Differential requirement for BMP signaling in atrial and ventricular lineages establishes cardiac chamber proportionality

The function of an organ relies upon the proper relative proportions of its individual operational components. For example, effective embryonic circulation requires the appropriate relative sizes of each of the distinct pumps created by the atrial and ventricular cardiac chambers. Although the differences between atrial and ventricular cardiomyocytes are well established, little is known about the mechanisms regulating production of proportional numbers of each cell type. We find that mutation of the zebrafish type I BMP receptor gene alk8 causes reduction of atrial size without affecting the ventricle. Loss of atrial tissue is evident in the lateral mesoderm prior to heart tube formation and results from the inhibition of BMP signaling during cardiac progenitor specification stages. Comparison of the effects of decreased and increased BMP signaling further demonstrates that atrial cardiomyocyte production correlates with levels of BMP signaling while ventricular cardiomyocyte production is less susceptible to manipulation of BMP signaling. Additionally, mosaic analysis provides evidence for a cell-autonomous requirement for BMP signaling during cardiomyocyte formation and chamber fate assignment. Together, our studies uncover a new role for BMP signaling in the regulation of chamber size, supporting a model in which differential reception of cardiac inductive signals establishes chamber proportion.     

Alk3/Alk3b and Smad5 Mediate BMP Signaling during Lymphatic Development in Zebrafish

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.     

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.     

GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses

Within the TGF-β superfamily, there are approximately forty ligands divided into two major branches: the TGF-β/Activin/Nodal ligands and the BMP/GDF ligands. We studied the ligand GDF3 and found that it inhibits signaling by its co-family members, the BMPs; however, GDF3 has been described by others to have Nodal-like activity. Here, we show that GDF3 can activate Nodal signaling, but only at very high doses and only upon mRNA over-expression. In contrast, GDF3 inhibits BMP signaling upon over-expression of GDF3 mRNA, as recombinant protein, and regardless of its dose. We therefore further characterized the mechanism through which GDF3 protein acts as a specific BMP inhibitor and found that the BMP inhibitory activity of GDF3 resides redundantly in the unprocessed, predominant form and in the mature form of the protein. These results confirm and extend the activity that we described for GDF3 and illuminate the experimental basis for the different observations of others. We suggest that GDF3 is either a bi-functional TGF-β ligand, or, more likely, that it is a BMP inhibitor that can artificially activate Nodal signaling under non-physiological conditions.     

CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways

We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.     

HLA-B-associated transcript 3 (Bat3/Scythe) negatively regulates Smad phosphorylation in BMP signaling

Members of the transforming growth factor-β (TGF-β) superfamily participate in numerous biological phenomena in multiple tissues, including in cell proliferation, differentiation, and migration. TGF-β superfamily proteins therefore have prominent roles in wound healing, fibrosis, bone formation, and carcinogenesis. However, the molecular mechanisms regulating these signaling pathways are not fully understood. Here, we describe the regulation of bone morphogenic protein (BMP) signaling by Bat3 (also known as Scythe or BAG6). Bat3 overexpression in murine cell lines suppresses the activity of the Id1 promoter normally induced by BMP signaling. Conversely, Bat3 inactivation enhances the induction of direct BMP target genes, such as Id1, Smad6, and Smad7. Consequently, Bat3 deficiency accelerates the differentiation of primary osteoblasts into bone, with a concomitant increase in the bone differentiation markers Runx2, Osterix, and alkaline phosphatase. Using biochemical and cell biological analyses, we show that Bat3 inactivation sustains the C-terminal phosphorylation and nuclear localization of Smad1, 5, and 8 (Smad1/5/8), thereby enhancing biological responses to BMP treatment. At the mechanistic level, we show that Bat3 interacts with the nuclear phosphatase small C-terminal domain phosphatase (SCP) 2, which terminates BMP signaling by dephosphorylating Smad1/5/8. Notably, Bat3 enhances SCP2–Smad1 interaction only when the BMP signaling pathway is activated. Our results demonstrate that Bat3 is an important regulator of BMP signaling that functions by modulating SCP2–Smad interaction.     

Gbb/BMP signaling is required to maintain energy homeostasis in Drosophila

The coordination of animal growth and development requires adequate nutrients. During times of insufficient food, developmental progression is slowed and stored energy is utilized to ensure that cell and tissue survival are maintained. Here, we report our finding that the Gbb/BMP signaling pathway, known to play an important role in many developmental processes in both vertebrates and invertebrates, is critical in the Drosophila larval fat body for regulating energy homeostasis. Animals with mutations in the Drosophila BMP-5,7 orthologue, glass bottom boat (gbb), or in its signaling components, display phenotypes similar to nutrient-deprived and Tor mutant larvae. These phenotypes include a developmental delay with reduced overall growth, a transparent appearance, and altered total lipid, glucose and trehalose levels. We find that Gbb/BMP signaling is required in the larval fat body for maintaining proper metabolism, yet interestingly, following nutrient deprivation larvae in turn show a loss of BMP signaling in fat body cells indicating that Gbb/BMP signaling is a central player in homeostasis. Finally, despite strong phenotypic similarities between nutrient-compromised animals and gbb mutants, distinct differences are observed in the expression of a group of starvation responsive genes. Overall, our results implicate Gbb/BMP signaling as a new pathway critical for positive regulation of nutrient storage and energy homeostasis during development.     

