Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.

Pax genes have been shown to play important roles in mammalian development and organogenesis. Pax9, a member of this transcription factor family, is expressed in somites, pharyngeal pouches, mesenchyme involved in craniofacial, tooth, and limb development, as well as other sites during mouse embryogenesis. To analyze its function in vivo, we generated Pax9 deficient mice and show that Pax9 is essential for the development of a variety of organs and skeletal elements. Homozygous Pax9-mutant mice die shortly after birth, most likely as a consequence of a cleft secondary palate. They lack a thymus, parathyroid glands, and ultimobranchial bodies, organs which are derived from the pharyngeal pouches. In all limbs, a supernumerary preaxial digit is formed, but the flexor of the hindlimb toes is missing. Furthermore, craniofacial and visceral skeletogenesis is disturbed, and all teeth are absent. In Pax9-deficient embryos tooth development is arrested at the bud stage. At this stage, Pax9 is required for the mesenchymal expression of Bmp4, Msx1, and Lef1, suggesting a role for Pax9 in the establishment of the inductive capacity of the tooth mesenchyme. In summary, our analysis shows that Pax9 is a key regulator during the development of a wide range of organ primordia.

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, we now show that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, we find that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources.

Bone Morphogenetic Proteins (BMPs) play crucial roles in a variety of developmental processes, but their functions during early vertebrate brain development are largely unknown. To investigate this problem, we have compared by in situ hybridization the expression of five Bmp genes belonging to the Drosophila Decapentaplegic (Bmp2 and Bmp4) and 60A subgroups (Bmp5, Bmp6 and Bmp7). Striking co-expression of these Bmps is observed within the dorsomedial telencephalon, coincident with a future site of choroid plexus development. Bmp co-expression overlaps that of Msx1 and Hfh4, and is complementary to that of Bf1. The domain of Bmp co-expression is also associated with limited growth of the neuroectoderm, as revealed by morphological observation, reduced cell proliferation, and increased local programmed cell death. In vitro experiments using explants from the embryonic lateral telencephalic neuroectoderm reveal that exogenous BMP proteins (BMP4 and BMP2) induce expression of Msx1 and inhibit Bf1 expression, a finding consistent with their specific expression patterns in vivo. Moreover, BMP proteins locally inhibit cell proliferation and increase apoptosis in the explants. These results provide evidence that BMPs function during regional morphogenesis of the dorsal telencephalon by regulating specific gene expression, cell proliferation and local cell death.

Nanog, Oct4, and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated, the mechanistic functions are not well defined. Here, we identify general and cell-line-specific requirements for NANOG, OCT4, and SOX2 in hESCs. We show that OCT4 regulates, and interacts with, the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages, whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs.

BACKGROUND

The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods.

RESULTS

We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.

CONCLUSIONS

This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/beta-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for beta-catenin in this disease has not been reported. Because tissue-specific activation of the beta-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the beta-catenin gene in articular chondrocytes in adult mice by generating beta-catenin conditional activation (cAct) mice through breeding of beta-catenin(fx(Ex3)/fx(Ex3)) mice with Col2a1-CreER(T2) transgenic mice. Deletion of exon 3 of the beta-catenin gene results in the production of a stabilized fusion beta-catenin protein that is resistant to phosphorylation by GSK-3beta. In this study, tamoxifen was administered to the 3- and 6-mo-old Col2a1-CreER(T2);beta-catenin(fx(Ex3)/wt) mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of beta-catenin protein was detected by immunostaining in articular cartilage tissues of beta-catenin cAct mice. In 5-mo-old beta-catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8-mo-old beta-catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in beta-catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp-9, Mmp-13, Alp, Oc, and colX, was significantly increased (3- to 6-fold) in articular chondrocytes derived from beta-catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6-fold increase) in these cells. In addition, we also observed overexpression of beta-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of beta-catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA-like phenotype. This study provides direct and definitive evidence about the role of beta-catenin in the development of OA.

We investigated the interaction between angiogenic and osteogenic factors in bone formation and bone healing with ex vivo gene therapy using muscle-derived stem cells genetically engineered to express human bone morphogenetic protein-4 (BMP4), VEGF, or VEGF-specific antagonist (soluble Flt1). Our results show that although VEGF alone did not improve bone regeneration, it acted synergistically with BMP4 to increase recruitment of mesenchymal stem cells, to enhance cell survival, and to augment cartilage formation in the early stages of endochondral bone formation. These early effects, coupled with accelerated cartilage resorption, eventually led to a significant enhancement of bone formation and bone healing. The beneficial effect of VEGF on bone healing elicited by BMP4 depends critically on the ratio of VEGF to BMP4, with an improper ratio leading to detrimental effects on bone healing. Finally, we show that soluble Flt1 inhibits bone formation elicited by BMP4. Thus, VEGF plays an important role in bone formation elicited by BMP4, and it can significantly enhance BMP4-elicited bone formation and regeneration through multiple mechanisms. This study has important implications for the formulation of new strategies to improve bone healing through increasing mesenchymal stem cell recruitment and survival, in combination with muscle-derived stem cell-based gene therapy.

