Endogenous Bone Morphogenetic Protein Signaling Research Articles

BMP4 signaling plays critical roles in self-renewal of R2i mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state under R2i culture conditions that inhibit the TGF-β and ERK signaling pathways. BMP4 is another member of the TGF-β family that plays a crucial role in maintaining the pluripotency state of mESCs. It has been reported that inhibition of BMP4 caused the death of R2i-grown cells. In this study, we used the loss-of-function approach to investigate the role of BMP4 signaling in mESC self-renewal. Inhibition of this pathway with Noggin and dorsomorphin, two bone morphogenetic protein (BMP) antagonists, elicited a quick death of the R2i-grown cells. We showed that the canonical pathway of BMP4 (BMP/SMAD) was dispensable for self-renewal and maintaining pluripotency of these cells. Transcriptome analysis of the BMPi-treated cells revealed that the p53 signaling and two adhesion (AD) and apoptotic mitochondrial change (MT) pathways could be involved in the cell death of the BMPi-treated cells. According to our results, inhibition of BMP4 signaling caused a decrease in cell adhesion and ECM detachment, which triggered anoikis in the R2i-grown cells. Altogether, these findings demonstrate that endogenous BMP signaling is required for the survival of mESCs under the R2i condition.

Phenotypic screen identifies calcineurin-sparing FK506 analogs as BMP potentiators for treatment of acute kidney injury

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile invivo relative to FK506. OxtFK stimulated BMP signaling invitro and invivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.

Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial-Like Cells.

Cornea is a clear outermost layer of the eye which enables transmission of light onto the retina. The transparent corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in LSCs deficiency (LSCD). Ex vivo expansion of autologous LSCs obtained from patient's healthy eye followed by transplantation onto the LSCs damaged/deficient eye, has provided a successful treatment for unilateral LSCD. However, this is not applicable to patient with total bilateral LSCD, where LSCs are lost/damaged from both eyes. We investigated the potential of human induced pluripotent stem cell (hiPSC) to differentiate into corneal epithelial‐like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD. Our study showed that combined addition of bone morphogenetic protein 4 (BMP4), all trans‐retinoic acid and epidermal growth factor for the first 9 days of differentiation followed by cell‐replating on collagen‐IV‐coated surfaces with a corneal‐specific‐epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESC) to corneal epithelial progenitors and mature corneal epithelial‐like cells. We observed differences in the ability of hiPSC lines to undergo differentiation to corneal epithelial‐like cells which were dependent on the level of endogenous BMP signaling and could be restored via the activation of this signaling pathway by a specific transforming growth factor β inhibitor (SB431542). Together our data reveal a differential ability of hiPSC lines to generate corneal epithelial cells which is underlined by the activity of endogenous BMP signaling pathway. Stem Cells 2018;36:337–348

BMP signaling is essential in neonatal surfactant production during respiratory adaptation.

Deficiency in pulmonary surfactant results in neonatal respiratory distress, and the known genetic mutations in key components of surfactant only account for a small number of cases. Therefore, determining the regulatory mechanisms of surfactant production and secretion, particularly during the transition from prenatal to neonatal stages, is essential for better understanding of the pathogenesis of human neonatal respiratory distress. We have observed significant increase of bone morphogenetic protein (BMP) signaling in neonatal mouse lungs immediately after birth. Using genetically manipulated mice, we then studied the relationship between BMP signaling and surfactant production in neonates. Blockade of endogenous BMP signaling by deleting Bmpr1a (Alk3) or Smad1 in embryonic day 18.5 in perinatal lung epithelial cells resulted in severe neonatal respiratory distress and death, accompanied by atelectasis in histopathology and significant reductions of surfactant protein B and C, as well as Abca3, whereas prenatal lung development was not significantly affected. We then identified a new BMP-Smad1 downstream target, Nfatc3, which is known as an important transcription activator for surfactant proteins and Abca3. Furthermore, activation of BMP signaling in cultured lung epithelial cells was able to promote endogenous Nfatc3 expression and also stimulate the activity of an Nfatc3 promoter that contains a Smad1-binding site. Therefore, our study suggests that the BMP-Alk3-Smad1-Nfatc3 regulatory loop plays an important role in enhancing surfactant production in neonates, possibly helping neonatal respiratory adaptation from prenatal amniotic fluid environment to neonatal air breathing.

