Expression of activated MAP kinase in Xenopus laevis embryos: evaluating the roles of FGF and other signaling pathways in early induction and patterning

FGF signaling has been implicated in germ layer formation and axial determination. An antibody specific for the activated form of mitogen-activated protein kinase (MAPK) was used to monitor FGF signaling in vivo during early Xenopus development. Activation of MAPK in young embryos is abolished by injection of a dominant negative FGF receptor (XFD) RNA, suggesting that MAPK is activated primarily by FGF in this context. A transition from cytoplasmic to nuclear localization of activated MAPK occurs in morula/blastula stage embryo animal and marginal zones coinciding with the proposed onset of mesodermal competence. Activated MAPK delineates the region of the dorsal marginal zone before blastopore formation and persists in this region during gastrulation, indicating an early role for FGF signaling in dorsal mesoderm. Activated MAPK was also found in posterior neural tissue from late gastrulation onward. Inhibition of FGF signaling does not block posterior neural gene expression (HoxB9) or activation of MAPK; however, inhibition of FGF signaling does cause a statistically significant decrease in the level of activated MAPK. These results point toward the involvement of other receptor tyrosine kinase signaling pathways in posterior neural patterning.     

Arabidopsis MAP kinase phosphatase 1 and its target MAP kinases 3 and 6 antagonistically determine UV-B stress tolerance, independent of the UVR8 photoreceptor pathway

Plants perceive UV-B radiation as an informational signal by a pathway involving UVR8 as UV-B photoreceptor, activating photomorphogenic and acclimation responses. In contrast, the response to UV-B as an environmental stress involves mitogen-activated protein kinase (MAPK) signalling cascades. Whereas the perception pathway is plant specific, the UV-B stress pathway is more broadly conserved. Knowledge of the UV-B stress-activated MAPK signalling pathway in plants is limited, and its potential interplay with the UVR8-mediated pathway has not been defined. Here, we show that loss of MAP kinase phosphatase 1 in the mutant mkp1 results in hypersensitivity to acute UV-B stress, but without impairing UV-B acclimation. The MKP1-interacting proteins MPK3 and MPK6 are activated by UV-B stress and are hyperactivated in mkp1. Moreover, mutants mpk3 and mpk6 exhibit elevated UV-B tolerance and partially suppress the UV-B hypersensitivity of mkp1. We show further that the MKP1-regulated stress-response MAPK pathway is independent of the UVR8 photoreceptor, but that MKP1 also contributes to survival under simulated sunlight. We conclude that, whereas UVR8-mediated acclimation in plants promotes UV-B-induced defence measures, MKP1-regulated stress signalling results when UV-B protection and repair are insufficient and damage occurs. The combined activity of these two mechanisms is crucial to UV-B tolerance in plants.     

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.     

FGF receptor gene expression and its regulation by FGF signaling during early zebrafish development

The expression of all four fgfr genes was extensively examined throughout early embryogenesis of the zebrafish (Danio rerio). fgfr1 alone was expressed maternally throughout the blastoderm, and then zygotically in the anterior neural plate and presomitic mesoderm. fgfr4 expression was first detected in late blastulae and was gradually restricted to the brain. fgfr2 and fgfr3 expression were initiated in early and late gastrulae, respectively; fgfr2 was expressed in the anterior neural plate and somitic mesoderm, whereas fgfr3 was activated in the axial mesoderm and then in the midbrain and somitic mesoderm. During somitogenesis, each of these fgfr genes was expressed in a characteristic manner in the brain. Using an FGF signal inhibitor, dominant-negative FGF receptors and fgf8.1/fgf8a mutants, we found that fgfr expression is directly or indirectly regulated by FGF signaling during epiboly and at the end of somitogenesis, revealing the presence of an autoregulatory mechanism.     

Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium.     

MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. Here we show that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI3K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.     

Gastrulation in zebrafish: what mutants teach us.

A major approach to the study of development is to compare the phenotypes of normal and mutant individuals for a given genetic locus. Understanding the development of a complex metazoan therefore requires examination of many mutants. Relatively few organisms are being studied this way, and zebrafish is currently the best example of a vertebrate for which large-scale mutagenesis screens have successfully been carried out. The number of genes mutated in zebrafish that have been cloned expands rapidly, bringing new insights into a number of developmental pathways operating in vertebrates. Here, we discuss work on zebrafish mutants affecting gastrulation and patterning of the early embryo. Gastrulation is orchestrated by the dorsal organizer, which forms in a region where maternally derived beta-catenin signaling is active. Mutation in the zygotic homeobox gene bozozok disrupts the organizer genetic program and leads to severe axial deficiencies, indicating that this gene is a functional target of beta-catenin signaling. Once established, the organizer releases inhibitors of ventralizing signals, such as BMPs, and promotes dorsoanterior fates within all germ layers. In zebrafish, several mutations affecting dorsal-ventral (D/V) patterning inactivate genes functioning in the BMP pathway, stressing the central role of this pathway in the gastrula embryo. Cells derived from the organizer differentiate into several axial structures, such as notochord and prechordal mesoderm, which are thought to induce various fates in adjacent tissues, such as the floor plate, after the completion of gastrulation. Studies with mutants in nodal-related genes, in one-eyed pinhead, which is required for nodal signaling, and in the Notch pathway reveal that midline cell fate specification is, in fact, initiated during gastrulation. Furthermore, the organizer coordinates morphogenetic movements, and zebrafish mutants in T-box mesoderm-specific genes help clarify the mechanism of convergence movements required for the formation of axial and paraxial mesoderm.     

Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis

Somites, the segmented building blocks of the vertebrate embryo, arise one by one in a patterning process that passes wavelike along the anteroposterior axis of the presomitic mesoderm (PSM). We have studied this process in Xenopus embryos by analyzing the expression of the bHLH gene, Thylacine1, which is turned on in the PSM as cells mature and segment, in a pattern that marks both segment boundaries and polarity. Here, we show that this segmental gene expression involves a PSM enhancer that is regulated by retinoic acid (RA) signaling at two levels. RA activates Thylacine1 expression in rostral PSM directly. RA also activates Thylacine1 expression in the caudal PSM indirectly by inducing the expression of MKP3, an inhibitor of the FGF signaling pathway. RA signaling is therefore a major contributor to segmental patterning by promoting anterior segmental polarity and by interacting with the FGF signaling pathway to position segmental boundaries.     

sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish

In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, we have isolated a zebrafish sprouty4 gene, based on its extensive similarities with the expression patterns of both fgf8 and fgf3. Through gain- and loss-of-function experiments, we demonstrate that Fgf8 and Fgf3 act in vivo to induce the expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses, Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, an opposite phenotype to the dorsalisation induced by overexpression of Fgf8 or Fgf3. Conversely we have shown that inhibition of Spry4 function through injection of antisense morpholino oligonucleotide leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, we show that Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, our analysis reveals that signaling through FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the regulation of bone morphogenetic protein (BMP) expression.     

The role of NADPH oxidase and MAP kinase phosphatase in UV-B-dependent gene expression in Arabidopsis

Plant responses to supplementary UV-B irradiation have been reported to include formation of reactive oxygen species (ROS), hydrogen peroxide, in particular, and regulation by mitogen-activated protein kinase (MAPK) cascades which in turn are fine-tuned by MAPK phosphatases (MKPs). Here we present direct genetic evidence for the involvement of plasma membrane NADPH oxidase, a source of superoxide and hydrogen peroxide in the apoplasts, in UV-B signalling in Arabidopsis thaliana, by analysis of gene expression of the UV-B molecular markers in NADPH oxidase (atrbohD, F and DF) and MAP kinase phosphatase 1 (MKP1) knockout mutants (mkp1). Whereas the NADPH oxidase mutants were affected in UV-B-dependent CHS, PYROA and MEB5.2 gene expression, the mkp1 mutant was affected in the general expression pattern of the pathogenesis-related (PR) and PDF1.2 genes. The results indicate involvement of MKP1 in repressive action on gene expression of more general stress response pathways, similar to those activated by pathogen attack, while NADPH oxidase is involved in quantitative (rather than absolute) regulation of more UV-B-specific genes. The expressions of the molecular markers in the knockout mutant mkp1 and in its complemented lines (lines 6 and 10) were similar, as opposed to the responses of the corresponding wild-type Wassilewskija-4 (Ws-4). Lines 6 and 10 showed much higher MKP1 mRNA than Ws-4 but did not complement the mutant. This suggests a complex dependency of the MAPK phosporylation level of the PR and PDF1.2 genes. Both NADPH oxidase mutants and the mkp1 mutant phenotypically responded to UV-B by growth retardation.     

Fgf signals from a novel signaling center determine axial patterning of the prospective neural retina

Axial eye patterning determines the positional code of retinal ganglion cells (RGCs), which is crucial for their topographic projection to the midbrain. Several asymmetrically expressed determinants of retinal patterning are known, but it is unclear how axial polarity is first established. We find that Fgf signals, including Fgf8, determine retinal patterning along the nasotemporal (NT) axis during early zebrafish embryogenesis: Fgf8 induces nasal and/or suppresses temporal retinal cell fates; and inhibition of all Fgf-receptor signaling leads to complete retinal temporalization and concomitant loss of all nasal fates. Misprojections of RGCs with Fgf-dependent alterations in retinal patterning to the midbrain demonstrate the importance of this early patterning process for late topographic map formation. The crucial period of Fgf-dependent patterning is at the onset of eye morphogenesis. Fgf8 expression, the restricted temporal requirement for Fgf-receptor signaling and target gene expression at this stage suggests that the telencephalic primordium is the source of Fgf8 and acts as novel signaling center for non-autonomous axial patterning of the prospective neural retina.     

A role for maternal beta-catenin in early mesoderm induction in Xenopus

Mesoderm formation results from an inducing process that requires maternal and zygotic FGF/MAPK and TGFbeta activities, while maternal activation of the Wnt/beta-catenin pathway determines the anterior-dorsal axis. Here, we show a new role of Wnt/beta-catenin signaling in mesoderm induction. We find that maternal beta-catenin signaling is not only active dorsally but also all around the equatorial region, coinciding with the prospective mesoderm. Maternal beta-catenin function is required both for expression of dorsal genes and for activation of MAPK and the mesodermal markers Xbra and eomesodermin. beta-catenin acts in a non- cell-autonomous manner upstream of zygotic FGF and nodal signals. The Wnt/beta-catenin activity in the equatorial region of the early embryo is the first example of a maternally provided mesoderm inducer restricted to the prospective mesoderm.     

Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos

BACKGROUND: The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS: We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS: Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.