Morphogenesis of the mouse lung involves reciprocal interactions between the epithelial endoderm and the surrounding mesenchyme, leading to an invariant early pattern of branching that forms the basis of the respiratory tree. There is evidence that Fibroblast growth factor 10 (Fgf10) and Bone Morphogenetic Protein 4 (Bmp4), expressed in the distal mesenchyme and endoderm, respectively, play important roles in branching morphogenesis. To examine these roles in more detail, we have exploited an in vitro culture system in which isolated endoderm is incubated in Matrigel(TM) substratum with Fgf-loaded beads. In addition, we have used a Bmp4(lacZ) line of mice in which lacZ faithfully reports Bmp4 expression. Analysis of lung endoderm in vivo shows a dynamic pattern of Bmp4(lacZ) expression during bud outgrowth, extension and branching. In vitro, Fgf10 induces both proliferation and chemotaxis of isolated endoderm, whether it is derived from the distal or proximal lung. Moreover, after 48 hours, Bmp4(lacZ) expression is upregulated in the endoderm closest to the bead. Addition of 30-50 ng/ml of exogenous purified Bmp4 to the culture medium inhibits Fgf-induced budding or chemotaxis, and inhibits overall proliferation. By contrast, the Bmp-binding protein Noggin enhances Fgf-induced morphogenesis. Based on these and other results, we propose a model for the combinatorial roles of Fgf10 and Bmp4 in branching morphogenesis of the lung.

The molecular mechanisms dictating the morphogenesis and differentiation of the mammalian inner ear are largely unknown. To better elucidate the normal development of this organ, two approaches were taken. First, the membranous labyrinths of mouse inner ears ranging from 10.25 to 17 d postcoitum (dpc) were filled with paint to reveal their gross development. Particular attention was focused on the developing utricle, saccule, and cochlea. Second, we used bone morphogenetic protein 4 (BMP4) and lunatic fringe (Fng) as molecular markers to identify the origin of the sensory structures. Our data showed that BMP4 was an early marker for the superior, lateral, and posterior cristae, whereas Fng served as an early marker for the macula utriculi, macula sacculi, and the sensory portion of the cochlea. The posterior crista was the first organ to appear at 11.5 dpc and was followed by the superior crista, the lateral crista, and the macula utriculi at 12 dpc. The macula sacculi and the cochlea were present at 12 dpc but became distinguishable from each other by 13 dpc. Based on the gene expression patterns, the anterior and lateral cristae may share a common origin. Similarly, three sensory organs, the macula utriculi, macula sacculi, and cochlea, seem to arise from a single region of the otocyst.

Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes Msx-1 and Msx-2 and the proto-oncogene JunB, whereas c-jun transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include Id3, which showed strong mRNA induction, and the moderately induced Cyr61, DEK, and eIF4AII genes, as well as a gene encoding a GC-binding protein. Besides Id3, also the Id1 and Id2 genes were activated by BMP4 in both ES cells and a range of different cell lines. Id genes encode negative regulators of basic helix-loop-helix transcription factors. In vivo we observed local ectopic expression of Id3 and Msx-2 mRNAs in Ft/+ embryos at overlapping regions of ectopic Bmp4 misexpression. We therefore propose that the Msx and Id genes are direct target genes of embryonic BMP4 signaling in vivo.

Primordial ovarian follicles in mice form when somatic cells surround individual oocytes. We show that lack of Nobox, an oocyte-specific homeobox gene, accelerates postnatal oocyte loss and abolishes the transition from primordial to growing follicles in mice. Follicles are replaced by fibrous tissue in female mice lacking Nobox in a manner similar to nonsyndromic ovarian failure in women. Genes preferentially expressed in oocytes, including Oct4 and Gdf9, are down-regulated in Nobox-/- mice, whereas ubiquitous genes such as Bmp4, Kit, and Bax remain unaffected. Therefore, Nobox is critical for specifying an oocyte-restricted gene expression pattern essential for postnatal follicle development.

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1(-/-) palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.

Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. By combining use of a hypomorphic Bone morphogenetic protein 4 (Bmp4) allele with conditional gene inactivation, we here identify Bmp4 as a signal from the myocardium directly mediating atrioventricular septation. Defects in this process cause one of the most common human congenital heart abnormalities, atrioventricular canal defect (AVCD). The spectrum of defects obtained through altering Bmp4 expression in the myocardium recapitulates the range of AVCDs diagnosed in patients, thus providing a useful genetic model with AVCD as the primary defect.

Accumulating evidence suggests that mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment; however, controversy exists regarding their role in solid tumors. In this study, we identified and confirmed the presence of carcinoma-associated MSCs (CA-MSCs) in the majority of human ovarian tumor samples that we analyzed. These CA-MSCs had a normal morphologic appearance, a normal karyotype, and were nontumorigenic. CA-MSCs were multipotent with capacity for differentiating into adipose, cartilage, and bone. When combined with tumor cells in vivo, CA-MSCs promoted tumor growth more effectively than did control MSCs. In vitro and in vivo studies suggested that CA-MSCs promoted tumor growth by increasing the number of cancer stem cells. Although CA-MSCs expressed traditional MSCs markers, they had an expression profile distinct from that of MSCs from healthy individuals, including increased expression of BMP2, BMP4, and BMP6. Importantly, BMP2 treatment in vitro mimicked the effects of CA-MSCs on cancer stem cells, while inhibiting BMP signaling in vitro and in vivo partly abrogated MSC-promoted tumor growth. Taken together, our data suggest that MSCs in the ovarian tumor microenvironment have an expression profile that promotes tumorigenesis and that BMP inhibition may be an effective therapeutic approach for ovarian cancer.

Mouse embryos bearing hypomorphic and conditional null Fgf8 mutations have small and abnormally patterned telencephalons. We provide evidence that the hypoplasia results from decreased Foxg1 expression, reduced cell proliferation and increased cell death. In addition, alterations in the expression of Bmp4, Wnt8b, Nkx2.1 and Shh are associated with abnormal development of dorsal and ventral structures. Furthermore, nonlinear effects of Fgf8 gene dose on the expression of a subset of genes, including Bmp4 and Msx1, correlate with a holoprosencephaly phenotype and with the nonlinear expression of transcription factors that regulate neocortical patterning. These data suggest that Fgf8 functions to coordinate multiple patterning centers, and that modifications in the relative strength of FGF signaling can have profound effects on the relative size and nature of telencephalic subdivisions.

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.

Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H2O2 and O2- production in ECs. Next, we used ECs obtained from p47phox-/- mice (MAE-p47-/-), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47-/-, but it was restored when the cells were transfected with p47phox plasmid. Moreover, OS-induced O2- production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step.

Bone morphogenetic protein (BMP) signaling inhibits the generation of oligodendroglia and enhances generation of astrocytes by neural progenitor cells both in vitro and in vivo. This study examined the mechanisms underlying the effects of BMP signaling on glial lineage commitment. Treatment of cultured neural progenitor cells with BMP4 induced expression of all four members of the inhibitor of differentiation (ID) family of helix-loop-helix transcriptional inhibitors and blocked oligodendrocyte (OL) lineage commitment. Overexpression of Id4 or Id2 but not Id1 or Id3 in cultured progenitor cells reproduced both the inhibitory effects of BMP4 treatment on OL lineage commitment and the stimulatory effects on astrogliogenesis. Conversely, decreasing the levels of Id4 mRNA by RNA interference enhanced OL differentiation and inhibited the effects of BMP4 on glial lineage commitment. This suggests that induction of Id4 expression mediates effects of BMP signaling. Bacterial two-hybrid and co-immunoprecipitation studies demonstrated that ID4, and to a lesser extent ID2, complexed with the basic-helix-loop-helix transcription (bHLH) factors OLIG1 and OLIG2, which are required for the generation of OLs. By contrast, ID1 and ID3 did not complex with the OLIG proteins. In addition, the OLIG and ID proteins both interacted with the E2A proteins E12 and E47. Further, exposure of cultured progenitor cells to BMP4 changed the intracellular localization of OLIG1 and OLIG2 from a predominantly nuclear to a predominantly cytoplasmic localization. These observations suggest that the induction of ID4 and ID2, and their sequestration of both OLIG proteins and E2A proteins mediate the inhibitory effects of BMP signaling on OL lineage commitment and contribute to the generation of astrocytes.

