Table of contents
Other Drosophila MAP kinases
A mitogen-activated protein kinase (MAPK) has been cloned and sequenced from a Drosophila neoplasmic l(2)mbn cell line. This Drosophila kinase is a homologue of mammalian p38 MAPK and the yeast HOG1 gene and thus is referred to as Dp38. A distinguishing feature of all MAPKs is the conserved sequence TGY in the activation domain. In Drosophila l(2)mbn and Schneider cell lines, Dp38 is rapidly tyrosine 186-phosphorylated in response to osmotic stress, heat shock, serum starvation, and H2O2. However, unlike mammalian p38 MAPK, the addition of lipopolysaccharide (LPS) does not significantly affect the phosphorylation of Dp38 in the LPS-responsive l(2)mbn cell line. Following osmotic stress, tyrosine 186-phosphorylated forms of Dp38 MAPK are detected exclusively in nuclear regions of Schneider cells. Yeast complementation studies demonstrate that the Saccharomyces cerevisiae HOG1 mutant strain JBY10 (hog1-Delta1) is functionally complemented by Dp38 cDNA in hyperosmolar medium. These findings demonstrate that similar osmotic stress-responsive signal transduction pathways are conserved in yeast, Drosophila, and mammalian cells, whereas LPS signal transduction pathways appear to be different (Han, 1998).
Yeast Map kinases
Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner. The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1. Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis. Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade. Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation. Both proteins also affect the timing and extent of Mpk1 activation. This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation. Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2. Surprisingly, Mpk1 activation by pheromone is only partially impaired in cells lacking the MEK kinase Bck1. This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions (Buehrer, 1997).
Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules that regulate responses to diverse extracellular stimuli, developmental cues and environmental stresses. A MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is activated by an upstream protein kinase, such as Raf, Mos, or a MAPKK kinase. Ste7, a MAPKK in the yeast Saccharomyces cerevisiae, is required for two developmental pathways: mating and invasive (filamentous) growth. Kss1 and Fus3, the MAPK targets of Ste7, are required for mating, but their role in invasive growth has been unclear. Because no other S. cerevisiae MAPK has been shown to function in invasive growth, it was proposed that Ste7 may have non-MAPK targets. Instead, Kss1 is the principal target of Ste7 in the invasive-growth response in both haploids and diploids. Kss1 in its inactive form is a potent negative regulator of invasive growth. Ste7 acts to relieve this negative regulation by switching Kss1 from an inhibitor to an activator. These results indicate that this MAPK has a physiologically important function in its unactivated state. Comparison of normal and MAPK-deficient cells indicates that nitrogen starvation and activated Ras stimulate filamentous growth through both MAPK-independent and MAPK-dependent means (Cook, 1997).
In yeast, an overlapping set of mitogen-activated protein kinase (MAPK) signaling components controls mating, haploid invasion, and pseudohyphal development. Paradoxically, a single downstream transcription factor, Ste12, is necessary for the execution of these distinct programs. Ste12's DNA-binding domain contains similarites to the homeodomain. To direct pheromone-responsive transcription, Ste12 acts as a homomultimer or as a heteromultimer with the Mcm 1 protein. Developmental specificity requires a transcription factor of the TEA/ATTS family, Tec1, which cooperates with Ste12 during filamentous and invasive growth. The TEA domain is found in SV40 enhancer factor TEF-1 as well as an Aspergillus abaA regulatory gene product, which is necessary for spore differentiation. Drosophila Scalloped is a related protein. Purified derivatives of Ste12 and Tec1 bind cooperatively to enhancer elements called filamentation and invasion response elements (FREs), which program transcription that is specifically responsive to the MAPK signaling components required for filamentous growth. An FRE in the TEC1 promoter functions in a positive feedback loop required for pseudohyphal development (Madhani, 1997a).
Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which the mating pheromone now activates filamentation-specific gene expression, using the Kss1 MAPK (Madhani, 1997b).
The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither. This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition. Unphosphorylated Kss1 binds directly to the transcription factor Ste12. This binding is necessary for Kss1-mediated repression of Ste12: Ste7-mediated phosphorylation of Kss1 weakens Kss1-Ste12 interaction and relieves Kss1-mediated repression. Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth. Analysis of Kss1 mutants indicates that the activation loop of Kss1 controls binding to Ste12. Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression (Bardwell, 1998).
The mitogen-activated protein kinase (MAPK) pathway is a highly conserved eukaryotic signaling cascade that converts extracellular signals into various outputs, such as cell growth and differentiation. MAPK is phosphorylated and activated by a specific MAPK kinase (MAPKK): MAPKK is therefore considered to be an activating regulator of MAPK. Pmk1 is a MAPK that regulates cell integrity and which, with calcineurin phosphatase, antagonizes chloride homeostasis in fission yeast. Pek1, a MAPKK for Pmk1 MAPK, has now been identified. Pek1, in its unphosphorylated form, acts as a potent negative regulator of Pmk1 MAPK signaling. Mkh1, an upstream MAPKK kinase (MAPKKK), converts Pek1 from being an inhibitor to an activator. These results indicate that Pek1 has a dual stimulatory and inhibitory function that depends on its phosphorylation state. This switch-like mechanism could contribute to the all-or-none physiological response mediated by the MAPK signaling pathway (Sugiura, 1999).
In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival. Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Kss1 and Fus3 are shown to act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks (Cherkasova, 2003).
Specialized gene expression programs are induced by signaling pathways that act on transcription factors. Whether these transcription factors can function in multiple developmental programs through a global switch in promoter selection is not known. Genome-wide location analysis has been used to show that the yeast Ste12 transcription factor, which regulates mating and filamentous growth, is bound to distinct program-specific target genes dependent on the developmental condition. This condition-dependent distribution of Ste12 requires concurrent binding of the transcription factor Tec1 during filamentation and is differentially regulated by the MAP kinases Fus3 and Kss1. Program-specific distribution across the genome may be a general mechanism by which transcription factors regulate distinct gene expression programs in response to signaling (Zeitlinger, 2003).
