In C. elegans, the early embryo contains five somatic founder cells (known as AB, MS, E, C and D) which give rise to very different lineages. Two simply produce twenty intestinal (E) or muscle (D) cells each, whereas the remainder produce a total of 518 cells, which collectively contribute in a complex pattern to a variety of tissues. A central problem in embryonic development is to understand how the developmental potential of blastomeres is restricted to permit the terminal expression of such complex differentiation patterns. lit-1 appears to play a central role in controlling the asymmetry of cell division during embryogenesis in C. elegans. Mutants in lit-1 suggest that its product controls up to six consecutive binary switches which cause one of the two equivalent cells produced at each cleavage to assume a posterior fate. Most blastomere identities in C. elegans may therefore stem from a process of stepwise binary diversification (Kaletta, 1997).
During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. The WRM-1 protein binds to LIT-1 in vivo and WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. These findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway (Rocheleau, 1999).
The signaling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signalling downregulates POP-1 activity in one posterior daughter cell called E. The genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1: Drosophila homolog TGF-ß activated kinase 1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. It is concluded that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, since TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1 (Meneghini, 1999).
In C. elegans, a Wnt/WG-like signaling pathway down-regulates the TCF/LEF- related protein, POP-1, to specify posterior cell fates. Effectors of this signaling pathway include a beta-catenin homolog, WRM-1, and a conserved protein kinase, LIT-1. WRM-1 and LIT-1 form a kinase complex that can directly phosphorylate POP-1, but how signaling activates WRM-1/LIT-1 kinase is not yet known. mom-4, a genetically defined effector of polarity signaling, encodes a MAP kinase kinase kinase-related protein that stimulates the WRM-1/LIT-1-dependent phosphorylation of POP-1. LIT-1 kinase activity requires a conserved residue analogous to an activating phosphorylation site in other kinases, including MAP kinases. These findings suggest that anterior/posterior polarity signaling in C. elegans may involve a MAP kinase-like signaling mechanism (Shin, 1999).
Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. Nlk, a murine homolog of the Drosophila nemo (nmo) gene, has been cloned. The Nlk amino acid sequence is 54.5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4.0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60%-70% of Nlk is localized to the nucleus, whereas 30%-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks (Brott, 1998).
The stromal compartment of the bone marrow is composed of various cell types that provide trophic and instructive signals for hematopoiesis. The mesenchymal stem cell is believed to give rise to all major cellular components of the bone marrow microenvironment. Nemo-like kinase, Nlk, is a serine-threonine kinase that connects MAP kinase and Wnt signaling pathways; its in vivo function in mouse is unknown. Mice have been generated with a targeted disruption of Nlk; the complex phenotype significantly varies with the genetic background. Whereas C57BL/6 mice lacking Nlk die during the third trimester of pregnancy, the 129/Sv background supports survival into adolescence; such mice are growth retarded and suffer from various neurological abnormalities. The Nlk deficiency syndrome includes aberrant differentiation of bone marrow stromal cells. Varying degrees of morphological abnormality, such as increased numbers of adipocytes, large blood sinuses and absence of bone-lining cells are observed in the bone marrow of mutant mice. Nlk deficient mice thus provide a novel model to study the genetic requirements for bone marrow stromal differentiation (Kortenjann, 2001).
The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and C. elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. This study shows that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway (Ishitani, 1999).
Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor [beta(2)AR-Rfz-2] containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor [beta(2)AR] and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling (Ishitani, 2003a; full text of article).
The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. A specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. This study investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. It was found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity (Ishitani, 2003b; full text of article).
The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. This study reports that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps (Kanei-Ishii, 2004; full text of article).
Genetic studies on endoderm-mesoderm specification in C. elegans have demonstrated a role for several Wnt cascade components as well as for a MAPK-like pathway in this process. The latter pathway includes the MAPK kinase kinase-like MOM-4/Tak1, its adaptor TAP-1/Tab1, and the MAPK-like LIT-1/Nemo-like kinase. A model has been proposed in which the Tak1 kinase cascade counteracts the Wnt cascade at the level of beta-catenin/TCF phosphorylation. In this model, the signal that activates the Tak1 kinase cascade is unknown. As an alternative explanation of these genetic data, whether Tak1 is directly activated by Wnt was explored. It was found that Wnt1 stimulation results in autophosphorylation and activation of MOM-4/Tak1 in a TAP-1/Tab1-dependent fashion. Wnt1-induced Tak1 stimulation activates Nemo-like kinase, resulting in the phosphorylation of TCF. These results combined with the genetic data from C. elegans imply a mechanism whereby Wnt directly activates the MOM-4/Tak1 kinase signaling pathway. Thus, Wnt signal transduction through the canonical pathway activates beta-catenin/TCF, whereas Wnt signal transduction through the Tak1 pathway phosphorylates and inhibits TCF, which might function as a feedback mechanism (Smit, 2004; full text of article).
Wnt/ß-catenin signaling regulates many aspects of early vertebrate development, including patterning of the mesoderm and neurectoderm during gastrulation. In zebrafish, Wnt signaling overcomes basal repression in the prospective caudal neurectoderm by Tcf homologs that act as inhibitors of Wnt target genes. The vertebrate homolog of Drosophila nemo, nemo-like kinase (Nlk), can phosphorylate Tcf/Lef proteins and inhibit the DNA-binding ability of ß-catenin/Tcf complexes, thereby blocking activation of Wnt targets. By contrast, mutations in a C. elegans homolog show that Nlk is required to activate Wnt targets that are constitutively repressed by Tcf. Overexpressed zebrafish nlk, in concert with wnt8, can downregulate two tcf3 homologs, tcf3a and tcf3b, that repress Wnt targets during neurectodermal patterning. Inhibition of nlk using morpholino oligos reveals essential roles in regulating ventrolateral mesoderm formation in conjunction with wnt8, and in patterning of the midbrain, possibly functioning with wnt8b. In both instances, nlk appears to function as a positive regulator of Wnt signaling. Additionally, nlk strongly enhances convergent/extension phenotypes associated with wnt11/silberblick, suggesting a role in modulating cell movements as well as cell fate (Thorpe, 2004).
These results support a role for nlk in the activation of Wnt targets during zebrafish embryogenesis. Overexpressed nlk downregulates two tcf3 homologs, tcf3a and tcf3b, that repress activation of Wnt target genes during neural patterning. This functional interaction with Tcf3 homologs requires wnt8 signaling, and thus probably ß-catenin, consistent with previous data indicating that Nlk specifically interferes with the DNA-binding ability of ß-catenin/Tcf complexes, not that of Tcf alone. Interference with endogenous nlk function reveals important roles in two processes that are regulated by canonical Wnts, mesoderm patterning by wnt8, and patterning of midbrain and forebrain by wnt8b. Since loss of nlk enhances or phenocopies loss of function of these two Wnts, it is concluded that nlk functions as an activator of some canonical Wnt targets in zebrafish. nlk also interacts, directly or indirectly, with non-canonical Wnt pathways, since inhibition of nlk strongly enhances convergent extension phenotypes associated with loss of wnt11 function. A role was uncovered for an unusual wnt8 homolog, wnt8 ORF2, in regulating cell movements during gastrulation (Thorpe, 2004).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.