Rieger syndrome, an autosomal dominant disorder, includes ocular, craniofacial and umbilical abnormalities. The pitx2 homeobox gene, which is mutated in Rieger syndrome, has been proposed to be the effector molecule interpreting left-right axial information from the early embryonic trunk to each organ. Here we have used gene targeting in mice to generate a loss-of-function allele that would be predicted to result in organ randomization or isomerization. Although pitx2-/- embryos had abnormal cardiac morphogenesis, mutant hearts looped in the normal direction. Pitx2-/- embryos had correctly oriented, but arrested, embryonic rotation and right pulmonary isomerism. They also had defective development of the mandibular and maxillary facial prominences, regression of the stomodeum and arrested tooth development. Fgf8 expression was absent, and Bmp4 expression was expanded in the branchial-arch ectoderm. These data reveal a critical role for pitx2 in left-right asymmetry but indicate that pitx2 may function at an intermediate step in cardiac morphogenesis and embryonic rotation.

In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.

Branching morphogenesis of the embryonic lung requires interactions between the epithelium and the mesenchyme. Previously, we reported that Sonic hedgehog (Shh) transcripts are present in the epithelium of the developing mouse lung, with highest levels in the terminal buds. Here, we report that transcripts of mouse patched (Ptc), the homologue of a Drosophila gene encoding a putative transmembrane protein required for hedgehog signaling, are expressed at high levels in the mesenchyme adjacent to the end buds. To investigate the function of SHH in lung development, Shh was overexpressed throughout the distal epithelium, using the surfactant protein-C (SP-C)-enhancer/promoter. Beginning around 16.5 dpc, when Shh and Ptc RNA levels are normally both declining, this treatment caused an increase in the ratio of interstitial mesenchyme to epithelial tubules in transgenic compared to normal lungs. Transgenic newborn mice die soon after birth. Histological analysis of the lungs at the light and electron microscope level shows an abundance of mesenchyme and the absence of typical alveoli. In vivo BrdU labeling indicates that Shh overexpression results in increased mesenchymal and epithelial cell proliferation at 16.5 and 17.5 dpc. However, analysis of CC-10 and SP-C expression reveals no significant inhibition in the differentiation of proximal and distal epithelial cells. The expression of genes potentially regulated by SHH was also examined. No difference could be observed between transgenic and control lungs in either the level or distribution of Bmp4, Wnt2 and Fgf7 RNA. By contrast, Ptc is clearly upregulated in the transgenic lung. These results thus establish a role for SHH in lung morphogenesis, and suggest that SHH normally regulates lung mesenchymal cell proliferation in vivo.

Vertebrate lens development is a classical model system for studying embryonic tissue interactions. Little is known, however, about the molecules mediating such inductive events. Here, we show that Bmp4, which is expressed strongly in the optic vesicle and weakly in the surrounding mesenchyme and surface ectoderm, has crucial roles during lens induction. In Bmp4(tm1) homozygous null mutant embryos, lens induction is absent, but the process can be rescued by exogenous BMP4 protein applied into the optic vesicle in explant cultures. This is associated with rescue of ectodermal expression of Sox2, an early lens placode marker. Substituting the optic vesicle in explant cultures with BMP4-carrying beads, however, does not lead to lens induction, indicating that other factors produced by the optic vesicle are involved. BMP4 appears to regulate expression of a putative downstream gene, Msx2, in the optic vesicle. No change in Pax6 expression is seen in Bmp4(tm1) mutant eyes, and Bmp4 expression appears unaffected in the eyes of homozygous Pax6(Sey-1Neu), suggesting that PAX6 and BMP4 function independently. Based on these results we propose that BMP4 is required for the optic vesicle to manifest its lens-inducing activity, by regulating downstream genes and/or serving as one component of multiple inductive signals.

The induction of developmental structures derived from the ectoderm, such as the neural tube or tooth, occurs through neutralization of the inhibitory activity of members of the bone-morphogenetic protein (BMP) family by BMP antagonists. Here we show that, during hair-follicle development, the neural inducer and BMP-neutralizing protein Noggin is expressed in the follicular mesenchyme, that noggin-knockout mice show significant retardation of hair-follicle induction, and that Noggin neutralizes the inhibitory action of BMP-4 and stimulates hair-follicle induction in embryonic skin organ culture. As a crucial mesenchymal signal that stimulates hair-follicle induction, Noggin operates through antagonistic interactions with BMP-4, which result in upregulation of the transcription factor Lef-1 and the cell-adhesion molecule NCAM, as well as through BMP4-independent downregulation of the 75 kD neurotrophin receptor in the developing hair follicle.