In response to pheromone from a mating partner, Ste12 induces the expression of mating gene, whereas under certain starvation conditions, Ste12 induces genes involved in filamentous growth, which facilitates foraging for nutrients. Ste12 can activate transcription alone or in partnership with other transcription factors. Some mating genes are induced in response to pheromone by Ste12 alone, whereas other mating genes are regulated by Ste12 together with Mcm1. For its role in filamentous growth, Ste12 requires the function of Tec1, a well-conserved transcription factor of the ATTS/TEA (AbaA, TEF-1, TEC1, Scalloped/TEF-1, TEC1, AbaA) family. Ste12 binds cooperatively with Tec1 at filamentation-responsive elements (FREs) in vitro. These studies suggest that differential gene expression by Ste12 might be achieved through selective partnership with other transcription factors. It is not known, however, whether the two MAPK pathways that induce mating and filamentation differentially regulate Ste12 by activating distinct complexes already bound to DNA or by regulating the partnership and DNA binding behaviors of these complexes (Zeitlinger, 2003 and references therein).
Activation of Ste12 during mating and filamentation involves two different MAPK signal transduction pathways that share components. Since they share the MAPK kinase and the MAPK kinase, two MAPKs, Fus3 and Kss1, may be activated by the mating and filamentation pathways. Genetic studies indicate that Fus3 and Kss1 act in a partially redundant fashion during mating. Deletion of both MAPKs, but not either one alone, abolishes Ste12-dependent induction of mating genes in response to pheromone. The two MAPKs, however, have different effects on Ste12-dependent induction of filamentous growth. The kinase activity of Kss1 increases filamentation, whereas the kinase activity of Fus3 appears to suppress filamentation. The mechanism by which Fus3 and Kss1 might differentially regulate Ste12 is not clear. Genetic assays indicate that both MAPKs activate Ste12 through regulation of two inhibitors, Dig1 and Dig2. Ste12 can form a complex with these two proteins and is released from the complex upon activation by pheromone. Both MAPKs phosphorylate Dig1 and Dig2, as well as Ste12 (Zeitlinger, 2003 and references therein).
A genome-wide binding assay that combines chromatin immunoprecipitation (ChIP) with DNA microarray technology was used to study the binding behavior of Ste12 during mating and filamentation. Ste12 binds to distinct promoters in vivo under different conditions and the different sets of genes specify distinct developmental programs. The global switch in target gene specificity of Ste12 depends on the transcription factor Tec1 during filamentation and is differentially regulated by the two MAPKs. Both MAPKs have the ability to induce mating genes in response to pheromone, but Fus3 has an additional activity that inhibits Ste12 binding to filamentation genes under the same condition. Thus, regulation of Ste12 binding, and not selective activation of transcription factor complexes already bound to DNA, is the mechanism by which the two MAPKs regulate distinct gene expression programs and direct cells toward specific developmental fates (Zeitlinger, 2003 and references therein).
Regulation of gene expression by mitogen-activated protein kinases (MAPKs) is essential for proper cell adaptation to extracellular stimuli. Exposure of yeast cells to high osmolarity results in rapid activation of the MAPK Hog1, which coordinates the transcriptional program required for cell survival on osmostress. The mechanisms by which Hog1 and MAPKs in general regulate gene expression are not completely understood, although Hog1 can modify some transcription factors. It is proposed that Hog1 induces gene expression by a mechanism that involves recruiting a specific histone deacetylase complex to the promoters of genes regulated by osmostress. Cells lacking the Rpd3-Sin3 histone deacetylase complex are sensitive to high osmolarity and show compromised expression of osmostress genes. Hog1 interacts physically with Rpd3 in vivo and in vitro and, on stress, targets the deacetylase to specific osmostress-responsive genes. Binding of the Rpd3-Sin3 complex to specific promoters leads to histone deacetylation, entry of RNA polymerase II and induction of gene expression. Together, these data indicate that targeting of the Rpd3 histone deacetylase to osmoresponsive promoters by the MAPK Hog1 is required to induce gene expression on stress (De Nadal, 2004).
Signaling molecules such as Cdc42 and mitogen-activated protein kinases (MAPKs) can function in multiple pathways in the same cell. Proposed here is one mechanism by which such factors may be directed to function in a particular pathway, such that a specific response is elicited. Using genomic approaches, a new component of the Cdc42- and MAPK-dependent signaling pathway has been identified that regulates filamentous growth (FG) in yeast. This factor, called Msb2, is a FG-pathway-specific factor that promotes differential activation of the MAPK for the FG pathway, Kss1. Msb2 is localized to polarized sites on the cell surface and interacts with Cdc42 and with the osmosensor for the high osmolarity glycerol response (HOG) pathway, Sho1. Msb2 is glycosylated and is a member of the mucin family, proteins that in mammalian cells promote disease resistance and contribute to metastasis in cancer cells. Remarkably, loss of the mucin domain of Msb2 causes hyperactivity of the FG pathway, demonstrating an inhibitory role for mucin domains in MAPK pathway activation. Taken together, these data suggest that Msb2 is a signaling mucin that interacts with general components, such as Cdc42 and Sho1, to promote their function in the FG pathway (Cullen, 2004).
