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.     

The β-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans

The Wnt and Src pathways are widely used signal transduction pathways in development. β-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans β-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.     

MES-1, a protein required for unequal divisions of the germline in early C. elegans embryos, resembles receptor tyrosine kinases and is localized to the boundary between the germline and gut cells

During Caenorhabditis elegans embryogenesis the primordial germ cell, P(4), is generated via a series of unequal divisions. These divisions produce germline blastomeres (P(1), P(2), P(3), P(4)) that differ from their somatic sisters in their size, fate and cytoplasmic content (e.g. germ granules). mes-1 mutant embryos display the striking phenotype of transformation of P(4) into a muscle precursor, like its somatic sister. A loss of polarity in P(2) and P(3) cell-specific events underlies the Mes-1 phenotype. In mes-1 embryos, P(2) and P(3) undergo symmetric divisions and partition germ granules to both daughters. This paper shows that mes-1 encodes a receptor tyrosine kinase-like protein, though it lacks several residues conserved in all kinases and therefore is predicted not to have kinase activity. Immunolocalization analysis shows that MES-1 is present in four- to 24-cell embryos, where it is localized in a crescent at the junction between the germline cell and its neighboring gut cell. This is the region of P(2) and P(3) to which the spindle and P granules must move to ensure normal division asymmetry and cytoplasmic partitioning. Indeed, during early stages of mitosis in P(2) and P(3), one centrosome is positioned adjacent to the MES-1 crescent. Staining of isolated blastomeres demonstrated that MES-1 was present in the membrane of the germline blastomeres, consistent with a cell-autonomous function. Analysis of MES-1 distribution in various cell-fate and patterning mutants suggests that its localization is not dependent on the correct fate of either the germline or the gut blastomere but is dependent upon correct spatial organization of the embryo. Our results suggest that MES-1 directly positions the developing mitotic spindle and its associated P granules within P(2) and P(3), or provides an orientation signal for P(2)- and P(3)-specific events.     

Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos

BACKGROUND: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P(1). How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated.RESULTS: We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. We demonstrate that preferential activation of this checkpoint in the P(1) blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Galpha signaling.CONCLUSION: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.     

Functional analyses of vertebrate TCF proteins in C. elegans embryos

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.     

Wnt signaling specifies and patterns intestinal endoderm

Wnt signaling has been implicated in many developmental processes, but its role in early endoderm development is not well understood. Wnt signaling is active in posterior endoderm as early as E7.5. Genetic and chemical activation show that the Wnt pathway acts directly on endoderm to induce the intestinal master regulator Cdx2, shifting global gene away from anterior endoderm and toward a posterior, intestinal program. In a mouse embryonic stem cell differentiation platform that yields pure populations of definitive endoderm, Wnt signaling induces intestinal gene expression in all cells. We have identified a set of genes specific to the anterior small intestine, posterior small intestine, and large intestine during early development, and show that Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses. These findings shed light on the mechanism of embryonic intestinal induction and provide a method to manipulate intestinal development from embryonic stem cells.     

LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling

G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. In this report, we show that the asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99. Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that LET-99 and Galpha/GPR-1/2 signaling function in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division.     

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation,results in delayed degradation of maternal proteins and embryonic lethality

In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.     

Chato, a KRAB zinc-finger protein, regulates convergent extension in the mouse embryo

In Xenopus and zebrafish embryos, elongation of the anterior-posterior body axis depends on convergent extension, a process that involves polarized cell movements and is regulated by non-canonical Wnt signaling. The mechanisms that control axis elongation of the mouse embryo are much less well understood. Here, we characterize the ENU-induced mouse mutation chato, which causes arrest at midgestation and defects characteristic of convergent extension mutants, including a shortened body axis, mediolaterally extended somites and an open neural tube. The chato mutation disrupts Zfp568, a Krüppel-associated box (KRAB) domain zinc-finger protein. Morphometric analysis revealed that the definitive endoderm of mouse wild-type embryos undergoes cell rearrangements that lead to convergent extension during early somite stages, and that these cell rearrangements fail in chato embryos. Although non-canonical Wnt signaling is important for convergent extension in the mouse notochord and neural plate, the results indicate that chato regulates body axis elongation in all embryonic tissues through a process independent of non-canonical Wnt signaling.     

SRC-1, a non-receptor type of protein tyrosine kinase, controls the direction of cell and growth cone migration in C. elegans

Src family tyrosine kinase (SFK) has been implicated in the regulation of cell adhesion and migration during animal development. We show that SRC-1, an ortholog of SFK, plays an essential role in directing cell migration in Caenorhabditis elegans. The mutation in the src-1 gene results in defective distal tip cell (DTC)-directed gonad morphogenesis in an activity-dependent and DTC cell-autonomous manners. In the src-1 mutants, DTCs fail to turn and continue their centrifugal migration along the ventral muscles. The effect of the src-1 mutation is suppressed by mutations in genes that function in the CED/Rac pathway, suggesting that SRC-1 in DTCs is an upstream regulator of a Rac pathway that controls cytoskeletal remodeling. In the src-1 mutant, the expression of unc-5/netrin receptor is normally regulated, and neither the precocious expression of UNC-5 nor the mutation in the unc-5 gene significantly affects the DTC migration defect. These data suggest that SRC-1 acts in the netrin signaling in DTCs. The src-1 mutant also exhibits cell-autonomous defects in the migration and growth cone path-finding of Q neuroblast descendants AVM and PVM. However, these roles of SRC-1 do not appear to involve the CED/Rac pathway. These findings show that SRC-1 functions in responding to various extracellular guidance cues that direct the cell migration via disparate signaling pathways in different cell types.     

Mouse homologues of Shisa antagonistic to Wnt and Fgf signalings

In an effort to identify Otx2 targets in mouse anterior neuroectoderm we identified a gene, mShisa, which is homologous to xShisa1 that we previously reported as a head inducer in Xenopus. mShisa encodes an antagonist against both Wnt and Fgf signalings; it inhibits these signalings cell-autonomously as xShisa1 does. The mShisa expression is lost or greatly reduced in Otx2 mutant visceral endoderm, anterior mesendoderm and anterior neuroectoderm. However, mShisa mutants exhibited no defects in head development. Shisa is composed of five subfamilies, but normal head development in mShisa mutants is unlikely to be explained in terms of the compensation of mShisa deficiency by its paralogues or by known Wnt antagonists in anterior visceral endoderm and/or anterior mesendoderm.