Role of FGFs in the control of programmed cell death during limb development

We have investigated the role of FGFs in the control of programmed cell death during limb development by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered as the signals responsible for cell death. Here we show that FGF signaling is also necessary for apoptosis and that the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. As previously demonstrated, cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and that mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together our findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although we provide evidence for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. We have identified MSX2 and Snail as candidate genes associated with apoptosis the expression of which requires the combined action of FGFs and BMPs.     

The BMP signaling and in vivo bone formation

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smads 1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and their signaling molecules have shown that BMP signaling plays critical roles in bone and cartilage development and postnatal bone formation. BMP activities are regulated at different molecular levels. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue.     

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.     

Fibroblast growth factor interactions in the developing lung.

Cellular activities that lead to organogenesis are mediated by epithelial-mesenchymal interactions, which ultimately result from local activation of complex gene networks. Fibroblast growth factor (FGF) signaling is an essential component of the regulatory network present in the embryonic lung, controlling proliferation, differentiation and pattern formation. However, little is known about how FGFs interact with other signaling molecules in these processes. By using cell and organ culture systems, we provide evidence that FGFs, Sonic hedgehog (Shh), bone morphogenetic protein 4 (BMP-4), and TGFbeta-1 form a regulatory circuit that is likely relevant for lung development in vivo. Our data show that FGF-10 and FGF-7, important for patterning and growth of the lung bud, are differentially regulated by FGF-1, -2 and Shh. In addition, we show that FGFs regulate expression of Shh, BMP-4 and other FGF family members. Our data support a model in which Shh, TGFbeta-1 and BMP-4 counteract the bud promoting effects of FGF-10, and where FGF levels are maintained throughout lung development by other FGFs and Shh.     

Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

Cell surface heparan sulfate (HS) not only binds several major classes of growth factors but also sometimes potentiates their activities—an effect usually termed “coreception.” A view that coreception is due to the stabilization of growth factor–receptor interactions has emerged primarily from studies of the fibroblast growth factors (FGFs). Recent in vivo studies have strongly suggested that HS also plays an important role in regulating signaling by the bone morphogenetic proteins (BMPs). Here, we provide evidence that the mechanism of coreception for BMPs is markedly different from that established for FGFs. First, we demonstrate a direct, stimulatory role for cell surface HS in the immediate signaling activities of BMP2 and BMP4, and we provide evidence that HS–BMP interactions are required for this effect. Next, using several independent assays of ligand binding and receptor assembly, including coimmunoprecipitation, cross-linking, and fluorescence fluctuation microscopy, we show that HS does not affect BMP binding to type I receptor subunits but instead enhances the subsequent recruitment of type II receptor subunits to BMP-type I receptor complexes. This suggests a view of HS as a catalyst of the formation of signaling complexes, rather than as a stabilizer of growth factor binding.     

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.     

BMPs are mediators in tissue crosstalk of the regenerating musculoskeletal system

The musculoskeletal system is a tight network of many tissues. Coordinated interplay at a biochemical level between tissues is essential for development and repair. Traumatic injury usually affects several tissues and represents a large challenge in clinical settings. The current demand for potent growth factors in such applications thus accompanies the keen interest in molecular mechanisms and orchestration of tissue formation. Of special interest are multitasking growth factors that act as signals in a variety of cell types, both in a paracrine and in an autocrine manner, thereby inducing cell differentiation and coordinating not only tissue assembly at specific sites but also maturation and homeostasis. We concentrate here on bone morphogenetic proteins (BMPs), which are important crosstalk mediators known for their irreplaceable roles in vertebrate development. The molecular crosstalk during embryonic musculoskeletal tissue formation is recapitulated in adult repair. BMPs act at different levels from the initiation to maturation of newly formed tissue. Interestingly, this is influenced by the spatiotemporal expression of different BMPs, their receptors and co-factors at the site of repair. Thus, the regenerative potential of BMPs needs to be evaluated in the context of highly connected tissues such as muscle and bone and might indeed be different in more poorly connected tissues such as cartilage. This highlights the need for an understanding of BMP signaling across tissues in order to eventually improve BMP regenerative potential in clinical applications. In this review, the distinct members of the BMP family and their individual contribution to musculoskeletal tissue repair are summarized by focusing on their paracrine and autocrine functions.     

