Function of FGF-4 in limb development

The apical ectodermal ridge plays a central role in limb development through its interactions with the underlying mesenchyme. Removal of the AER results in cessation of limb outgrowth and leads to truncation of the limb along the proximo-distal axis. The many functions attributed to the ridge include maintenance of the progress zone mesenchyme. Here, cells are stimulated to proliferate, are maintained in an undifferentiated state, and are assigned progressively more distal positional values as the limb grows. The AER also functions to maintain the activity of the polarizing region, a region of mesenchyme which is thought to provide the primary signal for patterning along the antero-posterior axis. We have begun to explore the function of fibroblast growth factor-4 (FGF-4) during limb development. FGF-4, which encodes an efficiently secreted protein, is expressed in the AER. We have previously demonstrated that FGF-4 protein can stimulate limb mesenchyme proliferation and can induce the expression of a downstream homeobox gene, Evx-1 (homologue of the Drosophila even-skipped gene), that is normally regulated by a signal from the AER. To determine to what extent FGF-4 protein can substitute for the AER to allow normal limb outgrowth, we performed experiments on the developing chick limb in ovo. Remarkably, we find that after AER removal, the FGF-4 protein can provide all the signals required for virtually normal outgrowth and patterning of the limb. Further studies indicate that proliferation of progress zone cells is not sufficient, and that an additional signal is produced by the posterior mesenchyme in response to FGF-4 which enables progress zone cells to acquire progressively more distal fates. Thus FGF-4 maintains progress zone activity through a combination of at least two signals—one that acts directly on progress zone cells to stimulate their proliferation, and one that acts indirectly by maintaining the production of patterning signal(s) by the posterior mesenchyme. We further show that failure of the posterior mesenchyme to produce this signal correlates with failure to maintain polarizing activity. This raises the possibility that the signal produced by the posterior mesenchyme and required for progressive proximo-distal limb patterning is identical to the polarizing activity. Further experiments demonstrate that retinoic acid, which mimics the activity of the polarizing region, can supply this signal. In conclusion, the finding that a single growth factor can serve as both the direct and indirect signals required to maintain progress zone activity provides a simple mechanism for ensuring that growth and pattern formation are linked in the developing limb. © 1994 Wiley-Liss, Inc.     

Inhibition of BMPs by follistatin is required for FGF3 expression and segmental patterning of the hindbrain

A network of molecular interactions is required in the developing vertebrate hindbrain for the formation and anterior-posterior patterning of the rhombomeres. FGF signaling is required in this network to upregulate the expression of the Krox20 and Kreisler segmentation genes, but little is known of how FGF gene expression is regulated in the hindbrain. We show that the dynamic expression of FGF3 in chick hindbrain segments and boundaries is similar to that of the BMP antagonist, follistatin. Consistent with a regulatory relationship between BMP signaling and FGF3 expression, we find that an increase in BMP activity due to blocking of follistatin translation by morpholino antisense oligonucleotides or overexpression of BMP results in strong inhibition of FGF3 expression. Conversely, addition of follistatin leads to an increase in the level of FGF3 expression. Furthermore, the segmental inhibition of BMP activity by follistatin is required for the expression of Krox20, Hoxb1 and EphA4 in the hindbrain. In addition, we show that the maintenance of FGF3 gene expression requires FGF activity, suggestive of an autoregulatory loop. These results reveal an antagonistic relationship between BMP activity and FGF3 expression that is required for correct segmental gene expression in the chick hindbrain, in which follistatin enables FGF3 expression by inhibiting BMP activity.     

