C. elegans LEF-1 (POP-1), the WNT pathway and cell polarity
In a 4-cell stage C. elegans embryo, signaling by the P2 (posterior) blastomere induces anterior-posterior polarity in the adjacent EMS blastomere, leading to endoderm formation. Genetic and reverse genetic approaches have been taken toward understanding the molecular basis for this induction. These studies have identified a set of genes with sequence similarity to genes that have been shown to be, or that are implicated in, Wnt/Wingless signaling pathways in other systems. P2-EMS signaling may induce the E (endoderm) fate by lowering the amount or activity of POP-1 protein in the E blastomere. POP-1 is present at a high level in the MS nucleus and at a lower level in the E nucleus. In a mutant lacking detectable POP-1 in both MS and E, both blastomeres adopt E-like fates and produce endoderm. POP-1 is anHMG-domain protein similar to the vertebrate Tcf-1 and Lef-1 proteins and to Drosophila Pangolin. The C. elegans genes described here are related to wnt/wingless, porcupine, frizzled, beta-catenin/armadillo, and the human adenomatous polyposis coli gene, APC. The mom-1 gene encodes a gene related to Drosophila porcupine, and the mom-5 gene encodes a member of the frizzled gene family. The MOM-2 protein is homologous to Wingless. There may be partially redundant inputs into endoderm specification and a subset of these genes also appears to function in determining cytoskeletal polarity in certain early blastomeres (Rocheleau, 1997).
During the first few cleavages of the Caenorhabditis elegans embryo, localized expression of factors that regulate transcription or that mediate cell-cell interactions results in each blastomere acquiring a distinct identity, or potential to differentiate. Each blastomere then executes a unique and nearly invariant lineage, producing numerous cell types through a series of predominantly anterior/posterior (a/p) cleavages. Because blastomere lineages are essentially invariant, this means that patterns of cell division are correlated reproducibly with specific patterns of cell differentiation. For example, in the lineage of a blastomere called MS, the MS descendant born from the division sequence p-a-a-p-p invariably undergoes programmed cell death or apoptosis; none of the other MS descendants born at the same time, but from different division sequences, undergo apoptosis. Within a lineage, how is cell type differentiation reproducibly matched with division sequence? Invariant cleavage patterns could place cells consistently in the same position with respect to determinative environmental signals in the embryo. However, several studies have shown that, after about the 12-cell stage of embryogenesis, blastomeres have remarkable abilities to execute their normal lineages even after neighboring blastomeres are killed or removed. For example, the MS descendant born from the division sequence p-a-a-p-p undergoes apoptosis even if every blastomere except for MS is killed. Thus, in some lineages, cell fates do not appear to be determined by external, environmental cues within the embryo (Lin, 1998).
The pop-1 gene, coding a Tcf-1 and Lef-1 related protein, is part of a general system for transducing information about division sequences into changes in the cell nucleus that affect differentiation. The pop-1 gene was identified originally because of its role in the development of the MS blastomere. MS normally produces mesodermal tissues, and its sister E produces only endoderm. In a pop-1 mutant, MS adopts an E-like fate and produces endoderm. A signaling pathway similar to the Wnt pathway of vertebrates and Drosophila melanogaster has been shown to be required for MS and E to have different fates. In models for Wnt signaling, reception of the Wnt signal results in the nuclear localization of a beta-catenin such as the Armadillo protein in Drosophila; a C. elegans homolog WRM-1 is required for MS and E differences, but the localization of WRM-1 has not yet been determined. Once in the nucleus, beta-catenin is thought to interact with constitutive nuclear proteins such as Tcf-1 in vertebrates or the related Pangolin in Drosophila to regulate transcription; the POP-1 protein in C. elegans has sequence similarity to Tcf-1 and acts downstream in the Wnt-like pathway. A polyclonal antiserum raised against the POP-1 protein shows a slightly lower level of staining in the E nucleus than in the MS nucleus in most, but not all, C. elegans embryos. A monoclonal antibody specific to the POP-1 protein shows different levels of nuclear staining in almost all a/p pairs of sister blastomeres in the early embryo, including the MS/E pair. In each of these a/p pairs, a higher level of POP-1 staining is detected in the anterior sister than in the posterior sister. Loss of pop-1(+) activity causes several anterior cells to adopt fates similar to the fates of their posterior sisters. These studies show that the Wnt-like signaling pathway is required for generating or interpreting a/p polarity throughout the early C. elegans embryo and that POP-1 appears to be part of a general mechanism that couples division sequence to different patterns of gene expression in sister cells born from a/p cleavages (Lin, 1998).
