Essential role of Bmp signaling and its positive feedback loop in the early cell fate evolution of chordates

In chordates, early separation of cell fate domains occurs prior to the final specification of ectoderm to neural and non-neural as well as mesoderm to dorsal and ventral during development. Maintaining such division with the establishment of an exact border between the domains is required for the formation of highly differentiated structures such as neural tube and notochord. We hypothesized that the key condition for efficient cell fate separation in a chordate embryo is the presence of a positive feedback loop for Bmp signaling within the gene regulatory network (GRN), underlying early axial patterning. Here, we therefore investigated the role of Bmp signaling in axial cell fate determination in amphioxus, the basal chordate possessing a centralized nervous system. Pharmacological inhibition of Bmp signaling induces dorsalization of amphioxus embryos and expansion of neural plate markers, which is consistent with an ancestral role of Bmp signaling in chordate axial patterning and neural plate formation. Furthermore, we provided evidence for the presence of the positive feedback loop within the Bmp signaling network of amphioxus. Using mRNA microinjections we found that, in contrast to vertebrate Vent genes, which promote the expression of Bmp4, amphioxus Vent1 is likely not responsible for activation of cephalochordate ortholog Bmp2/4. Cis-regulatory analysis of amphioxus Bmp2/4, Admp and Chordin promoters in medaka embryos revealed remarkable conservation of the gene regulatory information between vertebrates and basal chordates. Our data suggest that emergence of a positive feedback loop within the Bmp signaling network may represent a key molecular event in the evolutionary history of the chordate cell fate determination.     

The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

Background  

Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse.     

Gdf6a is required for the initiation of dorsal–ventral retinal patterning and lens development

Dorsal–ventral patterning of the vertebrate retina is essential for accurate topographic mapping of retinal ganglion cell (RGC) axons to visual processing centers. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling and the relative roles of individual Bmps remain unclear. In this study, we investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity, and subsequently during lens differentiation. Knockdown of zebrafish Gdf6a blocks initiation of retinal Smad phosphorylation and dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity. We note a profound expansion of ventral retinal identity in gdf6a morphants, demonstrating that dorsal BMP signaling antagonizes ventral marker expression. Finally, we demonstrate a role for Gdf6a in non-neural ocular tissues. Knockdown of Gdf6a leads to defects in lens-specific gene expression, and when combined with Bmp signaling inhibitors, disrupts lens fiber cell differentiation. Taken together, these data indicate that Gdf6a initiates dorsal retinal patterning independent of Bmp4, and regulates lens differentiation.     

Wnt Signaling Interacts with Bmp and Edn1 to Regulate Dorsal-Ventral Patterning and Growth of the Craniofacial Skeleton

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.     

BMP and BMP receptor expression during murine organogenesis

Cell–cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell–cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.     

Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.     

Development and characterization of a mouse floxed <Emphasis Type="Italic">Bmp2</Emphasis> osteoblast cell line that retains osteoblast genotype and phenotype

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.     

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.     

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated.     

A Bmp Reporter with Ultrasensitive Characteristics Reveals That High Bmp Signaling Is Not Required for Cortical Hem Fate

Insights into Bone morphogenetic protein (Bmp) functions during forebrain development have been limited by a lack of Bmp signaling readouts. Here we used a novel Bmp signaling reporter (“BRE-gal” mice) to study Bmp signaling in the dorsal telencephalon. At early stages, BRE-gal expression was restricted to the dorsal telencephalic midline. At later stages, strong BRE-gal expression occurred in neurons of the marginal zone and dentate gyrus. Comparisons to nuclear phospho-Smad1/5/8 (pSmad) and Msx1 indicated that BRE-gal expression occurred exclusively in neural cells with high-level Bmp signaling. BRE-gal responsiveness to Bmps was confirmed in reporter-negative cortical cells cultured with Bmp4, and both in vivo and in vitro, BRE-gal expression was switch-like, or ultrasensitive. In the early dorsal telencephalon, BRE-gal expression negatively correlated with the cortical selector gene Lhx2, indicating a BRE-gal expression border that coincides with the cortex-hem boundary. However, in Lhx2 null chimeras, neither BRE-gal nor nuclear pSmad increases were observed in ectopic hem cells. These findings establish BRE-gal as an ultrasensitive reporter of Bmp signaling in the dorsal telencephalon, imply that hem fate can be specified at different Bmp signaling intensities, and suggest that Lhx2 primarily regulates the responses to – rather than the intensity of – Bmp signaling in dorsal telencephalic cells.     