Msb2 is a member of the mucin family of proteins, which are glycosylated cell-surface adhesion proteins. In mammalian cells, mucins act as barriers to pathogen infection and are key factors in metastasis in a variety of human cancers. In addition, two membrane-spanning mucins in humans, MUC1 and MUC4, function as signaling molecules. Like Msb2, MUC1 and MUC4 are cell-surface integral-membrane proteins whose cytoplasmic tails interact with signaling molecules at the head of a cascade. MUC1 is a docking protein for ß-catenin, and tyrosine phosphorylation of the cytoplasmic domain of MUC1 activates a MAPK pathway, the Grb2-Sos-Ras-MEK-ERK2 pathway. MUC4 binds to the tyrosine kinase ErbB2/HER2/Neu, to trigger phosphorylation of ErbB2 and potentiate signaling through the ErbB2/ErbB3 heterodimeric receptor complex (Cullen, 2004 and references therein).
Several findings from this study may be extrapolated to signaling mucins in general. First, as for Msb2, the mucin domains of MUC1 and MUC4 (and others) may have inhibitory roles. Hence, mutation of mucin domains may cause pathway activation and contribute to cancer progression in mammalian cells. The sequence similarity of mucin tandem repeats makes them highly susceptible to recombination-mediated deletion, as can occur for Msb2. Moreover, if, as for Msb2, the hyperactivity is dominant, then inappropriate pathway activation in mucin-deleted receptors may be prevalent among human cancers. The duality of mucin function in signaling pathway function should be a consideration in studies of adhesion-dependent developmental responses in normal mammalian cells and for appropriate drug design in human tumors. Indeed, MUC1 has been reported to have positive and negative roles in signaling. A second consequence of this study comes from the finding that Msb2 interacts with Cdc42 to redirect cell polarity. This finding suggests a role for signaling mucins in regulating polarized growth. Intriguingly, both MUC1 and MUC4 are localized to the apical surfaces of epithelial cells. Elucidation of the roles of signaling mucins in signaling pathway activation and polarized growth will help define the mechanisms by which these molecules induce metastasis in human cancers (Cullen, 2004 and references therein).
Signaling specificity is fundamental for parallel mitogen-activated protein kinase (MAPK) cascades that control growth and differentiation in response to different stimuli. In Saccharomyces cerevisiae, components of the pheromone-responsive MAPK cascade activate Fus3 and Kss1 MAPKs to induce mating and Kss1 to promote filamentation. Active Fus3 is required to prevent the activation of the filamentation program during pheromone response. How Fus3 prevents the crossactivation is not clear. Tec1, a cofactor of Ste12 for the expression of filamentation genes, is rapidly degraded during pheromone response. Fus3 but not Kss1 induces Tec1 ubiquination and degradation through the SCFCdc4 ubiquitin ligase. T273, in a predicted high-affinity Cdc4 binding motif, is phosphorylated by Fus3 both in vitro and in vivo. Tec1T273V blocks Tec1 ubiquitination and degradation and allows the induction of filamentation genes in response to pheromone. Thus, Fus3 inhibits filamentous growth during mating by degrading Tec1 (Chou, 2004).
The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Pheromone signaling is shown to induce Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway. Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273. Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro. Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF ubiquitin ligases. Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth. Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity (Bao, 2004).
In yeast, hyperosmotic stress causes an immediate dissociation of most proteins from chromatin, presumably because cells are unprepared for, and initially unresponsive to, increased ion concentrations in the nucleus. Osmotic stress activates Hog1 MAP kinase, which phosphorylates at least two proteins located at the plasma membrane, the Nha1 Na+/H+ antiporter and the Tok1 potassium channel. Hog1 phosphorylation stimulates Nha1 activity, and this is crucial for the rapid reassociation of proteins with their target sites in chromatin. This initial response to hyperosmolarity precedes and temporally regulates the activation of stress-response genes that depends on Hog1 phosphorylation of transcription factors in the nucleus. Thus, a single MAP kinase coordinates temporally, spatially, and mechanistically distinct responses to stress, thereby providing very rapid stress relief that facilitates subsequent changes in gene expression that permit long-term adaptation to harsh environmental conditions (Proft, 2004).
MAP kinase in C. elegans
In Caenorhabditis elegans, the MAP kinase signaling pathway is required for the development of multiple tissues: the hermaphrodite vulva, the male tail, the excretory system, the germline, the sex myoblasts, and possibly the posterior ectoderm as well. Of these tissues, the function of MAP kinase in the vulva has been the best characterized. During the L1 larval stage, six vulval precursor cells are generated along the ventral midline of the worm. LIN-3, a protein similar to epidermal growth factor, is produced by the gonadal anchor cell and initiates vulval development by activating the EGF receptor tyrosine kinase homolog LET-23 in the closest vulval precursor cell. Activation of LET-23 RTK triggers a conserved Grb2/Ras/Raf/MEK/MAP kinase cascade. Thus, the let-23 receptor stimulated mpk-1 MAP kinase signaling pathway functions to induce the vulva in C. elegans. MPK-1 directly regulates both the LIN-31 winged-helix transcription factor (a homolog of Drosophila Forkhead) and the LIN-1 Ets transcription factor (a homolog of Drosophila Pointed) to specify the vulval cell fate. lin-31 and lin-1 act genetically downstream of mpk-1, and both proteins can be directly phosphorylated by MAP kinase. LIN-31 binds to LIN-1, and the LIN-1/LIN-31 complex inhibits vulval induction. Phosphorylation of LIN-31 by MPK-1 disrupts the LIN-1/LIN-31 complex, relieving vulval inhibition. Phosphorylated LIN-31 may also act as a transcriptional activator, promoting vulval cell fates. LIN-31 is a vulval-specific effector of MPK-1, while LIN-1 acts as a general effector. The partnership of tissue-specific and general effectors may confer specificity onto commonly used signaling pathways, creating distinct tissue-specific outcomes (Tan, 1998).