The MS/E decision requires components of a Wnt-like signaling pathway. Studies in vertebrates and Drosophila have led to a model in which Wnt signaling regulates an interaction between beta-catenin and POP-1-related proteins, such as Tcf-1 or Lef-1. Additional studies indicate that the APC (human adenomatous polyposis) protein also can regulate beta-catenin, but it has not been resolved whether APC acts downstream, or in parallel to, Wnt. In C. elegans, the loss of wild-type activity of the wrm-1 (beta-catenin) gene alone, or the simultaneous loss of mom-2 (Wnt) and apr-1 (APC) activities, prevents the MS/E decision and causes MS and E to have similar levels of POP-1. Therefore, it was asked whether these genes are required for POP-1 asymmetry in other a/p pairs of sisters. All a/p pairs of sister blastomeres appear to have equivalent levels of POP-1 in wrm-1(RNAi) embryos (RNAi refers to RNA interference, which produces mutant-like phenotypes on treated embryos) and in mom-2(or9);apr-1(RNAi) embryos. Surprisingly, mom-2(or9) single mutants retain POP-1 asymmetry in AB descendants, though they lacked POP-1 asymmetry in the MS/E blastomeres. Thus, WRM-1 is essential for all POP-1 asymmetry (Lin, 1998).
During C. elegans embryonic development, many of the cell divisions in all regions of the embryo are oriented along the anterior/posterior (AP) axis: essentially all of these divisions result in AP daughter cells with different fates. Genetic studies of this polarity signaling process have defined a mechanism that involves several proteins with similarities to known Wnt/WG signaling components. For example, MOM-2 is related to the secreted protein Wnt/WG, and MOM-5 is related to the membrane protein Frizzled, a candidate Wnt/WG receptor. As in other Wnt/WG systems, these upstream factors appear to act through a beta-catenin-related protein, WRM-1. A key difference between polarity signaling in C. elegans and Wnt/WG signaling in vertebrates and Drosophila concerns the relationship of WRM-1 and a protein related to vertebrate TCF (T cell factor)/LEF (lymphoid enhancer factor) transcription factors, POP-1. In vertebrates and Drosophila, the WRM-1-related proteins, beta-catenin and Armadillo, are thought to enter the nucleus in response to signaling, where they bind to and activate TCF/LEF-related factors. In C. elegans, although WRM-1 is an effector of signaling, WRM-1 appears to have the opposite downstream activity, downregulating rather than activating POP-1. In studies on wild-type embryos, POP-1 exhibits a lower level of nuclear immunofluorescence staining in the posterior daughters of many AP divisions than in the anterior daughters. Genetic studies have shown that WRM-1 and other signaling components are required for this POP-1 asymmetry between AP sister cells. Thus, in C. elegans, Wnt/WG signaling through WRM-1 leads to downregulation of POP-1 (Shin, 1999).
Possible insights into POP-1 regulation by WRM-1 have come from analysis of the gene lit-1. Mutations in lit-1 result in a loss of AP cell fate asymmetries. Phenotypic and genetic analysis of lit-1 place this gene in both the MOM-2 and LIN-44 Wnt/WG signaling systems in C. elegans. In the embryo, LIT-1 appears to function along with WRM-1 in a process that reduces POP-1 levels or activity in posterior daughters of AP divisions. The LIT-1 protein is related to serine/threonine protein kinases and is most similar to the Drosophila tissue polarity protein, Nemo, and to the mouse protein Nlk. WRM-1 and LIT-1 appear to form a stable protein complex in vivo in C. elegans and in transfected vertebrate cells. In vertebrate cells, WRM-1 activates the LIT-1 protein kinase leading to phosphorylation of WRM-1, LIT-1, and POP-1. These observations support a model in which signaling activates the WRM-1/LIT-1 kinase complex. This complex then directly phosphorylates POP-1, leading to its downregulation in posterior daughters of AP divisions (Shin, 1999).
