FRS2 family docking proteins with overlapping roles in activation of MAP kinase have distinct spatial-temporal patterns of expression of their transcripts

FRS2alpha and FRS2beta, two members of the FRS2 family of docking proteins, become tyrosine phosphorylated in response to fibroblast growth factor (FGF) or nerve growth factor (NGF) stimulation. Tyrosine phosphorylated FRS2alpha serves as a platform for the recruitment of multiple signaling proteins for activation of the Ras-mitogen-activated protein (MAP) kinase signaling cascade. We report that Frs2alpha and Frs2beta have distinct spatio-temporal expression patterns in mouse embryos. We further show that FRS2beta can compensate for the loss of FRS2alpha for activation of MAP kinase when expressed in fibroblasts from Frs2alpha(-/-) mouse embryos. We propose that the FRS2 family proteins have distinct roles in vivo through activation of common signaling proteins including MAP kinase.     

Generation of an Frs2alpha conditional null allele

The fibroblast growth factor (FGF) signaling family controls a broad spectrum of cellular processes in development and adult tissue homeostasis and function, which is expressed in almost all tissues at all stages. FGF receptor substrate 2 alpha (FRS2alpha) is an adaptor protein that recruits downstream substrates to the FGF receptor (FGFR) tyrosine kinase. Disruption of Frs2alpha gene in mice abrogates activation of the mitogen-activated protein kinase pathway by the FGFR and leads to embryonic lethality at day E7.5 post copulation. To circumvent the embryonic lethality resulting from disruption of the Frs2alpha gene, which hinders further characterization of the role of FRS2alpha in adult tissue function and homeostasis, we generated an Frs2alpha conditional null allele for temporally- and tissue-specific disruption of the Frs2alpha gene. Using gene targeting in mouse embryonic stem cells, we introduced two loxP sites flanking the largest coding exon, exon 5, in the Frs2alpha allele. Our results indicate that the floxed Frs2alpha (Frs2alpha(flox)) allele is a true conditional null allele that encodes wildtype activity and is converted to a null allele after Cre recombinase mediated recombination.     

Role of epithelial cell fibroblast growth factor receptor substrate 2alpha in prostate development, regeneration and tumorigenesis

The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2alpha (FRS2alpha) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2alpha is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2alpha was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2alpha function in the prostate, the Frs2alpha alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2alpha disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2alpha had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2alpha inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2alpha-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2alpha(cn) mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.     

Frs2alpha-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis

The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2alpha (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2alpha in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2alpha mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.     

The docking protein Gab1 is an essential component of an indirect mechanism for fibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/Akt antiapoptotic pathway

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.     

Bone morphogenetic protein 4 signaling regulates development of the anterior visceral endoderm in the mouse embryo

The extraembryonic ectoderm (ExE) of the mouse conceptus is known to play a role in embryo patterning by signaling to the underlying epiblast and surrounding visceral endoderm. Bmp4 is one of the key ExE signaling molecules and has been recently implicated to participate in regulating development and migration of the anterior visceral endoderm (AVE). However, it remains unclear when exactly BMP4 signaling starts to regulate AVE positioning. To examine this, we have chosen to affect BMP4 function at two different time points, at embryonic day 5.25 (E5.25), thus before AVE migration, and E5.75, just after AVE migration. To this end, an RNAi technique was used, which consisted of the injection of Bmp4 dsRNA into the proamniotic cavity of the egg cylinder followed by its targeted electroporation into the ExE. This resulted in specific knockdown of Bmp4 . It was found that Bmp4 RNAi at E5.25, but not at E5.75, led to an abnormal pattern of expression of the AVE marker Cerberus-like . Thus, BMP4 signaling appears to affect the expression of Cer1 at a specific time window. This RNAi approach provides a convenient means to study spatial and temporal function of genes shortly after embryo implantation.     

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.     