The MAP kinase phosphorylation of general factors is well known, but only recently have tissue-specific effectors been found. In mammals, the bHLH transcription factor Microphthalmia and the PPAR nuclear receptor are tissue-specific effectors of MAP kinase signaling in melanocyte and adipocyte differentiation, respectively. Like LIN-31, these tissue-specific effectors may interact with general effectors (e.g., Elk-1) to initiate new programs of gene expression. In Drosophila, the MAP kinase Rolled may directly regulate at least three transcription factors: Aop/Yan, PointedP2, and dJun. However, none of these transcription factors is tissue specific. Thus, the mechanism of signaling specificity in Drosophila is currently unknown. Tissue-specific effectors of MAP kinase signaling may form an important class of proteins that confer specificity onto generally used signaling pathways so that diverse cellular outcomes can ultimately be generated (Tan, 1998).
Early C. elegans embryos exhibit protein asymmetries that allow rapid diversification of cells. Establishing these asymmetries requires the novel protein MEX-5. Mutations in the efl-1 and dpl-1 genes cause defects in protein localization resembling defects caused by mutations in mex-5. efl-1 and dpl-1 encode homologs of vertebrate E2F and DP proteins that regulate transcription as a heterodimer. efl-1 and dpl-1 mutants have elevated levels of activated Map kinase in oocytes. Their mutant phenotype and that of mex-5 mutants can be suppressed by reducing Ras/Map kinase signaling. It is proposed that this signaling pathway has a role in embryonic asymmetry and that EFL-1/DPL-1 control the level of Map kinase activation. The biochemical function of MEX-5 has not been established; however, PIE-1 localization is controlled by protein degradation. MEX-5 could have an important role in this process because MEX-5 expression is reciprocal to PIE-1 (a protein involved in embryonic polarity) in wild-type embryos, and ectopic expression of MEX-5 is sufficient to repress the expression of proteins similar to PIE-1. If MEX-5 controls PIE-1 degradation, this activity must be stimulated by events that occur after fertilization; oocytes show high, uniform levels of both PIE-1 and MEX-5 prior to fertilization (Page, 2001).
MAPK activation in C. elegans oocytes appears to depend on the presence of sperm or seminal fluid. Since MAPK becomes activated in oocytes that do not physically contact the spermatheca, where sperm are stored, an activating signal may either diffuse from the spermatheca or be relayed from the oocytes or somatic cells that contact the spermatheca. Once MAPK is activated in oocytes, it is proposed that the level of activation is determined by one or more transcriptional targets of EFL-1 and DPL-1. MAPK appears to be activated to an abnormally high level in efl-1 and dpl-1 mutants, and the phenotypic defects of efl-1 and dpl-1 mutants can be suppressed by reduction-of-function mutations in the Ras/MAPK pathway. Analysis of MAPK activation in oocytes from dpl-1;fem-1 mutants suggests two general models: dpl-1 oocytes either lack negative regulators of MAPK or contain excessive amounts of positive regulators. Wild-type oocytes that are near the spermatheca appear to be transcriptionally quiescent, suggesting that the level of MAPK activation is determined by gene products that are transcribed in another region of the syncytial gonad. EFL-1 and DPL-1 are both expressed in a band of syncytial germ nuclei before the transition zone where nuclei first cellularize and become oocytes, consistent with the view that EFL-1 and DPL-1 function in the transcription of gene products important for oogenesis (Page, 2001).
The region where EFL-1 is expressed in adult gonads corresponds closely to the region of MAPK activation in pachytene-stage nuclei. This localization pattern, combined with the analysis of MAPK in oocytes, suggests the possibility that MAPK activation might stimulate EFL-1 expression; transcriptional targets of EFL-1 might then, in turn, repress MAPK activation. Gonads in old fem-1 adults eventually lose all detectable activated MAPK but continue to express EFL-1. These results suggest that MAPK activation is not required to maintain EFL-1 expression but do not address the possibility that MAPK activation is involved in initiating EFL-1 expression (Page, 2001).
In C. elegans embryos, a Wnt/MAPK signaling pathway downregulates the TCF/LEF transcription factor POP-1, resulting in a lower nuclear level in signal-responsive cells compared to their sisters. Although the ß-catenin WRM-1 is required for POP-1 downregulation, a direct interaction between these two proteins does not seem to be required, since the ß-catenin-interacting domain of POP-1 is dispensable for both POP-1 downregulation and function in early embryos. WRM-1 downregulates POP-1 by promoting its phosphorylation by the MAP kinase LIT-1 and subsequent nuclear export via a 14-3-3 protein, PAR-5. In signal-responsive cells, a concurrent upregulation of nuclear LIT-1 that is dependent on Wnt/MAPK signaling is also detected. These results suggest a model whereby Wnt/MAPK signaling downregulates POP-1 levels in responsive cells, in part by increasing nuclear LIT-1 levels, thereby increasing POP-1 phosphorylation and PAR-5-mediated nuclear export (Lo, 2004).
Is the Wnt/MAPK-induced nuclear export of a TCF protein described in this study a C. elegans-specific phenomenon? C. elegans POP-1 is the only TCF protein known to undergo nucleocytoplasmic redistribution upon Wnt signaling. TCF/LEF proteins appear to be constitutive nuclear proteins in all other organisms examined so far. In addition, the canonical Wnt signaling pathway results in the activation of Wnt-responsive genes via a TCF/ß-catenin complex. It would seem counterintuitive to lower the level of nuclear TCF/LEF proteins in order to activate transcription in this model. Therefore, it is possible that the Wnt-induced nuclear export of TCF proteins only occurs in C. elegans embryos where POP-1 functions mainly as a repressor. However, two results suggest that Wnt signaling-induced nuclear export of TCF proteins may not be limited to C. elegans embryos. (1) It has been shown in flies that reduction of dTcf (Pangolin) partially suppresses, whereas its overexpression enhances, the wingless mutant phenotype. This is consistent with a model where Wnt signaling lowers the level of TCF proteins. (2) In the development of C. elegans male tail, Wnt signaling lowers the nuclear level of POP-1 in the cell T.p, whose fate is specified by POP-1. LIT-1 homologs have been shown to regulate the activity of TCF/LEF proteins in a variety of organisms, and 14-3-3 proteins are highly conserved among eukaryotes. Therefore, it is an intriguing possibility that LIT-1 homologs and 14-3-3 proteins may also regulate nuclear export of TCF/LEF in other organisms (Lo, 2004).