How upstream signaling events lead to activation of the WRM-1/LIT-1 kinase is not understood. LIT-1/Nemo/Nlk kinases make up a small subfamily of protein serine/threonine kinases distinct from, but closely related to, MAP kinases (MAPK). MAPK signaling pathways, which involve sequential activation of protein kinases called MAPK kinase kinases and MAPK kinases, are highly conserved from yeast to metazoans and regulate many developmental decisions in C. elegans, Drosophila, and vertebrates. In the present study, the cloning of the AP polarity gene, mom-4 is reported. The mom-4 locus was previously identified by a set of maternal mutations that cause defects in polarity signaling in the early embryo. This study shows that mom-4 activity is required for POP-1 asymmetries between anterior and posterior daughters of AP divisions. mom-4 encodes a C. elegans homolog of mammalian TAK1 (TGF-beta activated kinase), which is thought to function as a MAPK kinase kinase. When expressed in cultured mammalian cells, both MOM-4 and TAK1 are able to stimulate WRM-1/LIT-1 kinase activity leading to the increased phosphorylation of POP-1. This activation is dependent on the putative kinase activation loop of LIT-1 that serves as a target for activating phosphorylation in related kinases. The structural similarities of LIT-1 to MAPK and of MOM-4 to MAPK kinase kinase raise the possibility that a MAPK-like kinase cascade contributes to AP polarity signaling in C. elegans. Thus 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).
Genetic studies suggest that the signaling pathway is branched upstream of WRM-1 and LIT-1 and may have polarity inputs from sources other than the MOM-2/Wnt-related protein. For example, a large percentage of embryos from mutant strains carrying apparent null alleles of both mom-2(Wnt) and mom-5(Frizzled) nevertheless exhibit proper specification of posterior cell fates, strongly suggesting that alternative polarity signals must be able to activate the WRM-1/LIT-1 kinase. In the present study, mutations in mom-4 are shown to strongly synergize with mutations in mom-2 and mom-5, raising the possibility that mom-4(+) activity is required for Wnt/WG independent polarity signaling. Within its kinase domain, LIT-1 is approximately 45% identical (132 out of 292 residues) to human p38 MAP kinase and 43% identical (126 out of 292 residues) to human ERK1, respectively. Furthermore, LIT-1 activation appears to require a conserved motif analogous to a site required for activating phosphorylation by MAPK kinases. Thus, MOM-4 is similar to MAPK kinase kinase and LIT-1, which is similar to MAPK in amino acid sequence, is also similar to MAPK in its activation. These observations together with the genetic synergy between mom-4 mutants and Wnt/WG pathway mutants suggest that a MAP kinase-like cascade may function in parallel with Wnt/WG signaling to specify AP cell fate differences during C. elegans development. In the future, understanding how MOM-4 is activated and how MOM-4 in turn activates LIT-1 is likely to shed light on how Wnt/WG signals interact with other signaling pathways to control cell polarity and cell fate (Shin, 1999).
In Caenorhabditis elegans, Wnt signaling is involved in several aspects of development such as cell fate specification during embryogenesis, vulval formation, somatic gonad development, and tail patterning. Some components are shared by several or all of these processes. During embryogenesis, Wnt signaling pathway genes, mom-2 (Wnt), mom-5 (Fz), wrm-1 (beta-catenin), and pop-1 (TCF/LEF), mediate interactions between the P2 and EMS cells. Signaling from the P2 cell promotes asymmetric division of the EMS cell to produce an anterior MS fate and a posterior E fate. Inactivation of the Wnt signaling pathway results in an E-to-MS fate transformation. The Wnt pathway also plays a role in vulval development. Mutants of several Wnt pathway components show various vulva lineage defects: for example, lin-17 (Frizzled), bar-1 (beta-catenin), and mom-1 and mom-3 mutants. In somatic gonad development, lin-17 has also been shown to control several asymmetric cell divisions. In the tail region, a Wnt signal LIN-44 and its putative receptor LIN-17 control the polarity of several asymmetric cell divisions, including B, T, F, and U. lin-44, lin-17, and bar-1 also are involved in the specification of P12 fate in the tail region. A common feature of most of these Wnt signaling events in C. elegans appears to be that Wnt signaling is required for posterior cell fates, and inactivation of signaling results in posterior to anterior fates transformation (Jiang, 1999 and references).