Autopodial development is selectively impaired by misexpression of chordin-like 1 in the chick limb

Chordin-like 1 (CHRDL1) is a secreted bone morphogenetic protein (BMP) antagonist expressed in mesenchymal tissues whose function in development of the skeleton has not been examined in detail. Here we show Chrdl1 is dynamically expressed in the early distal limb bud mesenchyme, with expression becoming downregulated as development proceeds. Chrdl1 expression is largely excluded from the critical signaling center of the posterior limb bud, the Zone of Polarizing Activity (ZPA), as has been described for the BMP antagonist Gremlin (GREM1) ( Scherz et al., 2004, Science, 305, 396–399). Unlike Grem1, Chrdl1 is expressed in the hindlimb by a small subset of ZPA cells and their descendants suggesting divergent regulation and function between the various BMP antagonists. Ectopic expression of Chrdl1 throughout the avian limb bud using viral misexpression resulted in an oligodactyly phenotype with loss of digits from the anterior limb, although the development of more proximal elements of the zeugopod and stylopod were unaffected. Overgrowths of soft tissue and syndactyly were also observed, resulting from impaired apoptosis and failure of the anterior mesenchyme to undergo SOX9-dependent chondrogenesis, instead persisting as an interdigital-like soft tissue phenotype. Sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling were upregulated and persisted later in development, however these changes were only detected late in limb development at timepoints when endogenous Grem1 would normally be downregulated and increasing BMP signaling would cause termination of Shh and Fgf expression. Our results suggest that the early stages of the GREM1–SHH–FGF signaling network are resistant to Chrdl1-overexpression, leading to normal formation of proximal limb structures, but that later Bmp expression, impaired by ectopic CHRDL1, is essential for formation of the correct complement of digits.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Synergistic Signaling of Tumor Cell Invasiveness by Hepatocyte Growth Factor and Hypoxia

Hepatocyte growth factor (HGF) signaling promotes tumor invasiveness in renal cell carcinoma (RCC) and other cancers. In clear cell RCC, VHL loss generates pseudohypoxia that exacerbates HGF-driven invasion through β-catenin deregulation. Hypoxia also enhances HGF-driven invasiveness by papillary RCC cells, but in the absence of VHL, loss signaling integration involves three parallel routes: 1) hypoxia-induced reactive oxygen species production and decreased DUSP2 expression, leading to enhanced mitogen-activated protein kinase (MAPK) cascade activation; 2) reactive oxygen species-induced diacylglycerol production by phospholipase Cγ, leading to protein kinase C activation and increased protein phosphatase-2A activity, thereby suppressing HGF-induced Akt activation; and 3) a profound shift from HGF-enhanced, proliferation-oriented metabolism to autophagy-dependent invasion and suppression of proliferation. This tripartite signaling integration was not unique to RCC or HGF; in RCC cells, invasive synergy induced by the combination of hypoxia and epidermal growth factor occurred through the same mechanism, and in estrogen receptor-positive breast cancer cells, this mechanism was suppressed in the absence of estrogen. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states, and provide signaling profiles by which they may be predicted.     

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.     

Co-expression of the HGF/SF and c-met genes during early mouse embryogenesis precedes reciprocal expression in adjacent tissues during organogenesis

Early experiments with cells in culture and recent targeting experiments have confirmed that the mesenchyme-derived growth factor hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine agent that regulates the development of several epithelial and myogenic precursor cells during organogenesis. Here, we report the expression pattern of HGF/SF and its receptor, the product of the proto-oncogene c-met, during gastrulation and early organogenesis in mouse embryo. During gastrulation, the expression of HGF/SF and c-met overlaps. Initially the two genes are expressed in the endoderm and in the mesoderm along the rostro-intermediate part of the primitive streak and, later, in the node and in the notochord. Neither HGF/SF nor c-met is expressed in the ectodermal layer throughout gastrulation. During early organogenesis, overlapping expression of HGF/SF and c-met is found in heart, condensing somites and neural crest cells. However, a second and distinct pattern of expression, characterized by the presence of the ligand in mesenchymal tissues and the receptor in the surrounding ectoderm, is seen in the branchial arches and in the limb buds. At 13 days postcoitum (d.p.c.), only this second pattern of expression is observed in differentiated somites and several major organs (i.e., lungs, liver, and gut. The expression of the HGF/SF and c-met genes throughout embryogenesis suggests a shift from an autocrine to a paracrine signaling system. The shift takes place in early organogenesis and implies different roles of HGF/SF in development. During gastrulation, HGF/SF may affect the fate of migrating mesodermal cells and may play a role in axis determination, whereas during organogenesis, the expression patterns of HGF/SF and its receptor reflect the recently established roles in the growth of certain epithelia and the migration of specific myogenic precursor cells. © 1996 Wiley-Liss, Inc.     