FRS2α is essential for the fibroblast growth factor to regulate the mTOR pathway and autophagy in mouse embryonic fibroblasts

Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/Akt pathway, and suppresses autophagy activity in MEFs. In addition, the PI3K/Akt pathway regulated mTOR is crucial for the FGF signaling axis to suppress autophagy in MEFs. Since autophagy has been proposed to play important roles in cell survival, proliferation, and differentiation, the findings suggest a novel mechanism for the FGF signaling axis to transmit regulatory signals to downstream effectors.     

Compartmentalizing Proximal FGFR1 Signaling in Ovine Placental Artery Endothelial Cell Caveolae

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.     

Developmental expression patterns of the signaling adapters FRS-2 and FRS-3 during early embryogenesis

Two fibroblast growth factor (FGF) receptor substrates (FRS2 and FRS3) are involved in downstream signaling from activated FGF receptors and neurotrophin-activated Trk receptors. Despite the importance of signaling from these factors in embryogenesis, FRS2 and FRS3 expression patterns during development are unknown. In this study we characterize the expression of FRS2 and FRS3 from E7 to parturition and in adult murine tissues. Both are first detected in whole E8.5 CD1 mouse embryos. FRS2 is detected as early as E7 in the developing syncytiotrophoblast, later in the neural tube (NT) and in many adult and fetal tissues. FRS3 is more restricted in location than FRS2 (fetal NT, heart, stomach, liver and some adult tissues), and is expressed predominantly in the ventricular layer of the developing NT and brains of murine embryos.     

A Highlights from MBoC Selection: Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency—fiber cell differentiation and gap junction–mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function.     

Reciprocal epithelial-mesenchymal FGF signaling is required for cecal development

Fibroblast growth factor (FGF) signaling mediates reciprocal mesenchymal-epithelial cell interactions in the developing mouse lung and limb. In the gastrointestinal (GI) tract, FGF10 is expressed in the cecal mesenchyme and signals to an epithelial splice form of FGF receptor (FGFR) 2 to regulate epithelial budding. Here, we identify FGF9 as a reciprocal epithelial-mesenchymal signal required for cecal morphogenesis. Fgf9 null (Fgf9(-/-)) mouse embryos have agenesis of the embryonic cecum, lacking both mesenchymal expansion and an epithelial bud. In the cecal region of Fgf9(-/-) embryos, mesenchymal expression of Fgf10 and Bmp4 is notably absent, whereas the expression of epithelial markers, such as sonic hedgehog, is not affected. Using epithelial and whole explant cultures, we show that FGF9 signals to mesenchymal FGFRs and that FGF10 signals to epithelial FGFRs. Taken together, these data show that an epithelial FGF9 signal is necessary for the expansion of cecal mesenchyme and the expression of mesenchymal genes that are required for epithelial budding. Thus, these data add to our understanding of FGF-mediated reciprocal epithelial-mesenchymal signaling.     

Essential roles of zebrafish bmp2a, fgf10, and fgf24 in the specification of the ventral pancreas

In vertebrates, pancreas and liver arise from bipotential progenitors located in the embryonic gut endoderm. Bone morphogenic protein (BMP) and fibroblast growth factor (FGF) signaling pathways have been shown to induce hepatic specification while repressing pancreatic fate. Here we show that BMP and FGF factors also play crucial function, at slightly later stages, in the specification of the ventral pancreas. By analyzing the pancreatic markers pdx1, ptf1a, and hlxb9la in different zebrafish models of BMP loss of function, we demonstrate that the BMP pathway is required between 20 and 24 h postfertilization to specify the ventral pancreatic bud. Knockdown experiments show that bmp2a, expressed in the lateral plate mesoderm at these stages, is essential for ventral pancreas specification. Bmp2a action is not restricted to the pancreatic domain and is also required for the proper expression of hepatic markers. By contrast, through the analysis of fgf10(-/-); fgf24(-/-) embryos, we reveal the specific role of these two FGF ligands in the induction of the ventral pancreas and in the repression of the hepatic fate. These mutants display ventral pancreas agenesis and ectopic masses of hepatocytes. Overall, these data highlight the dynamic role of BMP and FGF in the patterning of the hepatopancreatic region.     