TAK1 promotes BMP4/Smad1 signaling via inhibition of erk MAPK: a new link in the FGF/BMP regulatory network

FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-β Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.     

A protein canyon in the FGF-FGF receptor dimer selects from an à la carte menu of heparan sulfate motifs

Heparan sulfate (HS) is an essential and dynamic regulator of fibroblast growth factor (FGF) signaling. Two fundamentally different crystallographic models have been proposed to explain, at the molecular level, how HS/heparin enables FGF and FGF receptor (FGFR) to assemble into a functional dimer on the cell surface. In the symmetric 'two-end' model, the heparin-binding sites of FGF and FGFR merge to form a basic canyon that recruits two HS for binding. Within this canyon, the HS molecules primarily act to orchestrate and fortify multivalent and cooperative protein-protein contacts within the dimer that are the foundations of dimerization. In contrast, in the asymmetric model, which mechanistically resembles the previously proposed trans FGF dimer model, a single heparin molecule facilitates dimerization by cross-linking two FGFs into a trans dimer that brings together the two FGFRs. Interestingly, the crystal structure upon which the asymmetric model is based contains a symmetric dimer reminiscent of the symmetric two-end model, suggesting that a different interpretation of the crystal structure has led to the postulation of the asymmetric model. Importantly, the symmetric two-end model provides an intriguing solution to the problem of how HS selectivity is achieved in FGF signaling. The model reveals that, within the canyon, FGF and FGFR no longer adhere to their individual HS binding specificities, but instead act in unison to search for a unique HS motif from a plethora of HS epitopes that are expressed in a tissue-specific and developmentally regulated fashion. Primary sequence differences within the heparin-binding sites of FGFs and FGFRs, together with ligand-induced changes in FGFR conformation, lead to the formation of distinct canyons with unique HS specificity for individual FGF-FGFR complexes.     

The PTHrP–Ihh Feedback Loop in the Embryonic Growth Plate Allows PTHrP to Control Hypertrophy and Ihh to Regulate Proliferation

Growth plate and long bone development is governed by biochemical signaling pathways of which the PTHrP–Ihh system is the best known. Other factors, such as BMPs, FGFs and mechanical loading, may interact with this system. This study aims at elucidating the relative importance of PTHrP and Ihh for controlling proliferation, and hypertrophy in fetal growth plate cartilage. We assessed the question why reduced Ihh expression leads to more pronounced effects on the number of non-hypertrophic cells and total bone formation, compared to PTHrP down-regulation.Using few basic equations, constituted from literature data, this paper shows how the PTHrP–Ihh feedback system can control different aspects of tissue differentiation at distinct locations. In particular, it is shown that (mechanical or biochemical) perturbations will affect proliferation via Ihh-related parameters, whereas changes in PTHrP-related parameters selectively interact with hypertrophy. This is contra-intuitive, since PTHrP acts to keep cells proliferating. In this context, the critical PTHrP level for keeping cells proliferating has been reconsidered. In addition, an explanation is provided for the aforementioned difference in effect between reduced Ihh and PTHrP expression.     

Investigating the mechanism of the assembly of FGF1-binding heparan sulfate motifs

Heparan sulfate (HS) chains play crucial biological roles by binding to various signaling molecules including fibroblast growth factors (FGFs). Distinct sulfation patterns of HS chains are required for their binding to FGFs/FGF receptors (FGFRs). These sulfation patterns are putatively regulated by biosynthetic enzyme complexes, called GAGOSOMES, in the Golgi. While the structural requirements of HS-FGF interactions have been described previously, it is still unclear how the FGF-binding motif is assembled in vivo. In this study, we generated HS structures using biosynthetic enzymes in a sequential or concurrent manner to elucidate the potential mechanism by which the FGF1-binding HS motif is assembled. Our results indicate that the HS chains form ternary complexes with FGF1/FGFR when enzymes carry out modifications in a specific manner.     