The Caenorhabditis elegans mpk-1 gene encodes a MAP kinase protein that plays an important role in Ras-mediated induction of vulval cell fates. Mutations that eliminate mpk-1 activity result in a highly penetrant, vulvaless phenotype. A double mutant containing a gain-of-function mpk-1 mutation and a gain-of-function mek mutation (MEK phosphorylates and activates MPK-1) exhibits a multivulva phenotype. These results suggest that mpk-1 may transduce most or all of the anchor cell signal. Epistasis analysis suggests that mpk-1 acts downstream of mek-2 (encodes a MEK homolog) and upstream of lin-1 (encodes an Ets transcription factor) in the anchor cell signaling pathway. mpk-1 may act together with let-60 ras in multiple developmental processes, as mpk-1 mutants exhibit nearly the same range of developmental phenotypes as let-60 ras mutants (Lackner, 1998).
Six gain-of-function mutations were identified and characterized that define a new class of lin-1 allele. The new lin-1 mutants displayed protruding vulval tissue and defects in egg laying, possible indications of abnormalities in the vulval passageway. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting that this region is required for negative regulation. The C terminus of LIN-1 is a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, these results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, which is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases (Jacobs, 1998).
ß-Catenin can promote adhesion at the cell cortex and mediate Wnt signaling in the nucleus. In Caenorhabditis elegans, both WRM-1/ß-catenin and LIT-1 kinase localize to the anterior cell cortex during asymmetric cell division but to the nucleus of the posterior daughter afterward. Both the cortical and nuclear localizations are regulated by Wnts and are apparently coupled. The daughters show different nuclear export rates for LIT-1. These results indicate that Wnt signals release cortical WRM-1 from the posterior cortex to generate cortical asymmetry that may control WRM-1 asymmetric nuclear localization by regulating cell polarity (Takeshita, 2005).
After the four-cell stage of C. elegans development, the polarity of many cells, including the EMS blastomere and the T hypodermal cell, is regulated by the Wnt signaling pathway. Unlike the tissue-polarity Wnt pathway, which regulates cell polarity in Drosophila and mammals independent of ß-catenin, the Wnt pathway that controls the EMS polarity involves WRM-1/ß-catenin and the POP-1/TCF transcription factor and hence is related to the canonical Wnt pathway. Unlike ß-catenin in other organisms, WRM-1 does not bind to cadherins and functions in Wnt signaling only, but not in cell adhesion. In the canonical Wnt pathway, the Wnt signal regulates the stability and nuclear localization of ß-catenin. However, it is not known how the Wnt signal regulates WRM-1, especially because WRM-1 does not have the conserved phosphorylation sites of GSK3ß. Furthermore, the subcellular localization of WRM-1 has not been determined. Therefore, the function of WRM-1 in the regulation of cell polarity has remained obscure (Takeshita, 2005).
In addition to the components of the Wnt pathway, LIT-1/MAP kinase and MOM-4/MAPKKK are involved in the EMS division. MOM-4 activates the LIT-1 kinase, while LIT-1 binds to WRM-1 to phosphorylate POP-1. Activation of this Wnt/MAPK pathway results in the asymmetric distribution of POP-1 between the nuclei of the daughter cells (POP-1 asymmetry). Unlike the Numb and Prospero proteins in Drosophila, however, POP-1 does not localize to the cell cortex during division. Instead, POP-1 asymmetry is regulated by nuclear export. Although the nuclear export of POP-1 is regulated by phosphorylation by the LIT-1-WRM-1 complex, it is not clear how the Wnt signaling pathway determines the difference in the rate of nuclear export between the daughter cells (Takeshita, 2005).
This study shows that WRM-1 and LIT-1 localized asymmetrically to the anterior cell cortex before and during the division of post-embryonic cells. Surprisingly, after division, WRM-1 and LIT-1 localized preferentially to the nucleus of the posterior rather than anterior daughters. These results suggest a role for cortical ß-catenin in the regulation of cell polarity, and provide a novel link between cortical and nuclear ß-catenin (Takeshita, 2005).
ß-Catenin regulates cell adhesion and cellular differentiation during development, and misregulation of ß-catenin contributes to numerous forms of cancer in humans. This study describes C. elegans conditional alleles of mom-2/Wnt, mom-4/Tak1, and wrm-1/ß-catenin. These reagents were used to examine the regulation of WRM-1/ß-catenin during a Wnt-signaling-induced asymmetric cell division. While WRM-1 protein initially accumulates in the nuclei of all cells, signaling promotes the retention of WRM-1 in nuclei of responding cells. Both PRY-1/Axin and the nuclear exportin homolog IMB-4/CRM-1 antagonize signaling. These findings reveal how Wnt signals direct the asymmetric localization of ß-catenin during polarized cell division (Nakamura, 2005).