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

NDRG3-mediated lactate signaling in hypoxia

Hypoxia is associated with many pathological conditions as well as the normal physiology of metazoans. We identified a lactate-dependent signaling pathway in hypoxia, mediated by the oxygen- and lactate-regulated protein NDRG family member 3 (NDRG3). Oxygen negatively regulates NDRG3 expression at the protein level via the PHD2/VHL system, whereas lactate, produced in excess under prolonged hypoxia, blocks its proteasomal degradation by binding to NDRG3. We also found that the stabilized NDRG3 protein promotes angiogenesis and cell growth under hypoxia by activating the Raf-ERK pathway. Inhibiting cellular lactate production abolishes NDRG3-mediated hypoxia responses. The NDRG3-Raf-ERK axis therefore provides the genetic basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases in addition to advancing our understanding of the normal physiology of hypoxia responses. [BMB Reports 2015; 48(6): 301-302]     

Two distinct domains in pro-region of Nodal-related 3 are essential for BMP inhibition

The transforming growth factor-beta (TGF-beta) superfamily member, Xenopus nodal-related 3 (Xnr3), induces neural tissues through inhibition of bone morphogenetic proteins (BMPs). We recently identified an inhibitory mechanism in which the pro-region of Xenopus tropicalis nodal-related 3 (Xtnr3) physically interacts with BMP ligands. Here, we show that disulfide-linked heterodimerization does not contribute to BMP inhibition by Xtnr3 and that the Xtnr3 mature region, overexpression of which can induce the same phenotype as full-length Xtnr3, does not inhibit BMP signaling. Furthermore, we find that the BMP-inhibitory domains of Xtnr3 are separately located in the N- and C-terminal regions of the pro-region. These results indicate the pro-region of Nodal-related 3 is both necessary and sufficient for its BMP inhibition.     

Evidence for an early role for BMP4 signaling in thymus and parathyroid morphogenesis

The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene expression and functional analyses have suggested a role for BMP4 signaling in early thymus organogenesis. We have used conditional deletion of Bmp4 or Alk3 from the pharyngeal endoderm and/or the surrounding mesenchyme using Foxg1-Cre, Wnt1-Cre or Foxn1-Cre. Deleting Bmp4 from both neural crest cells (NCC) and early endoderm-derived epithelial cells in Foxg1-Cre;Bmp4 conditional mutants resulted in defects in thymus-parathyroid morphogenesis. Defects included reduced condensation of mesenchymal cells around the epithelium, partial absence of the thymic capsule, a delay in thymus and parathyroid separation, and failed or dramatically reduced organ migration. Patterning of the primordia and initial organ differentiation were not affected in any of the mutants. Deleting Bmp4 from NCC-derived mesenchyme or differentiating thymic epithelial cells (TECs) had no effects on thymus-parathyroid development, while loss of Alk3 from either neural crest cells or TECs resulted in only a mild thymic hypoplasia. these results show that the processes of cell specification and morphogenesis during thymus-parathyroid development are independently controlled, and suggest a specific temporal and spatial role for BMP4-mediated epithelial-mesenchymal interactions during early thymus and parathyroid morphogenesis.     

The effects of clozapine on the GSK-3-mediated signaling pathway

We investigated the effect of 10 μM clozapine on the activity of glycogen synthase kinase-3β (GSK-3β) and its upstream and downstream molecules in SH-SY5Y human neuroblastoma cells. Clozapine activates both Akt- and Dvl-mediated phosphorylation of GSK-3β through phosphorylation at Ser9, and increased total cellular and intranuclear levels of β-catenin. Pretreatment with the specific inhibitor of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, LY294002 (20 μM), prevented the phosphorylation of Akt but did not affect the phosphorylation of GSK-3β. These results suggest that clozapine regulates the phosphorylation of GSK-3β through Wnt signal pathways involving Dvl upstream but not through the PI3K-Akt pathway in SH-SY5Y cells.     

TGF-β type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-β superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-β type II receptor appears to bind only TGF-β, whereas TGF-β type I receptor (ALK5) also binds to ligands in addition to TGF-β. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-β type II receptor (Wnt1-Cre;Tgfbr2^fl/fl) and TGF-β type I receptor/Alk5 (Wnt1-Cre;Alk5^fl/fl) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Ectodermal Wnt signaling regulates abdominal myogenesis during ventral body wall development

Defects of the ventral body wall are prevalent birth anomalies marked by deficiencies in body wall closure, hypoplasia of the abdominal musculature and multiple malformations across a gamut of organs. However, the mechanisms underlying ventral body wall defects remain elusive. Here, we investigated the role of Wnt signaling in ventral body wall development by inactivating Wls or β-catenin in murine abdominal ectoderm. The loss of Wls in the ventral epithelium, which blocks the secretion of Wnt proteins, resulted in dysgenesis of ventral musculature and genito-urinary tract during embryonic development. Molecular analyses revealed that the dermis and myogenic differentiation in the underlying mesenchymal progenitor cells was perturbed by the loss of ectodermal Wls. The activity of the Wnt-Pitx2 axis was impaired in the ventral mesenchyme of the mutant body wall, which partially accounted for the defects in ventral musculature formation. In contrast, epithelial depletion of β-catenin or Wnt5a did not resemble the body wall defects in the ectodermal Wls mutant. These findings indicate that ectodermal Wnt signaling instructs the underlying mesodermal specification and abdominal musculature formation during ventral body wall development, adding evidence to the theory that ectoderm-mesenchyme signaling is a potential unifying mechanism for the origin of ventral body wall defects.