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.     

A role for MKP3 in axial patterning of the zebrafish embryo

Fibroblast growth factors (FGFs) are secreted molecules that can activate the RAS/mitogen-activated protein kinase (MAPK) pathway to serve crucial functions during embryogenesis. Through an in situ hybridization screen for genes with restricted expression patterns during early zebrafish development, we identified a group of genes that exhibit similar expression patterns to FGF genes. We report the characterization of zebrafish MAP kinase phosphatase 3 (MKP3; DUSP6 - Zebrafish Information Network), a member of the FGF synexpression group, showing that it has a crucial role in the specification of axial polarity in the early zebrafish embryo. MKP3 dephosphorylates the activated form of MAPK, inhibiting the RAS/MAPK arm of the FGF signaling pathway. Gain- and loss-of-function studies reveal that MKP3 is required to limit the extent of FGF/RAS/MAPK signaling in the early embryo, and that disturbing this inhibitory pathway disrupts dorsoventral patterning at the onset of gastrulation. The earliest mkp3 expression is restricted to the future dorsal region of the embryo where it is initiated by a maternal beta-catenin signal, but soon after its initiation, mkp3 expression comes under the control of FGF signaling. Thus, mkp3 encodes a feedback attenuator of the FGF pathway, the expression of which is initiated at an early stage so as to ensure correct FGF signaling levels at the time of axial patterning.     

Hindbrain patterning: FGFs regulate Krox20 and mafB/kr expression in the otic/preotic region

Krox20 and mafB/kr are regulatory genes involved in hindbrain segmentation and anteroposterior (AP) patterning. They are expressed in rhombomeres (r) r3/r5 and r5/r6 respectively, as well as in the r5/r6 neural crest. Since several members of the fibroblast growth factor (FGF) family are expressed in the otic/preotic region (r2-r6), we investigated their possible involvement in the regulation of Krox20 and mafB/kr. Application of exogenous FGFs to the neural tube of 4- to 7-somite chick embryos led to ectopic expression in the neural crest of the somitic hindbrain (r7 and r8) and to the extension of the Krox20- or mafB/kr-positive areas in the neuroepithelium. Application of an inhibitor of FGF signalling led to severe and specific downregulation of Krox20 and mafB/kr in the hindbrain neuroepithelium and neural crest. These data indicate that FGFs are involved in the control of regional induction and/or maintenance of Krox20 and mafB/kr expression, thus identifying a novel function for these factors in hindbrain development, besides their proposed more general role in early neural caudalisation.     

Analysis of mouse kreisler mutants reveals new roles of hindbrain-derived signals in the establishment of the otic neurogenic domain

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3−/−Fgf10−/− double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis.     

FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain

The segmentation of the vertebrate hindbrain into rhombomeres is highly conserved, but how early hindbrain patterning is established is not well understood. We show that rhombomere 4 (r4) functions as an early-differentiating signaling center in the zebrafish hindbrain. Time-lapse analyses of zebrafish hindbrain development show that r4 forms first and hindbrain neuronal differentiation occurs first in r4. Two signaling molecules, FGF3 and FGF8, which are both expressed early in r4, are together required for the development of rhombomeres adjacent to r4, particularly r5 and r6. Transplantation of r4 cells can induce expression of r5/r6 markers, as can misexpression of either FGF3 or FGF8. Genetic mosaic analyses also support a role for FGF signaling acting from r4. Taken together, our findings demonstrate a crucial role for FGF-mediated inter-rhombomere signaling in promoting early hindbrain patterning and underscore the significance of organizing centers in patterning the vertebrate neural plate.     

Physical and functional interactions between protein tyrosine phosphatase alpha, PI 3-kinase, and PKCdelta.

The somatostatin analogue, TT-232 inhibits cell proliferation and induces apoptosis in a variety of tumor cells both in vivo and in vitro. While the early transient activation of Erk/MAPK was found to be important for the induction of cell cycle arrest, the signaling pathway leading to the activation of Erk/MAPK had not been fully established. Here we present evidence that activation of the Erk/MAPK pathway by TT-232 involves PI 3-kinase, PKCdelta and the protein tyrosine phosphatase alpha (PTPalpha). We show a physical interaction of PI 3-kinase and PKCdelta with PTPalpha and show that the tyrosine phosphatase plays a role in the activation of MAPK. In this process, PTPalpha Ser-180 and Ser-204 phosphorylation is critical for the induction of phosphatase activity, which is required for dephosphorylation of pp60(c-src). Taken together, we demonstrate the physical and functional association between PI 3-kinase, PKCdelta and PTPalpha in a signaling complex that mediates the antitumor activity of the somatostatin analogue TT-232.     

FGF‐2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3‐K/GSK3β signaling

Fibroblast growth factor‐2 (FGF‐2) is widely used to culture human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells. Despite its importance in maintaining undifferentiated hESC phenotype, a lack of understanding in the role of FGF‐2 still exists. Here, we investigate the signaling events in hESC following the addition of exogenous FGF‐2. In this study, we show that hESC express all forms of fibroblast growth factor receptors (FGFRs) which co‐localize on Oct3/4 positive cells. Furthermore, downregulation of Oct3/4 in hESC occurs following treatment with an FGFR inhibitor, suggesting that FGF signaling may regulate Oct3/4 expression. This is also observed in iPS cells. Also, downstream of FGF signaling, both mitogen activated protein kinase (MAPK) and phosphoinositide 3‐kinase pathways (PI3‐K) are activated following FGF‐2 stimulation. Notably, inhibition of MAPK and PI3‐K signaling using specific kinase inhibitors revealed that activated PI3‐K, rather than MAPK, can mediate pluripotent marker expression. To understand the importance of PI3‐K activation, activation of Wnt/β‐catenin by FGF‐2 was investigated. Wnt signaling had been implicated to have a role in maintaining of pluripotent hESC. We found that upon FGF‐2 stimulation, GSK3β is phosphorylated following which nuclear translocation of β‐catenin and TCF/LEF activation occurs. Interestingly, inhibition of the Wnt pathway with Dikkopf‐1 (DKK‐1) resulted in only partial suppression of the FGF‐2 induced TCF/LEF activity. Prolonged culture of hESC with DKK‐1 did not affect pluripotent marker expression. These results suggest that FGF‐2 mediated PI3‐K signaling may have a direct role in modulating the downstream of Wnt pathway to maintain undifferentiated hESC. J. Cell. Physiol. 225: 417–428, 2010. © 2010 Wiley‐Liss, Inc.     

Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway

Sotos syndrome (SoS) is characterized by tall stature, characteristic craniofacial features and mental retardation. It is caused by haploinsufficiency of the NSD1 gene. In this study, our objective was to identify downstream effectors of NSD1 and to map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed NSD1 abnormality. To substantiate those results, phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen-Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as FGF4 and FGF13 contributed strongly to the differential expression in this pathway. In addition, a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. The Ras Interacting Protein 1 (RASIP1) was identified to exhibit upregulated expression in SoS. It was shown that RASIP1 dose-dependently potentiated bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. Additionally, we demonstrated NSD1 expression in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. In analogy, we propose that deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of NSD1 expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS.     

Bisindolylmaleimide I suppresses fibroblast growth factor-mediated activation of Erk MAP kinase in chondrocytes by preventing Shp2 association with the Frs2 and Gab1 adaptor proteins

Fibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCalpha, -gamma, -delta, and -epsilon) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKClambda/zeta pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC.     

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.