Noggin antagonism of BMP4 signaling controls development of the axial skeleton in the mouse

The interaction between bone morphogenetic proteins (BMPs) and their antagonist, Noggin, is critical for normal development. Noggin null mice die at birth with a severely malformed skeleton that is postulated to reflect the activity of unopposed BMP signaling. However, the widespread expression and redundancy of different BMPs have made it difficult to identify a specific role for individual BMPs during mammalian skeletal morphogenesis. Here, we report the effects of modifying Bmp4 dosage on the skeletal development of Noggin mutant mice. The reduction of Bmp4 dosage results in an extensive rescue of the axial skeleton of Noggin mutant embryos. In contrast, the appendicular skeletal phenotype of Noggin mutants was unchanged. Analysis of molecular markers of somite formation and somite patterning suggests that the loss of Noggin results in the formation of small mispatterned somites. Mis-specification and growth retardation rather than cell death most likely account for the subsequent reduction or loss of axial skeletal structures. The severe Noggin phenotype correlates with Bmp4-dependent ectopic expression of Bmp4 in the paraxial mesoderm consistent with Noggin antagonizing an auto-inductive feed-forward mechanism. Thus, specific interactions between Bmp4 and Noggin in the early embryo are critical for establishment and patterning of the somite and subsequent axial skeletal morphogenesis.     

Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene

Cartilage and bone are formed into a remarkable range of shapes and sizes that underlie many anatomical adaptations to different lifestyles in vertebrates. Although the morphological blueprints for individual cartilage and bony structures must somehow be encoded in the genome, we currently know little about the detailed genomic mechanisms that direct precise growth patterns for particular bones. We have carried out large-scale enhancer surveys to identify the regulatory architecture controlling developmental expression of the mouse Bmp5 gene, which encodes a secreted signaling molecule required for normal morphology of specific skeletal features. Although Bmp5 is expressed in many skeletal precursors, different enhancers control expression in individual bones. Remarkably, we show here that different enhancers also exist for highly restricted spatial subdomains along the surface of individual skeletal structures, including ribs and nasal cartilages. Transgenic, null, and regulatory mutations confirm that these anatomy-specific sequences are sufficient to trigger local changes in skeletal morphology and are required for establishing normal growth rates on separate bone surfaces. Our findings suggest that individual bones are composite structures whose detailed growth patterns are built from many smaller lineage and gene expression domains. Individual enhancers in BMP genes provide a genomic mechanism for controlling precise growth domains in particular cartilages and bones, making it possible to separately regulate skeletal anatomy at highly specific locations in the body.     

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.     

The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb

Mice carrying a targeted disruption of BmprIB were generated by homologous recombination in embryonic stem cells. BmprIB(-/-) mice are viable and, in spite of the widespread expression of BMPRIB throughout the developing skeleton, exhibit defects that are largely restricted to the appendicular skeleton. Using molecular markers, we show that the initial formation of the digital rays occurs normally in null mutants, but proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region are markedly reduced. Our results suggest that BMPRIB-mediated signaling is required for cell proliferation after commitment to the chondrogenic lineage. Analyses of BmprIB and Gdf5 single mutants, as well as BmprIB; Gdf5 double mutants suggests that GDF5 is a ligand for BMPRIB in vivo. BmprIB; Bmp7 double mutants were constructed in order to examine whether BMPRIB has overlapping functions with other type I BMP receptors. BmprIB; Bmp7 double mutants exhibit severe appendicular skeletal defects, suggesting that BMPRIB and BMP7 act in distinct, but overlapping pathways. These results also demonstrate that in the absence of BMPRIB, BMP7 plays an essential role in appendicular skeletal development. Therefore, rather than having a unique role, BMPRIB has broadly overlapping functions with other BMP receptors during skeletal development.     

BMP signaling in skeletal development

Development of the vertebrate skeleton, a complex biological event that includes diverse processes such as formation of mesenchymal condensations at the sites of future skeletal elements, osteoblast and chondrocyte differentiation, and three dimensional patterning, is regulated by many growth factors. Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, play a pivotal role in the signaling network and are involved in nearly all processes associated with skeletal morphogenesis. BMP signals are transduced from the plasma membrane receptors to the nucleus through both Smad pathway and non-Smad pathways, and regulated by many extracellular and intercellular proteins that interact with BMPs or components of the BMP signaling pathways. To gain a better understanding of the molecular mechanisms underlying the role of BMP in early skeletal development, it is necessary to elucidate the BMP signaling transduction pathways in chondrocytes and osteoblasts. The major objective of this review was to summarize BMP signaling pathways in the context of craniofacial, axial, and limb development. In particular, this discourse will focus on recent advances of the role of different ligands, receptors, Smads, and BMP regulators in osteoblast and chondrocyte differentiation during embryonic development.     