A possible insight into the connection between cortical and nuclear signaling events comes from preliminary findings on the cortical localization of WRM-1. In the course of these studies, a faint localization of WRM-1::GFP to the cell cortex was seen during each mitosis. Interestingly, in the EMS cell (the 4-cell stage blastomere in C. elegans ), WRM-1::GFP is lost along the posterior cortex proximal to the signaling cell P2, while staining is maintained along the anterior cortex of the dividing EMS cell. This cortical localization is also visible at later stages and in developing larvae. These preliminary studies suggest that MOM-5/Frizzled is required for cortical association, while cortical release correlates with signaling via MOM-2/Wnt. Although these observations require further investigation, they suggest an interesting model that could explain how signaling at the cortex could drive nuclear WRM-1 asymmetries. Importantly, this model could also explain the difference between the penetrance of the endoderm defects seen in mom-2/Wnt mutants (~60% gutless) and mom-5/Fz mutants (only 5% gutless), and the surprising finding that the lower penetrance gutless phenotype of mom-5 is epistatic to mom-2 (Nakamura, 2005).
According to this model, P2/EMS signaling alters the affinity of WRM-1 for the posterior cortex of EMS and simultaneously activates WRM-1 for downstream signaling. This activation could be direct (e.g., by phosphorylation of WRM-1) or indirect (e.g., by modification of a WRM-1-interacting protein such as LIT-1). For simplicity in this discussion, the direct activation model will be considered. At steady state, only a small percentage of WRM-1 protein localizes at the cortex and this level drops during signaling, suggesting that cortical association may reflect a dynamic process that is modulated by signaling. Cortical signaling events also ensure that the mitotic apparatus of the cell is oriented such that division produces one nucleus that is more proximal to the posterior cortex and thus exposed to higher concentrations of an activated and less cortically associated form of WRM-1. At the beginning of telophase, WRM-1 accumulates in both nascent nuclei via a mechanism that depends on the kinases MOM-4 and LIT-1. During late telophase, and shortly after cytokinesis, IMB-4/CRM-1-dependent export begins to reduce WRM-1 nuclear levels in MS. However, in E, the signal-dependent release of an activated form of WRM-1 from the cortex induces a net nuclear retention of WRM-1. Finally, retention of WRM-1 in the nascent E (endoderm-restricted precursor) nucleus correlates with a simultaneous CRM-1-dependent nuclear export of POP-1 (Nakamura, 2005).
This model explains the phenotypic differences between mom-2 and mom-5 mutants. In mom-2 mutants, MOM-5 sequesters WRM-1 at the posterior cortex, reducing WRM-1 nuclear retention in E, and resulting in the higher penetrance of the mom-2 endoderm defect. In mom-5 mutants or in mom-2; mom-5 double mutants, signaling from P2 via the parallel SRC-1 tyrosine kinase pathway can activate WRM-1, which is then free to enter the nucleus and promote POP-1 nuclear export. Since SRC-1 has little effect on WRM-1 localization, these findings suggest that SRC-1 may instead alter WRM-1 activity (Nakamura, 2005).
The details of the mechanism that drives the reciprocal nuclear accumulation of WRM-1 and POP-1 are still not clear. The finding that the nuclear accumulation of WRM-1 partially depends on POP-1 suggests that WRM-1 and POP-1 may directly compete for nuclear export factors or nuclear/cytoplasmic retention sites. For example, WRM-1-dependent phosphorylation of POP-1 might increase the affinity of POP-1 for CRM-1, perhaps by promoting the interaction of POP-1 with PAR-5/14-3-3. This could lead to a direct competition that displaces WRM-1 from the export machinery in responding cells. Alternatively, signaling may alter the relative affinity of WRM-1 and/or POP-1 for binding to mutually exclusive partners in the nucleus or in the cytoplasm, causing a simultaneous and codependent shift in the net balance of their nuclear/cytoplasmic retention (Nakamura, 2005).
In summary, this study has analyzed the regulation of a ß-catenin homolog, WRM-1, during a polarized cell division in C. elegans. The findings suggest that WRM-1 is subject to regulation at multiple levels, and begin to place the surprising genetic complexity of P2/EMS signaling into a cell-biological context. Furthermore, the findings suggest that Wnt signaling can control the nuclear accumulation of ß-catenin and may also influence its cortical distribution. These modes of regulation may be of particular importance when Wnt signaling induces a polarized, asymmetric cell division (Nakamura, 2005).
Coupling the production of mature gametes and fertilized zygotes to favorable nutritional conditions improves reproductive success. In invertebrates, the proliferation of female germline stem cells is regulated by nutritional status. However, in mammals, the number of female germline stem cells is set early in development, with oocytes progressing through meiosis later in life. Mechanisms that couple later steps of oogenesis to environmental conditions remain largely undefined. This study shows that, in the presence of food, the DAF-2 insulin-like receptor signals through the RAS-ERK pathway to drive meiotic prophase I progression and oogenesis; in the absence of food, the resultant inactivation of insulin-like signaling leads to downregulation of the RAS-ERK pathway, and oogenesis is stalled. Thus, the insulin-like signaling pathway couples nutrient sensing to meiotic I progression and oocyte production in C. elegans, ensuring that oocytes are only produced under conditions favorable for the survival of the resulting zygotes (Lopez, 2013).
MAP kinase in other invertebrates
The mechanism by which fertilization initiates S-phase in the zygote was examined by manipulating the activity of MAP kinase in mature starfish eggs. These unfertilized eggs, which are arrested at G1-phase after the completion of meiosis, evince high MAP kinase activity but with undetectable cdc2 kinase activity. Either the process of fertilization or the inhibition of protein synthesis causes a decrease in MAP kinase activity, which is followed by DNA synthesis. Inactivation of MAP kinase with its specific phosphatase, CL100, initiates DNA synthesis in the absence of fertilization, while constitutive activation of MAP kinase with MEK represses the initiation of DNA synthesis following fertilization. Thus, in unfertilized mature starfish eggs, a capacity for DNA replication is already acquired, but entry into S-phase is negatively regulated by MAP kinase activity that is supported by a continuously synthesized protein(s) but not by cdc2 kinase. Upon fertilization, downregulation of MAP kinase activity is both necessary and sufficient for triggering the G1/S-phase transition (Tachibana, 1997).