Spatially Resolved Genome-wide Transcriptional Profiling Identifies BMP Signaling as Essential Regulator of Zebrafish Cardiomyocyte Regeneration

In contrast to mammals, zebrafish regenerate heart injuries via proliferation of cardiomyocytes located near the wound border. To identify regulators of cardiomyocyte proliferation, we used spatially resolved RNA sequencing (tomo-seq) and generated a high-resolution genome-wide atlas of gene expression in the regenerating zebrafish heart. Interestingly, we identified two wound border zones with distinct expression profiles, including the re-expression of embryonic cardiac genes and targets of bone morphogenetic protein (BMP) signaling. Endogenous BMP signaling has been reported to be detrimental to mammalian cardiac repair. In contrast, we find that genetic or chemical inhibition of BMP signaling in zebrafish reduces cardiomyocyte dedifferentiation and proliferation, ultimately compromising myocardial regeneration, while bmp2b overexpression is sufficient to enhance it. Our results provide a resource for further studies on the molecular regulation of cardiac regeneration and reveal intriguing differential cellular responses of cardiomyocytes to a conserved signaling pathway in regenerative versus non-regenerative hearts.

Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis.

BackgroundEffective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis.MethodsLung fibrosis was induced in wild-type or noggin haploinsufficient (Nog+/LacZ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis.ResultsNog+/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity.ConclusionsUpon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog+/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.

Coordinated regulation of mesenchymal stem cell differentiation on microstructured titanium surfaces by endogenous bone morphogenetic proteins

Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4μm); sandblasted/acid-etched Ti (SLA, Ra=3.2μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment.

Glioma cancer stem cells secrete Gremlin1 to promote their maintenance within the tumor hierarchy.

Glioblastomas are the most prevalent and lethal primary brain tumor and are comprised of hierarchies with self-renewing cancer stem cells (CSCs) at the apex. Like neural stem cells (NSCs), CSCs reside in functional niches that provide essential cues to maintain the cellular hierarchy. Bone morphogenetic proteins (BMPs) instruct NSCs to adopt an astrocyte fate and are proposed as anti-CSC therapies to induce differentiation, but, paradoxically, tumors express high levels of BMPs. Here we demonstrate that the BMP antagonist Gremlin1 is specifically expressed by CSCs as protection from endogenous BMPs. Gremlin1 colocalizes with CSCs in vitro and in vivo. Furthermore, Gremlin1 blocks prodifferentiation effects of BMPs, and overexpression of Gremlin1 in non-CSCs decreases their endogenous BMP signaling to promote stem-like features. Consequently, Gremlin1-overexpressing cells display increased growth and tumor formation abilities. Targeting Gremlin1 in CSCs results in impaired growth and self-renewal. Transcriptional profiling demonstrated that Gremlin1 effects were associated with inhibition of p21(WAF1/CIP1), a key CSC signaling node. This study establishes CSC-derived Gremlin1 as a driving force in maintaining glioblastoma tumor proliferation and glioblastoma hierarchies through the modulation of endogenous prodifferentiation signals.

Treatment with Anti–Gremlin 1 Antibody Ameliorates Chronic Hypoxia/SU5416–Induced Pulmonary Arterial Hypertension in Mice

The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.

The BMP Coreceptor RGMb Promotes While the Endogenous BMP Antagonist Noggin Reduces Neurite Outgrowth and Peripheral Nerve Regeneration by Modulating BMP Signaling

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

Distinct bone morphogenetic proteins activate indistinguishable transcriptional responses in nephron epithelia including Notch target genes

Endogenous Bone Morphogenetic Protein (BMP) signaling plays a significant role in the kidney's recovery from acute injury and exogenous administration of BMP7 has therapeutic potential in numerous rodent models of renal injury and disease. However, in the healthy kidney endogenous BMP7 ligand is vigorously counteracted by extracellular antagonists such as USAG1 and CHRDL1. Little is known about the degree of BMP signaling and the ligands driving it in the healthy adult kidney. In this study we characterize basal BMP signaling in the healthy tubular nephron, and show that BMP2 is expressed in proximal nephron epithelial cells. Comparative gene profiling of proximal tubule cell responses to BMP2 and BMP7 does not reveal any qualitative difference, suggesting that identical BMP gene targets may be activated in healthy and injured organs. Interestingly, our gene profiling analysis shows that BMP signaling activates a number of Notch regulated transcription factors, including HEY1. As in other biological systems, HEY1 functions as a negative feedback regulator of BMP2 expression in the proximal tubule. In summary, this work reveals endogenous BMP signaling patterns in the healthy human and mouse kidneys, and identifies novel gene targets, some of which are involved in the complex regulation of BMP signaling in the adult kidney.

Acute Skeletal Injury Is Necessary for Human Adipose-Derived Stromal Cell–Mediated Calvarial Regeneration

Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects. Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction). Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression. Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.