Early embryonic lethality in Bmp5;Bmp7 double mutant mice suggests functional redundancy within the 60A subgroup

Members of the BMP family of signaling molecules display a high conservation of structure and function, and multiple BMPs are often coexpressed in a variety of tissues during development. Moreover, distinct BMP ligands are capable of activating common pathways. Here we describe the coexpression of two members of the 60A subfamily of BMPs, Bmp5 and Bmp7, at a number of different sites in the embryo from gastrulation onwards. Previous studies demonstrate that loss of either Bmp5 or Bmp7 has negligible effects on development, suggesting these molecules functionally compensate for each other at early stages of embryonic development. Here we show this is indeed the case. Thus we find that Bmp5;Bmp7 double mutants die at 10.5 dpc and display striking defects primarily affecting the tissues where these factors are coexpressed. The present analysis also uncovers novel roles for BMP signaling during the development of the allantois, heart, branchial arches, somites and forebrain. Bmp5 and Bmp7 do not appear to be involved in establishing pattern in these tissues, but are instead necessary for the proliferation and maintenance of specific cell populations. These findings are discussed with respect to potential mechanisms underlying cooperative signaling by multiple members of the TGF-beta superfamily.     

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.     

Control of digit formation by activin signalling

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or beta-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.     

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.     

The role of bone morphogenetic proteins in endochondral bone formation

Bone morphogenetic proteins (BMPs) were originally identified as proteins capable of inducing endochondral bone formation when implanted at extraskeletal sites. BMPs have diverse biological activities during early embryogenesis and various aspects of organogenesis. BMPs bind to BMP receptors on the cell surface, and these signals are transduced intracellularly by Smad proteins. BMP signal pathways can be inhibited by both extra- and intracellular mechanisms. As for skeletal development, genetic studies suggest that BMPs are skeletal mesoderm inducers. Recent studies of tissue-specific activation and inactivation of BMP signals have revealed that BMP signals control proliferation and differentiation of chondrocytes, differentiation of osteoblasts and bone quality. These findings may contribute not only to understanding of bone biology and pathology, but also to improvement of the clinical efficacy of BMPs.     

Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms

Analyses of gene expression profiles at five different stages of mouse submandibular salivary gland development provide insight into gland organogenesis and identify genes that may be critical at different stages. Genes with similar expression profiles were clustered, and RT-PCR was used to confirm the developmental changes. We focused on fibroblast growth factor receptor 1 (FGFR1), as its expression is highest early in gland development. We extended our array results and analyzed the developmental expression patterns of other FGFR and FGF isoforms. The functional significance of FGFR1 was confirmed by submandibular gland organ culture. Antisense oligonucleotides decreased expression of FGFR1 and reduced branching morphogenesis of the glands. Inhibiting FGFR1 signaling with SU5402, a FGFR1 tyrosine kinase inhibitor, reduced branching morphogenesis. SU5402 treatment decreased cell proliferation but did not increase apoptosis. Fgfr, Fgf and Bmp gene expression was localized to either the mesenchyme or the epithelium by PCR, and then measured over time by real time PCR after SU5402 treatment. FGFR1 signaling regulates Fgfr1, Fgf1, Fgf3 and Bmp7 expression and indirectly regulates Fgf7, Fgf10 and Bmp4. Exogenous FGFs and BMPs added to glands in culture reveal distinct effects on gland morphology. Glands cultured with SU5402 were then rescued with exogenous BMP7, FGF7 or FGF10. Taken together, our results suggest specific FGFs and BMPs play reciprocal roles in regulating branching morphogenesis and FGFR1 signaling plays a central role by regulating both FGF and BMP expression.     

Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton

Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains.     

Osteocyte control of bone formation via sclerostin, a novel BMP antagonist

There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss.     

Mice lacking Bmp6 function

Bmp6, a member of the 60A subgroup of bone morphogenetic proteins (BMPs), is expressed in diverse sites in the developing mouse embryo from preimplantation stages onwards. To evaluate roles for Bmp6 signaling in vivo, gene targeting was used to generate a null mutation at the Bmp6 locus. The resulting Bmp6 mutant mice are viable and fertile, and show no overt defects in tissues known to express Bmp6 mRNA. The skeletal elements of newborn and adult mutants are indistinguishable from wild-type. However, careful examination of skeletogenesis in late gestation embryos reveals a consistent delay in ossification strictly confined to the developing sternum. In situ hybridization studies in the developing long bones and sternum show that other BMP family members are expressed in overlapping domains. In particular we find that Bmp2 and Bmp6 are coexpressed in hypertrophic cartilage, suggesting that Bmp2 may functionally compensate in Bmp6 null mice. The defects in sternum development in Bmp6 null mice are likely to be associated with a transient early expression of Bmp6 in the sternal bands, prior to ossification. These sternal defects are slightly exacerbated in Bmp5/6 double mutant animals. Dev. Genet. 22:321–339, 1998. © 1998 Wiley-Liss, Inc.