Binary cell fate specification during C. elegans embryogenesis driven by reiterated reciprocal asymmetry of TCF POP-1 and its coactivator beta-catenin SYS-1

C. elegans embryos exhibit an invariant lineage comprised primarily of a stepwise binary diversification of anterior-posterior (A-P) blastomere identities. This binary cell fate specification requires input from both the Wnt and MAP kinase signaling pathways. The nuclear level of the TCF protein POP-1 is lowered in all posterior cells. We show here that the beta-catenin SYS-1 also exhibits reiterated asymmetry throughout multiple A-P divisions and that this asymmetry is reciprocal to that of POP-1. Furthermore, we show that SYS-1 functions as a coactivator for POP-1, and that the SYS-1-to-POP-1 ratio appears critical for both the anterior and posterior cell fates. A high ratio drives posterior cell fates, whereas a low ratio drives anterior cell fates. We show that the SYS-1 and POP-1 asymmetries are regulated independently, each by a subset of genes in the Wnt/MAP kinase pathways. We propose that two genetic pathways, one increasing SYS-1 and the other decreasing POP-1 levels, robustly elevate the SYS-1-to-POP-1 ratio in the posterior cell, thereby driving A-P differential cell fates.     

The C. elegans SYS-1 protein is a bona fide beta-catenin

C. elegans SYS-1 has key functional characteristics of a canonical beta-catenin, but no significant sequence similarity. Here, we report the SYS-1 crystal structure, both on its own and in a complex with POP-1, the C. elegans TCF homolog. The two structures possess signature features of canonical beta-catenin and the beta-catenin/TCF complex that could not be predicted by sequence. Most importantly, SYS-1 bears 12 armadillo repeats and the SYS-1/POP-1 interface is anchored by a conserved salt-bridge, the "charged button." We also modeled structures for three other C. elegans beta-catenins to predict the molecular basis of their distinct binding properties. Finally, we generated a phylogenetic tree, using the region of highest structural similarity between SYS-1 and beta-catenin, and found that SYS-1 clusters robustly within the beta-catenin clade. We conclude that the SYS-1 protein belongs to the beta-catenin family and suggest that additional divergent beta-catenins await discovery.     

Control of cell polarity by noncanonical Wnt signaling in C. elegans

The three Caenorhabditis elegans beta-catenin each function in distinct processes: BAR-1 in canonical Wnt signaling that controls cell fates and cell migrations, HMP-2 in cell adhesion and WRM-1 in Wnt signaling pathways that function in conjunction with a mitogen-activated kinase (MAPK) pathway to control the orientations, or cell polarities, of cells that undergo asymmetric cell divisions. In addition, WRM-1 does not interact with the canonical beta-catenin binding site in POP-1/Tcf. Thus, Wnt signaling through WRM-1 is noncanonical and, except for one division that might not include any of the three C. elegans beta-catenin, controls cell polarity in C. elegans.     

Crosstalk between a nuclear receptor and beta-catenin signaling decides cell fates in the C. elegans somatic gonad

beta-Catenin signaling determines the proximal-distal axis of the C. elegans gonad by promoting distal fate in asymmetrically dividing somatic gonad precursor cells (SGPs). Impaired function of the Wnt effector POP-1/TCF, its coactivator SYS-1/beta-catenin, and of upstream components including beta-catenin WRM-1 causes all SGP daughters to adopt the proximal fate. Consequently, no distal tip cells (DTCs) that would lead differentiation of gonad arms form in the affected hermaphrodites. Here, we show that deficiency of the nuclear receptor NHR-25 has the opposite effect: extra DTCs develop instead of proximal cells. NHR-25 knockdown restores DTC formation and fertility in pop-1 and sys-1 mutants, suggesting that a balance between NHR-25 and beta-catenin pathway activities is required to establish both proximal and distal fates. This balance relies on direct crossregulation between NHR-25 and the distinct beta-catenin proteins WRM-1 and SYS-1. The nuclear receptor-beta-catenin interaction may be an ancient mechanism of cell-fate decision.     

The long and the short of Wnt signaling in C. elegans

The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.     

MOM-4, a MAP kinase kinase kinase-related protein, activates WRM-1/LIT-1 kinase to transduce anterior/posterior polarity signals in C. elegans.

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. Here we show that 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.     

C. elegans POP-1/TCF functions in a canonical Wnt pathway that controls cell migration and in a noncanonical Wnt pathway that controls cell polarity

In Caenorhabditis elegans, Wnt signaling pathways are important in controlling cell polarity and cell migrations. In the embryo, a novel Wnt pathway functions through a (beta)-catenin homolog, WRM-1, to downregulate the levels of POP-1/Tcf in the posterior daughter of the EMS blastomere. The level of POP-1 is also lower in the posterior daughters of many anteroposterior asymmetric cell divisions during development. I have found that this is the case for of a pair of postembryonic blast cells in the tail. In wild-type animals, the level of POP-1 is lower in the posterior daughters of the two T cells, TL and TR. Furthermore, in lin-44/Wnt mutants, in which the polarities of the T cell divisions are frequently reversed, the level of POP-1 is frequently lower in the anterior daughters of the T cells. I have used a novel RNA-mediated interference technique to interfere specifically with pop-1 zygotic function and have determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans (beta)-catenin homologs appeared to function with POP-1 to control T cell polarity. Wnt signaling by EGL-20/Wnt controls the migration of the descendants of the QL neuroblast by regulating the expression the Hox gene mab-5. Interfering with pop-1 zygotic function caused defects in the migration of the QL descendants that mimicked the defects in egl-20/Wnt mutants and blocked the expression of mab-5. This suggests that POP-1 functions in the canonical Wnt pathway to control QL descendant migration and in novel Wnt pathways to control EMS and T cell polarities.     

Wnt activates the Tak1/Nemo-like kinase pathway

Genetic studies on endoderm-mesoderm specification in Caenorhabditis 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, we have explored whether Tak1 is directly activated by Wnt. We find 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. Our 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.     

A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans

The polarities of several cells that divide asymmetrically during Caenorhabditis elegans development are controlled by Wnt signaling. LIN-44/Wnt and LIN-17/Fz control the polarities of cells in the tail of developing C. elegans larvae, including the male-specific blast cell, B, that divides asymmetrically to generate a larger anterior daughter and a smaller posterior daughter. We determined that WRM-1 and the major canonical Wnt pathway components: BAR-1, SGG-1/GSK-3 and PRY-1/Axin were not involved in the control of B cell polarity. However, POP-1/Tcf is involved and is asymmetrically distributed to the B daughter nuclei, as it is in many cell divisions during C. elegans development. Aspects of the B cell division are reminiscent of the divisions controlled by the planar cell polarity (PCP) pathway that has been described in both Drosophila and vertebrate systems. We identified C. elegans homologs of Wnt/PCP signaling components and have determined that many of them appear to be involved in the regulation of B cell polarity. Specifically, MIG-5/Dsh, RHO-1/RhoA and LET-502/ROCK appear to play major roles, while other PCP components appear to play minor roles. We conclude that a noncanonical Wnt pathway, which is different from other Wnt pathways in C. elegans, regulates B cell polarity.     

WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.

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. We show here that lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. We demonstrate that the WRM-1 protein binds to LIT-1 in vivo and that 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. Our findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway.