In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth

The apical ectodermal ridge (AER) in the vertebrate limb is required for limb outgrowth and patterning. To investigate the role BMP ligands expressed in the AER play in limb development we selectively inactivated both Bmp2 and Bmp4 in this tissue. The autopods of mice lacking both of these genes contained extra digits, digit bifurcations and interdigital webbing due to a decrease in programmed cell death and an increase in cell proliferation in the underlying mesoderm. Upon removal of Bmp2 and Bmp4 in the AER, no defects in proximal-distal patterning were observed. At the molecular level, removal of Bmp2 and Bmp4 in the AER caused an increase in Fgf expression, which correlated with an increase in both the width and length of the AER. Investigation of Engrailed-1 (En1) expression in the AER of limb buds in which Bmp2 and Bmp4 had been removed indicated that En1 expression was absent from this tissue. Our data suggests that AER expression of Bmp2 and Bmp4 is required for digit and dorsal-ventral patterning but surprisingly not for limb outgrowth.     

Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.     

Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway

The eye field is initially a large single domain at the anterior end of the neural plate and is the first indication of optic potential in the vertebrate embryo. During the course of development, this domain is subject to interactions that shape and refine the organogenic field. The action of the prechordal mesoderm in bisecting this single region into two bilateral domains has been well described, however the role of signalling interactions in the further restriction and refinement of this domain has not been previously characterised. Here we describe a role for the rostral cephalic paraxial mesoderm in limiting the extent of the eye field. The anterior transposition of this mesoderm or its ablation disrupted normal development of the eye. Importantly, perturbation of optic vesicle development occurred in the absence of any detectable changes in the pattern of neighbouring regions of the neural tube. Furthermore, negative regulation of eye development is a property unique to the rostral paraxial mesoderm. The rostral paraxial mesoderm expresses members of the bone morphogenetic protein (BMP) family of signalling molecules and manipulation of endogenous BMP signalling resulted in abnormalities of the early optic primordia.     

BMP4 substitutes for loss of BMP7 during kidney development

Functional inactivation of divergent bone morphogenetic proteins (BMPs) causes discrete disturbances during mouse development. BMP4-deficient embryos display mesodermal patterning defects at early post-implantation stages, whereas loss of BMP7 selectively disrupts kidney and eye morphogenesis. Whether these distinct phenotypes simply reflect differences in expression domains, or alternatively intrinsic differences in the signaling properties of these ligands remains unknown. To address this issue, we created embryos exclusively expressing BMP4 under control of the BMP7 locus. Surprisingly, this novel knock-in allele efficiently rescues kidney development. These results demonstrate unequivocally that these structurally divergent BMP family members, sharing only minimal sequence similarity can function interchangeably to activate all the essential signaling pathways for growth and morphogenesis of the kidney. Thus, we conclude that partially overlapping expression patterns of BMPs serve to modulate strength of BMP signaling rather than create discrete fields of ligands with intrinsically different signaling properties.     

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1^Cre) mouse. Embryonic Wt1^Cre;Alk3^fl/fl specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1^Cre;Alk3^fl/fl mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1^Cre;Alk3^fl/fl specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.     

Expression of interleukin-17B in mouse embryonic limb buds and regulation by BMP-7 and bFGF

Interleukin-17B (IL-17B) is a member of interleukin-17 family that displays a variety of proinflammatory and immune modulatory activities. In this study, we found that IL-17B mRNA was maximally expressed in the limb buds of 14.5 days post coitus (dpc) mouse embryo and declined to low level at 19.5 dpc. By immunohistochemical staining, the strongest IL-17B signals were observed in the cells of the bone collar in the primary ossification center. The chondrocytes in the resting and proliferative zones were stained moderately, while little staining was seen in the hypertrophic zone. Furthermore, in both C3H10T1/2 and MC3T3-E1 cells, the IL-17B mRNA was up-regulated by recombinant human bone morphogenetic protein-7, but down-regulated by basic fibroblast growth factor via the extracellular signal-regulated kinase pathway. This study provides the first evidence that IL-17B is expressed in the mouse embryonic limb buds and may play a role in chondrogenesis and osteogenesis.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.     

Caveolin-1 regulates BMPRII localization and signaling in vascular smooth muscle cells

Recent studies demonstrate the interaction of BMPRII and caveolin-1 in various cell types. In this study we test the hypothesis that caveolin-1 interacts with and regulates BMPRII-dependent signaling in vascular smooth muscle cells. We demonstrate that BMPRII localizes to caveolae and directly interacts with caveolin-1 in mouse aortic smooth muscle cells. We demonstrate that this interaction is mediated by the caveolin-1 scaffolding domain and is regulated by caveolin-1 phosphorylation. Downregulation of caveolin-1 via siRNA resulted in a loss of BMP-dependent SMAD phosphorylation and gene regulation. Further studies revealed that loss of caveolin-1 results in decreased BMPRII membrane localization and decreased association of BMPRII with the type I BMP receptor BMPRIa. Dominant negative caveolin-1 decreased BMPRII membrane localization suggesting a role for caveolin-1 in BMPRII trafficking. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of BMPRII in vascular smooth muscle cells.     

glypican-3 controls cellular responses to Bmp4 in limb patterning and skeletal development

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.     

Influence of FGF4 on digit morphogenesis during limb development in the mouse

Much of what we currently know about digit morphogenesis during limb development is deduced from embryonic studies in the chick. In this study, we used ex utero surgical procedures to study digit morphogenesis during mouse embryogenesis. Our studies reveal some similarities; however, we have found considerable differences in how the chick and the mouse autopods respond to experimentation. First, we are not able to induce ectopic digit formation from interdigital cells as a result of wounding or TGFbeta-1 application in the mouse, in contrast to what is observed in the chick. Second, FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. We demonstrate with cell marking studies that this bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. We propose that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event. Finally, we discuss the relationship between the digit IV bifurcation restriction and the placement of the metapterygial axis in the evolution of the tetrapod limb.     

Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation

Adeno-associated virus (AAV) is so far the most valuable vehicle for gene therapy because it has no association with immune response and human disease. The present study was conducted to investigate the feasibility of AAV-mediated BMP4 gene transfer for bone formation. In vitro study suggested that AAV-BMP4 vectors could transduce myoblast C2C12 cells and produce osteogenic BMP4. In vivo study demonstrated that new bone formation could be induced by direct injection of AAV-BMP4 into the skeletal muscle of immunocompetent rats. Histological analysis revealed that the newly formed bone was induced through endochondral mechanism. Immunohistochemical staining further demonstrated that AAV-BMP4 gene delivery could mediate long-term transduction, and the involvement of BMP4 expression was responsible for the endochondral ossification. This study is, to our knowledge, the first report in the field of AAV-based BMP gene transfer and should be promising for clinical orthopaedic applications.     

Identification of novel genes affecting mesoderm formation and morphogenesis through an enhanced large scale functional screen in Xenopus

The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.     

Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos

In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.     

Role of RhoC in digit morphogenesis during limb development

Here we report a new role for the small GTPase RhoC in the control of limb chondrogenesis. Expression of rhoC is a precocious marker of the zeugopodial and digit blastemas and is induced by treatments with TGFbetas preceding the formation of ectopic digits. As development progresses, expression of rhoC outlines the growing distal tip of the digits, and marks the regions of interphalangeal joint formation. Functional experiments show that RhoC is a negative regulator of chondrogenesis, which controls digit outgrowth and joint segmentation. These functions appear to be mediated by reorganization of the actin cytoskeleton and modification of the adhesive properties of the mesenchymal cells.