During early development, gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs, which are translationally dormant or masked until meiotic maturation. Fertilization of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein: Drosophila homolog Orb) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilized eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. The protein kinase(s) that phosphorylates clam p82/CPEB were examined using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilization. p82/CPEB phosphorylation requires Ca2+ (<100 muM) in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. A model of clam p82/CPEB phosphorylation is proposed in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB (Katsu, 1999).
In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. The MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, the network was identified of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, it was show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. Ci-sushi encodes a protein containing domains known as complement control protein (CCP) modules, or short consensus repeats (SCR), which exist in a wide variety of complement and adhesion proteins. These observations have lead to proposal of a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi (Chambon, 2007).
The genes identified were separated for convenience as either upregulated or downregulated by JNK or ERK activity into four categories. There are genes involved in innate immunity, hormone signalling, metabolism of the extracellular matrix (ECM) or coding components of the ECM, and the remaining genes into one category that was term diverse genes. The screen identified genes that had previously been shown to be specifically expressed during ascidian metamorphosis, such as Ci-meta5, which is downregulated in response to JNK-inhibitor treatment. Ci-meta5 has been identified by differential screening of a cDNA library of swimming larvae and metamorphosing juveniles. Three genes (glutathione S-transferase, Cytochrome p450 and Gluthathione-requiring prostaglandin D synthase) were identified that are under the control of Ci-ERK and are orthologues of or closely related to genes expressed in papillae of the ascidians (Chambon, 2007).
Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein. Although it is not known whether Hemps activates ERK, it is tempting to speculate that it does, because activation of the Ras/Raf/ERK pathway by EGF is well described in many species. Moreover, the identification of genes controlled by Ci-ERK in papillae, the observation that metamorphosis does not occur with MEK inhibition, and data on Hemps that shows that it induces settlement and metamorphosis are consistent with that observations that one of the effects of the Hemps pathway is to activate the ERK cascade in papillae cells (Chambon, 2007).
Among the genes identified that are controlled by the JNK pathway, two are interesting: Ci-GNRH and Ci-oatp, which are involved, respectively, in the reproductive and thyroid axes. The mouse Oatp14 (also known as Slco1c1) was described in the transport of thyroxine across the blood-brain barrier. The role of thyroid hormones in metamorphosis had been reported previously in ascidians, and also in amphibians and lamprey. Moreover, in four ascidian species, thyroxin is present in larval mesenchyme and seems to be involved in the control of metamorphosis. The expression of Ci-oatp via JNK activation in the CNS may enhance thyroid signalling in larvae. Concerning GNRH, no report describes any function of this hormone in invertebrate metamorphosis. However, it is possible that GNRH may have a role in lamprey metamorphosis because, in sea lamprey, the level of GNRH increases throughout the stage of spontaneous metamorphosis (Chambon, 2007).
In addition to identifying genes involved in the immune system and hormonal signalling, a number of genes were identified coding for proteins involved in the composition or processing of the ECM. For example, Ci-LyOx, which is responsible for the cross linking and deposition of collagen fibres, elastin fibres and Ci-Mx, a matrix metalloprotease was identified. The regulation of matrix metalloprotease and the ECM remodelling have been shown to affect apoptosis in different systems, including the apoptotic remodelling of the intestine during Xenopus laevis metamorphosis and post-lactation involution of the mouse mammary gland. Anoikis is apoptosis induced by the loss of, or inappropriate, cell adhesion. It is tempting to hypothesize that one of the inductive signals from Ci-JNK in the CNS controls apoptosis by changing ECM composition. The role of JNK in ECM degradation has already been reported in rat aortic walls. In the tail of the tadpole, nerve corde is surrounded by matrix, which leads us to speculate that remodelling the ECM could provide a means to coordinate the response of tail cells in promoting either cell death or survival. In support of such a scenario, it was reported that, after a modification in ECM components, activation of the MAPK ERK leads to anoikis-type death. Because Ci-ERK activation precedes apoptosis in tail cells, this cell death could be regulated by JNK-controlled anoikis in the tail of ascidian tadpoles (Chambon, 2007).
Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. This study uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. The segregation of notochord and neural fates is an autonomous property of the mother cell, and the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. These cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. It has been demonstrated that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. A model is proposed whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification (Picco, 2007).
The ascidian neural plate has a grid-like organisation, with six rows and eight columns of aligned cells, generated by a series of stereotypical cell divisions. Unique molecular signatures have been defined for each of the eight cells in the posterior-most two rows of the neural plate - rows I and II. Using a combination of morpholino gene knockdown, dominant-negative forms and pharmacological inhibitors, the role of three signalling pathways was tested in defining these distinct cell identities. Nodal signalling at the 64-cell stage is required to define two different neural plate domains - medial and lateral - with Nodal inducing lateral and repressing medial identities. Delta2, an early Nodal target, then subdivides each of the lateral and medial domains to generate four columns. Finally, a separate signalling system along the anteroposterior axis, involving restricted ERK1/2 activation, was found to promote row I fates and repress row II fates. These results reveal how the sequential integration of three signalling pathways -- Nodal, Delta2/Notch and FGF/MEK/ERK -- defines eight different sub-domains that characterise the ascidian caudal neural plate. Most remarkably, the distinct fates of the eight neural precursors are each determined by a unique combination of inputs from these three signalling pathways (Hudson, 2007).
The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Using the planarian Dugesia japonica this study found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, is specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic RNA interference approaches provided evidence that ERK activity is required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induces an increased level of ERK activity and rescues the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration (Tasaki, 2011).
Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube (Shi, 2008).
This study presents evidence that competition between Eprhin and FGF signaling is important for the asymmetric specification of endoderm and mesoderm lineages from a common endomesoderm progenitor cell, the A6.3 blastomere. A similar mechanism was recently invoked to account for the asymmetric specification of the notochord and nerve cord from common A6.2 and A6.4 progenitors (Picco, 2007). In both cases, a localized Ephrin-Ad signal produced by the primitive ectoderm (the animal blastomeres) competes with FGF9 signals from the primitive gut. Those cells in extended contact with the ectoderm lack MAPK activation, whereas those cells in contact with the endoderm experience MAPK activation and follow a different fate. It is conceivable that this interplay of Ephrin and FGF signaling is used in other systems to produce asymmetric cell fates (Shi, 2008).
Ephrins have been implicated in a variety of cellular processes, including axonal guidance, repulsive cell-cell interactions, and adhesion. Different Ephrin family members can activate or inhibit RTK signaling in different cellular contexts. The present study, along with the recent analysis of Ci-Bra regulation (Picco, 2007), suggest that Ci-ephrin-Ad functions as a localized inhibitor of FGF signaling to produce asymmetric cell fates in Ciona. The presumptive endoderm/endomesoderm produces a localized source of FGF9/16/20, which induces the specification of diverse mesoderm lineages, including the notochord and mesenchyme. Evidence is presented that Ephrin also controls the subdivision of the A6.3 endomesoderm (Shi, 2008).
A model is presented for the specification of the A7.6 blastomere. Previous studies have shown that Nodal is essential for the expression of several A7.6 marker genes, including Hand-like (also known as NoTrlc), FGF8 and Delta-like. Nodal is expressed in the A6.3 blastomere of 32-cell embryos, as well as in the other progenitors of the endoderm. FGF/MAPK signaling is also active in the A6.3 at this stage, as judged by anti-dpERK staining. Ephrin-Ad produced by b6.5 (and other animal blastomeres) inhibits MAPK in A7.6, thereby permitting Nodal to activate the A7.6 group genes. Nodal signaling in A7.6 might be reinforced by Nodal expression in the b6.5 lineage. Thus, the inhibition of FGF signaling by Eprhin-Ad, along with augmented levels of Nodal signal, might be responsible for the activation of A7.6 group genes. However, evidence is presented that Nodal in b6.5 is not essential for A7.6 group gene expression. Instead, it would appear that the combination of endogenous Nodal in the A6.3 progenitor, along with the localized inhibition of MAPK in A7.6 by Eprhin-Ad, is the decisive determinant of A7.6 specification (Shi, 2008).
Inhibition of MAPK signaling via drug treatment or ectopic expression of Ephrin-Ad leads to misexpression of A7.6 marker genes in the anterior endoderm, where Nodal is normally inactive owing to FGF/MAPK signaling. Posterior endoderm cells also contain Nodal but fail to express A7.6 marker genes upon inhibition of MAPK. This might reflect the restricted distribution of additional activators required for A7.6 gene expression. For example, Hand-like is activated by the combination of Nodal signaling and the FoxA transcription factor. FoxA expression is restricted to the anterior endoderm, dorsal mesoderm and future CNS floorplate, but is absent from the posterior endoderm. This is consistent with the result of ectopic Hand-like and Delta-like activation in the A-line neural lineage by expression of the FoxD::Nodal transgene (Shi, 2008).
A7.6 expresses a number of localized determinants, including two crucial signaling molecules, FGF8 and Delta-like. A7.6 is located in a strategically important position within the vegetal hemisphere. It contacts components of all three germ layers: the endoderm, ectoderm and mesenchyme. The Delta-like ligand expressed in A7.6 induces the secondary notochord lineage via Notch signaling, and also induces the lateralmost neural fate. Similarly, FGF8 expression is required for maintaining the primary notochord fate. Because these signaling pathways require either direct cell-cell contact (Notch) or act over one or two cell diameters (FGF), it is crucial to activate the expression of Delta-like and FGF8 to A7.6, but not in its sibling A7.5 endoderm blastomere. The activities of three pathways, Ephrin, MAPK and Nodal signaling, are employed to achieve this precise asymmetric cell-fate specification event (Shi, 2008).
Recent phylogenetic analysis suggests that tunicates (e.g., Ciona) are the closest living relatives of the vertebrates. As a result, it is possible that vertebrates employ a mechanism for the specification and subdivision of the endomesoderm that is similar to the one used in Ciona. The A6.3 endomesoderm cell is established by the action of a localized maternal determinant, β-Catenin, which activates the expression of multiple signaling molecules including Nodal and FGF9. Nodal is required to activate A7.6-specific genes such as Hand-like, FGF8 and Delta-like. The failure of Nodal to activate A7.6 group genes in the endoderm is due to MAPK signaling. FGF signaling either directly or indirectly inhibits Nodal. As a result, Nodal signaling is blocked in A6.3, but is activated in A7.6 owing to the localized inhibition of FGF signaling by Eprhin-Ad (Shi, 2008).
Most or all metazoan embryos possess a transient endomesoderm that generates specific mesodermal derivatives. In vertebrates, the presumptive endomesoderm gives rise to blood, heart and muscle. Formation of the vertebrate endomesoderm depends on TGF-β signaling molecules such as Xnrs in Xenopus and Squint and Cyclops (Nodal-related 1 and 2, respectively; ZFIN) in zebrafish. The subsequent subdivision of the endomesoderm is not clearly understood, but might depend on FGF signaling. It remains to be seen if competitive interactions between Nodal (or some other TGF-β signaling molecule) and FGF lead to the subdivision of endomesoderm in vertebrate embryos (Shi, 2008).
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