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.     

BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions

Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER^-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER^-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER^-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.     

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.     

The pro-BMP activity of Twisted gastrulation is independent of BMP binding

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.     

Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted Gastrulation

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.     

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.     

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.     

An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.     

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.     

Targeted Inactivation of Cerberus Like-2 Leads to Left Ventricular Cardiac Hyperplasia and Systolic Dysfunction in the Mouse

Previous analysis of the Cerberus like 2 knockout (Cerl2^−/−) mouse revealed a significant mortality during the first day after birth, mostly due to cardiac defects apparently associated with randomization of the left-right axis. We have however, identified Cerl2-associated cardiac defects, particularly a large increase in the left ventricular myocardial wall in neonates that cannot be explained by laterality abnormalities. Therefore, in order to access the endogenous role of Cerl2 in cardiogenesis, we analyzed the embryonic and neonatal hearts of Cerl2 null mutants that did not display a laterality phenotype. Neonatal mutants obtained from the compound mouse line Cer2^−/−::Mlc1v-nLacZ24⁺, in which the pulmonary ventricle is genetically marked, revealed a massive enlargement of the ventricular myocardium in animals without laterality defects. Echocardiography analysis in Cerl2^−/− neonates showed a left ventricular systolic dysfunction that is incompatible with a long lifespan. We uncovered that the increased ventricular muscle observed in Cerl2^−/− mice is caused by a high cardiomyocyte mitotic index in the compact myocardium which is mainly associated with increased Ccnd1 expression levels in the left ventricle at embryonic day (E) 13. Interestingly, at this stage we found augmented left ventricular expression of Cerl2 levels when compared with the right ventricle, which may elucidate the regionalized contribution of Cerl2 to the left ventricular muscle formation. Importantly, we observed an increase of phosphorylated Smad2 (pSmad2) levels in embryonic (E13) and neonatal hearts indicating a prolonged TGFβs/Nodal-signaling activation. Concomitantly, we detected an increase of Baf60c levels, but only in Cerl2^−/− embryonic hearts. These results indicate that independently of its well-known role in left-right axis establishment Cerl2 plays an important role during heart development in the mouse, mediating Baf60c levels by exerting an important control of the TGFβs/Nodal-signaling pathway.     

BMPER-induced BMP signaling promotes coronary artery remodeling

The connection of the coronary vasculature to the aorta is one of the last essential steps of cardiac development. However, little is known about the signaling events that promote normal coronary artery formation. The bone morphogenetic protein (BMP) signaling pathway regulates multiple aspects of endothelial cell biology but has not been specifically implicated in coronary vascular development. BMP signaling is tightly regulated by numerous factors, including BMP-binding endothelial cell precursor-derived regulator (BMPER), which can both promote and repress BMP signaling activity. In the embryonic heart, BMPER expression is limited to the endothelial cells and the endothelial-derived cushions, suggesting that BMPER may play a role in coronary vascular development. Histological analysis of BMPER^−/− embryos at early embryonic stages demonstrates that commencement of coronary plexus differentiation is normal and that endothelial apoptosis and cell proliferation are unaffected in BMPER^−/− embryos compared with wild-type embryos. However, analysis between embryonic days 15.5–17.5 reveals that, in BMPER^−/− embryos, coronary arteries are either atretic or connected distal to the semilunar valves. In vitro tubulogenesis assays indicate that isolated BMPER^−/− endothelial cells have impaired tube formation and migratory ability compared with wild-type endothelial cells, suggesting that these defects may lead to the observed coronary artery anomalies seen in BMPER^−/− embryos. Additionally, recombinant BMPER promotes wild-type ventricular endothelial migration in a dose-dependent manner, with a low concentration promoting and high concentrations inhibiting migration. Together, these results indicate that BMPER-regulated BMP signaling is critical for coronary plexus remodeling and normal coronary artery development.     

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.     

Increased Sensitivity of DNA Damage Response-Deficient Cells to Stimulated Microgravity-Induced DNA Lesions

Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9 ^-/- MES and Mdc1 ^-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9 ^-/- MES. As the exposure to SMG was prolonged, Rad9 ^-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9 ^-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1 ^-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1 ^-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects.     

The Short Stature Homeobox 2 (Shox2)-bone Morphogenetic Protein (BMP) Pathway Regulates Dorsal Mesenchymal Protrusion Development and Its Temporary Function as a Pacemaker during Cardiogenesis

The atrioventricular (AV) junction plays a critical role in chamber septation and transmission of cardiac conduction pulses. It consists of structures that develop from embryonic dorsal mesenchymal protrusion (DMP) and the embryonic AV canal. Despite extensive studies on AV junction development, the genetic regulation of DMP development remains poorly understood. In this study we present evidence that Shox2 is expressed in the developing DMP. Intriguingly, this Shox2-expressing domain possesses a pacemaker-specific genetic profile including Hcn4 and Tbx3. This genetic profile leads to nodal-like electrophysiological properties, which is gradually silenced as the AV node becomes matured. Phenotypic analyses of Shox2^−/− mice revealed a hypoplastic and defectively differentiated DMP, likely attributed to increased apoptosis, accompanied by dramatically reduced expression of Bmp4 and Hcn4, ectopic activation of Cx40, and an aberrant pattern of action potentials. Interestingly, conditional deletion of Bmp4 or inhibition of BMP signaling by overexpression of Noggin using a Shox2-Cre allele led to a similar DMP hypoplasia and down-regulation of Hcn4, whereas activation of a transgenic Bmp4 allele in Shox2^−/− background attenuated DMP defects. Moreover, the lack of Hcn4 expression in the DMP of mice carrying Smad4 conditional deletion and direct binding of pSmad1/5/8 to the Hcn4 regulatory region further confirm the Shox2-BMP genetic cascade in the regulation of DMP development. Our results reveal that Shox2 regulates DMP fate and development by controlling BMP signaling through the Smad-dependent pathway to drive tissue growth and to induce Hcn4 expression and suggest a temporal pacemaking function for the DMP during early cardiogenesis.