). This explains the competition between modulator proteins and receptors for BMP binding and therefore the inhibition of BMP signaling. A subset of VWC domains from CHL2 binds to the Tsg (twisted gastrulation) protein similar to chordin. A stable ternary complex consisting of BMP-2, CHL2, and Tsg can be formed, thus making CHL2 a more efficient BMP-2 inhibitor. The VWCs of CV2, however, do not interact with Tsg. The present results show that chordin, CHL2, and CV2 regulate BMP-2 signaling by different recognition mechanisms.     

Twisted gastrulation limits apoptosis in the distal region of the mandibular arch in mice

The mandibular arch (BA1) is critical for craniofacial development. The distal region of BA1, which gives rise to most of the mandible, is dependent upon an optimal level of bone morphogenetic protein (BMP) signaling. BMP activity is modulated in the extracellular space by BMP-binding proteins such as Twisted gastrulation (TWSG1). Twsg1^−/− mice have a spectrum of craniofacial phenotypes, including mandibular defects that range from micrognathia to agnathia. At E9.5, the distal region of the mutant BA1 was prematurely and variably fused with loss of distal markers eHand and Msx1. Expression of proximal markers Fgf8 and Barx1 was expanded across the fused BA1. The expression of Bmp4 and Msx2 was preserved in the distal region, but shifted ventrally. While wild type embryos showed a gradient of BMP signaling with higher activity in the distal region of BA1, this gradient was disrupted and shifted ventrally in the mutants. Thus, loss of TWSG1 results in disruption of the BMP4 gradient at the level of signaling activity as well as mRNA expression. Altered distribution of BMP signaling leads to a shift in gene expression and increase in apoptosis. The extent of apoptosis may account for the variable degree of mandibular defects in Twsg1 mutants.     

Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.     

Drosophila S6 Kinase Like Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv.     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.