Ongoing sonic hedgehog signaling is required for dorsal midline formation in the developing forebrain

The division of the mammalian forebrain into distinct left and right hemispheres represents a critical step in neural development. Several signaling molecules including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and bone morphogenetic proteins (BMPs) have been implicated in dorsal midline development, and prior work suggests that the organizing centers from which these proteins are secreted mutually regulate one another during development. To explore the role of the ventral organizing center in the formation of two hemispheres, we assessed dorsal midline development in Shh mutant embryos and in wildtype embryos treated with the SHH signaling inhibitor HhAntag. Collectively, our findings demonstrate that SHH signaling plays an important role in maintaining the normal expression patterns of Fgf8 and Bmp4 in the developing forebrain. We further show that FGF8 can induce the expression of Zic2, which is normally expressed at the midline and is required in vivo for hemispheric cleavage, suggesting that FGF signaling may stimulate dorsal midline development by inducing Zic2 expression.     

BMP is an important regulator of proepicardial identity in the chick embryo

The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo.     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.     

Binding of Shp2 Tyrosine Phosphatase to FRS2 Is Essential for Fibroblast Growth Factor-Induced PC12 Cell Differentiation

FRS2 is a lipid-anchored docking protein that plays an important role in linking fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein (MAP) kinase signaling pathway. In this report, we demonstrate that FRS2 forms a complex with the N-terminal SH2 domain of the protein tyrosine phosphatase Shp2 in response to FGF stimulation. FGF stimulation induces tyrosine phosphorylation of Shp2, leading to the formation of a complex containing Grb2 and Sos1 molecules. In addition, a mutant FRS2 deficient in both Grb2 and Shp2 binding induces a weak and transient MAP kinase response and fails to induce PC12 cell differentiation in response to FGF stimulation. Furthermore, FGF is unable to induce differentiation of PC12 cells expressing an FRS2 point mutant deficient in Shp2 binding. Finally, we demonstrate that the catalytic activity of Shp2 is essential for sustained activation of MAP kinase and for potentiation of FGF-induced PC12 cell differentiation. These experiments demonstrate that FRS2 recruits Grb2 molecules both directly and indirectly via complex formation with Shp2 and that Shp2 plays an important role in FGF-induced PC12 cell differentiation.     

Zili antagonizes Bmp signaling to regulate dorsal-ventral patterning during zebrafish early embryogenesis

Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-βsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.     

Retinoic acid regulates morphogenesis and patterning of posterior foregut derivatives

Retinoic acid (RA) is an embryonic signaling molecule regulating a wide array of target genes, thereby being a master regulator of patterning and differentiation in a variety of organs. Here we show that mouse embryos deficient for the RA-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2), if rescued from early lethality by maternal RA supplementation between E7.5 and E8.5, lack active RA signaling in the foregut region. The resulting mutants completely fail to develop lungs. Development of more posterior foregut derivatives (stomach and duodenum), as well as liver growth, is also severely affected. A primary lung bud is specified in the RA-deficient embryos, which fails to outgrow due to defective FGF10 signaling and lack of activation of FGF-target genes, such as Pea3 and Bmp4 in the epithelium. Specific Hox and Tbx genes may mediate these RA regulatory effects. Development of foregut derivatives can be partly restored in mutants by extending the RA supplementation until at least E10.5, but lung growth and branching remain defective and a hypoplastic lung develops on the right side only. Such conditions poorly restore FGF10 signaling in the lung buds. Explant culture of RALDH2-deficient foreguts show a capacity to undergo lung budding and early branching in the presence of RA or FGF10. Our data implicate RA as a regulator of gene expression in the early embryonic lung and stomach region upstream of Hox, Tbx and FGF10 signaling.