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.     

HGF/SF-met signaling in the control of branching morphogenesis and invasion

Hepatocyte growth factor/Scatter factor (HGF/SF) is a multifunctional growth factor which can induce diverse biological events. In vitro, these include scattering, invasion, proliferation and branching morphogenesis. In vivo, HGF/SF is responsible for many processes during embryonic development and a variety of activities in adults, and many of these normal activities have been implicated in its role in tumorgenesis and metastasis. The c-Met receptor tyrosine kinase is the only known receptor for HGF/SF and mediates all HGF/SF induced biological activities. Upon HGF/SF stimulation, the c-Met receptor is tyrosine-phosphorylated which is followed by the recruitment of a group of signaling molecules and/or adaptor proteins to its cytoplasmic domain and its multiple docking sites. This action leads to the activation of several different signaling cascades that form a complete network of intra and extracellular responses. Different combinations of signaling pathways and signaling molecules and/or differences in magnitude of responses contribute to these diverse series of HGF/SF-Met induced activities and most certainly are influenced by cell type as well as different cellular environments. In this review, we focus on HGF/SF-induced branching morphogenesis and invasion, and bring together recent new findings which provide insight into how HGF/SF, via c-Met induces this response.     

TM4SF1, a binding protein of DVL2 in hepatocellular carcinoma,positively regulates beta-catenin/TCF signalling

The interaction between Axin and DVL2 is critical for the breaking down of the beta-catenin destruction complex and the activation of the Wnt/beta-catenin cascade. However, this biological process remains poorly understood. In the present study, TM4SF1 was identified as the interacting partner of DVL2 and positively regulated as Wnt/beta-catenin signalling by strengthening the DVL2-Axin interaction. The expression levels of TM4SF1 were elevated in hepatocellular carcinoma (HCC) and were induced by Kras signalling. The overexpression of TM4SF1 promoted the growth and motility of HCC cells, and up-regulated the target genes (Axin2 and cyclin D1). The down-regulation of TM4SF1 impaired the capability of HCC cells for growth, migration and metastasis. In addition, the down-regulation of TM4SF1 promoted the ubiquitination of beta-catenin. In summary, these results reveal the oncogenic functions of TM4SF1 in HCC progression and suggest that TM4SF1 might be a target for treatment.     

A new crystal form of the NK1 splice variant of HGF/SF demonstrates extensive hinge movement and suggests that the NK1 dimer originates by domain swapping

NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (N) and first kringle (K) domains and retains receptor binding and signalling. While NK1 behaves as a monomer in solution, two independent crystallographic structures have previously shown an identical, tightly packed dimer. Here we report a novel orthorhombic crystal form of NK1 at 2.5 A resolution in which four NK1 protomers are packed in two distinct dimers in the asymmetric unit. Although the basic architecture of the new NK1 dimers is similar to the two described earlier, the new crystal form demonstrates extensive hinge movement between the N and K domain that leads to re-orientation of the receptor-binding sites. The hinge bending is evidence of the paucity of strong interactions between domains within the protomer, in contrast to the extensive interactions between protomers in the dimer. These observations are consistent with domain swapping in the dimer, such that the interdomain interactions of the monomer are replaced by equivalent interprotomer interactions in the dimer and offer a route for protein engineering of NK1 variants which may act as receptor antagonists.