Expression and Functional Study of Extracellular BMP Antagonists during the Morphogenesis of the Digits and Their Associated Connective Tissues

The purpose of this study is to gain insight into the role of BMP signaling in the diversification of the embryonic limb mesodermal progenitors destined to form cartilage, joints, and tendons. Given the importance of extracellular BMP modulators in in vivo systems, we performed a systematic search of those expressed in the developing autopod during the formation of the digits. Here, we monitored the expression of extracellular BMP modulators including: Noggin, Chordin, Chordin-like 1, Chordin-like 2, Twisted gastrulation, Dan, BMPER, Sost, Sostdc1, Follistatin, Follistatin-like 1, Follistatin-like 5 and Tolloid. These factors show differential expression domains in cartilage, joints and tendons. Furthermore, they are induced in specific temporal patterns during the formation of an ectopic extra digit, preceding the appearance of changes that are identifiable by conventional histology. The analysis of gene regulation, cell proliferation and cell death that are induced by these factors in high density cultures of digit progenitors provides evidence of functional specialization in the control of mesodermal differentiation but not in cell proliferation or apoptosis. We further show that the expression of these factors is differentially controlled by the distinct signaling pathways acting in the developing limb at the stages covered by this study. In addition, our results provide evidence suggesting that TWISTED GASTRULATION cooperates with CHORDINS, BMPER, and NOGGIN in the establishment of tendons or cartilage in a fashion that is dependent on the presence or absence of TOLLOID.     

Zili antagonizes Bmp signaling to regulate dorsal-ventral patterning during zebrafish early embryogenesis

Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-βsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.     

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.     

BMP4-BMPR1A signaling in beta cells is required for and augments glucose-stimulated insulin secretion

Impaired glucose-stimulated insulin secretion (GSIS) and perturbed proinsulin processing are hallmarks of beta cell dysfunction in type 2 diabetes. Signals that can preserve and/or enhance beta cell function are therefore of great therapeutic interest. Here we show that bone morphogenetic protein 4 (Bmp4) and its high-affinity receptor, Bmpr1a, are expressed in beta cells. Mice with attenuated BMPR1A signaling in beta cells show decreased expression of key genes involved in insulin gene expression, proinsulin processing, glucose sensing, secretion stimulus coupling, incretin signaling, and insulin exocytosis and develop diabetes due to impaired insulin secretion. We also show that transgenic expression of Bmp4 in beta cells enhances GSIS and glucose clearance and that systemic administration of BMP4 protein to adult mice significantly stimulates GSIS and ameliorates glucose tolerance in a mouse model of glucose intolerance. Thus, BMP4-BMPR1A signaling in beta cells plays a key role in GSIS.     

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.     

Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis

Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the anterior foregut where the tracheal primordium originates and targeted ablation of Bmp4 (Bmp4^cko) resulted in a loss-of-trachea phenotype that closely resembles the Floyd type II pathology, the most common form of TA in humans. In Bmp4^cko embryos, tracheal specification was not affected; however, its outgrowth was severely impaired due to reduced epithelial and mesenchymal proliferation. In agreement, we also observed significant reduction in the expression of Cyclin D1, a key cell cycle regulator associated with cellular proliferation. However, the proliferative effect of Bmp signaling appears to be independent of Wnt signaling. Interestingly, we found significantly reduced expression of activated extracellular signal-regulated kinase (Erk) in the Bmp4^cko ventral foregut, suggesting that Bmp signaling promotes Erk phosphorylation which has been associated with cellular proliferation. This study provides the first evidence linking Bmp signaling to tracheal formation by regulating the proliferative response of the anterior ventral foregut. Our finding sheds light on human tracheal malformations by providing a novel mouse model implicating Bmp signaling, non-canonical Erk activation and cellular proliferation.