Growth factor regulation of lens development

Lens arises from ectoderm situated next to the optic vesicles. By thickening and invaginating, the ectoderm forms the lens vesicle. Growth factors are key regulators of cell fate and behavior. Current evidence indicates that FGFs and BMPs are required to induce lens differentiation from ectoderm. In the lens vesicle, posterior cells elongate to form the primary fibers whereas anterior cells differentiate into epithelial cells. The divergent fates of these embryonic cells give the lens its distinctive polarity. There is now compelling evidence that, at least in mammals, FGF is required to initiate fiber differentiation and that progression of this complex process depends on the synchronized and integrated action of a number of distinct growth factor-induced signaling pathways. It is also proposed that an antero-posterior gradient of FGF stimulation in the mammalian eye ensures that the lens attains and maintains its polarity and growth patterns. Less is known about differentiation of the lens epithelium; however, recent studies point to a role for Wnt signaling. Multiple Wnts and their receptors are expressed in the lens epithelium, and mice with impaired Wnt signaling have a deficient epithelium. Recent studies also indicate that other families of molecules, that can modulate growth factor signaling, have a role in regulating the ordered growth and differentiation of the lens.     

The mechanisms of dorsoventral patterning in the vertebrate neural tube

We describe the essential features of and the molecules involved in dorsoventral (DV) patterning in the neural tube. The neural tube is, from its very outset, patterned in this axis as there is a roof plate, floor plate, and differing numbers and types of neuroblasts. These neuroblasts develop into different types of neurons which express a different range of marker genes. Early embryological experiments identified the notochord and the somites as being responsible for the DV patterning of the neural tube and we now know that 4 signaling molecules are involved and are generated by these surrounding structures. Fibroblast growth factors (FGFs) are produced by the caudal mesoderm and must be down-regulated before neural differentiation can occur. Retinoic acid (RA) is produced by the paraxial mesoderm and is an inducer of neural differentiation and patterning and is responsible for down-regulating FGF. Sonic hedgehog (Shh) is produced by the notochord and floor plate and is responsible for inducing ventral neural cell types in a concentration-dependent manner. Bone morphogenetic proteins (BMPs) are produced by the roof plate and are responsible for inducing dorsal neural cell types in a concentration-dependent manner. Subsequently, RA is used twice more. Once from the somites for motor neuron differentiation and secondly RA is used to define the motor neuron subtypes, but in the latter case it is generated within the neural tube from differentiating motor neurons rather than from outside. These 4 signaling molecules also interact with each other, generally in a repressive fashion, and DV patterning shows how complex these interactions can be.     

The signaling and functions of heterodimeric bone morphogenetic proteins

Heterodimeric bone morphogenetic proteins (BMPs) consist of disulfide-linked dimeric monomers derived from different BMP members. Owing to this specific constitution pattern, they bear high affinity to both type I and type II BMP receptors simultaneously. Meanwhile, the antagonism efficiency of extracellular antagonists to heterodimeric BMPs is also significantly lower than that to homodimeric ones. All these specific properties confer heterodimeric BMPs with distinct signaling and bio-functions that are characterized by more speediness, lower concentration/dose threshold and higher efficiency than homodimeric BMPs. Consequently, heterodimeric BMPs bear promising application potential in inducing osteogenesis. In addition, they may play indispensible roles in organogenesis. In this review, we summarize the current knowledge of heterodimeric BMPs in their signaling pathways and bio-functions.     

The role of fibroblast growth factors in vascular development

Fibroblast growth factors (FGFs) are considered angiogenic factors, yet the exact relationship between FGF and vascular development in normal and pathological tissue has long remained elusive. However, recent results from gene inactivation and transgenic studies in mice and in culture systems have demonstrated the role of FGFs in vessel assembly and sprouting. FGFs also promote blood-vessel branching and induce lymphangiogenesis. Novel players in FGF-mediated angiogenesis have been identified, such as p38 mitogen-activated protein kinase. Tumour angiogenesis is regulated by FGFs directly or indirectly via secondary angiogenesis factors, such as vascular endothelial growth factor. The newly established angiogenic role of FGFs makes FGF or molecules targeting FGF and its receptor promising candidates for the development of novel therapeutics.