BMP Signaling Promotes the Growth of Primary Human Colon Carcinomas in vivo

Human colon carcinomas (CCs) represent a growing worldwide problem. One of the pathways that has been negatively implicated in the genesis of CCs is triggered by bone morphogenetic protein (BMP) ligands, which activate BMP receptors leading to the function of SMAD proteins in the nucleus. BMP signaling is altered in familial human polyposis, and mice with compromised BMP signaling in the intestine develop tumors. Here, we have re-evaluated the presence and roles of BMP signaling in advanced sporadic human CCs, using both primary tumors and established cell lines, and directly modulating BMP pathway activity in a cell-autonomous manner using constitutively active and dominant-negative BMP receptor Ib forms. We find evidence for active endogenous BMP signaling in all primary CC samples and for its role in promoting primary CC tumor growth and CC cell survival and proliferation in vivo in xenografts. In vitro, we also document autonomous and non-autonomous effects of enhanced BMP receptor activity on gap closure in culture, suggesting possible roles in invasion. Caution should thus be exerted in trying to augment or restore its activity for therapeutic purposes. In contrast, we raise the possibility that blockade of BMP signaling might have beneficial effects against at least a subset of advanced colon cancers.

Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation

Extracellular regulation of signaling by transforming growth factor (TGF)-β family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-β and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2-null (Fbn2(-/-)) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2(-/-) phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1(-/-) mice exhibit improper latent TGF-β activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

Regulation of Neural Specification from Human Embryonic Stem Cells by BMP and FGF

Inhibition of bone morphogenetic protein (BMP) signaling is required for vertebrate neural induction, and fibroblast growth factors (FGFs) may affect neural induction through phosphorylation at the linker region of Smad1, thus regulating BMP signaling. Here we show that human embryonic stem cells efficiently convert to neuroepithelial cells in the absence of BMP antagonists, or even when exposed to high concentrations of exogenous BMP4. Molecular and functional analyses revealed multiple levels of endogenous BMP signaling inhibition that may account for the efficient neural differentiation. Blocking FGF signaling inhibited neural induction, but did not alter the phosphorylation of the linker region of Smad1, suggesting that FGF enhances human neural specification independently of BMP signaling.

Follistatin preferentially antagonizes activin rather than BMP signaling in Drosophila

Ligands of the transforming growth factor-beta (TGF-beta) superfamily play important roles in embryonic patterning and development throughout the animal kingdom. Consequently, extracellular factors that affect ligand stability, mobility, and receptor interaction also have profound effects on development. One such regulator, Follistatin (Fst), functions as an inhibitor of both activin and bone morphogenetic protein (BMP) subfamilies of TGF-beta ligands in vertebrates. Drosophila follistatin (fs) encodes a Fst homolog that is broadly expressed throughout development, but the in vivo function of the protein remains unclear. We show that overexpression of fs affects prepupal to pupal transition and morphogenesis, highlighting a novel requirement for TGF-beta signaling in metamorphosis. In addition, fs expression disrupts various aspects of neuronal morphogenesis, mimicking mutant phenotypes of the activin ligands, Dawdle (Daw) and Activin-beta. In assays targeting endogenous BMP signaling, we find no evidence that fs can antagonize BMP activity. We conclude that fs functions primarily as an inhibitor of activin rather than BMP ligands.

The Xenopus POU class V transcription factor XOct-25 inhibits ectodermal competence to respond to bone morphogenetic protein-mediated embryonic induction

Bone morphogenetic proteins (BMPs) have been shown to play a key role in controlling ectodermal cell fates by inducing epidermis at the expense of neural tissue during gastrulation. Here, we present evidence that the Xenopus POU class V transcription factor XOct-25 regulates ectodermal cell fate decisions by inhibiting the competence of ectodermal cells to respond to BMP during Xenopus embryogenesis. When overexpressed in the ectoderm after the blastula stage, XOct-25 suppressed early BMP responses of ectodermal cells downstream of BMP receptor activation and promoted neural induction while suppressing epidermal differentiation. In contrast, inhibition of XOct-25 function in the prospective neuroectoderm resulted in expansion of epidermal ectoderm at the expense of neuroectoderm. The reduction of neural tissue by inhibition of XOct-25 function could be rescued by decreasing endogenous BMP signaling, suggesting that XOct-25 plays a role in the formation of neural tissue at least in part by inhibiting BMP-mediated epidermal induction (neural inhibition). This hypothesis is supported by the observation that ectodermal cells from XOct-25 morphants were more sensitive to BMP signaling than cells from controls in inducing both immediate early BMP target genes and epidermis at the expense of neural tissue, while cells overexpressing XOct-25 are less competent to respond to BMP-mediated induction. These results document an essential role for XOct-25 in commitment to neural or epidermal cell fates in the ectoderm and highlight the importance of a regulatory mechanism that limits competence to respond to BMP-mediated embryonic induction.

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian-specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers.

Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.

Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex

Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. We used mouse in utero electroporation and mutant analysis to test whether cortical signaling sources interact to regulate one another. We identified interactions between the cortical hem, rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression reduced, and the hippocampus defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, we examined noggin and BMP signaling in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression was expanded and BMP activity reduced, potentially leading to FGF8 upregulation. Our findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon, integrated by EMX2, and required for normal cortical development.