The TCF/LEF class HMG box proteins have been shown to regulate Wnt target gene expression on pathway activation. The C. elegans TCF/LEF class protein is encoded by the pop-1 gene. pop-1 negatively regulates Wnt signaling, as loss-of-function mutation in pop-1 confers a phenotype opposite that of inactivation of Wnt signaling. Moreover, the expression level of POP-1 protein appears to be up-regulated on inactivation of Wnt signaling. Therefore, it was proposed that activation of the Wnt signaling pathway relieves the inhibitory effects of POP-1 on gene transcriptional regulation. Yet it is not clear how Wnt target gene expression is regulated and what other unidentified factors are involved in this process (Jiang, 1999 and references).
Cell-cell interactions and cell fate specification during C. elegans male spicule development has been studied. Cell fate specification in spicule development is mediated by multiple signaling pathways. The C. elegans male spicule is generated by a single male specific B blast cell. The first division of B cell is asymmetric and is controlled by the Wnt signaling pathway genes, lin-44/Wnt and lin-17/Frizzled. At the early third larval stage (L3; B lineage 10-cell stage), the specification of B cell progeny involves a RAS signaling pathway and a C. elegans Notch homolog lin-12. During the L3 stage, those cells continue to divide to generate spicule neurons, non-neuronal sheath and socket cells, and connective tissues. For example, zeta sublineage generates the neuron SPD and its associated sheath (SPDsh) and socket (SPDso) cells (Jiang, 1999 and references).
Using a molecular marker that is specifically expressed in zeta-derived SPD neurons, neuronal versus non-neuronal cell fate specification during male spicule development has been studied. Although the cell division pattern of the zeta sublineage resembles the Drosophila sensory organ precursor (SOP) lineage in which the Notch pathway plays a crucial role, lin-12/Notch is not involved in the specification of the SPD neuron fate. Rather, the lin-17-mediated Wnt signaling pathway plays an important role in this neuronal versus non-neuronal fate decision in zeta lineage. Loss-of-function mutants of lin-17 display an SPDsh (SPD associated sheath cell) to neuron fate transformation. To identify new genes involved in this cell fate decision, a genetic screen was carried out and a mutant, son-1(sy549), was discovered which shows the same cell fate transformation. Moreover the son-1 (sheath-to-neuron fate transformation) mutation enhances the phenotypes of a lin-17 hypomorph, n698, to lin-17 null-like, with respect to vulval development, somatic gonad development, and male tail patterning. The son-1 locus was cloned and found to encode an HMG1/2-like DNA-binding protein, which is distinct from the TCF/LEF class HMG proteins. Disruption of son-1 function through RNA-mediated interference (RNAi) leads to defects in several Wnt pathway-mediated developmental processes. Overexpression of POP-1 causes the same spicule defect as loss-of-function son-1. These results provide in vivo evidence that the HMG1/2-like protein encoded by son-1 plays a specific role in Wnt signaling. SON-1 and POP-1 may both act in the Wnt-responding cells, but in opposite directions, to regulate gene transcription. Taken together, these data provide evidence for further complexity in Wnt responses during development (Jiang, 1999).
Although the HMG1/2 class proteins are traditionally considered as architectural components of chromatin, the specific phenotypes of son-1(sy549) suggest that this gene may be specifically involved in Wnt signaling for four reasons: (1) son-1(sy549) causes a similar SPDsh to neuron fate transformation as does a lin-17 hypomorph. The vulva and gonad defects observed in son-1(sy549) mutants, although at low penetrance, do resemble what are seen in lin-17 mutants. (2) Specific postembryonic phenotypes have been observed that resemble lin-17 mutant phenotypes from son-1 RNAi experiments. (3) son-1(sy549) enhances lin-17(n698) phenotypes to lin-17 null-like but does not enhance phenotypes of other unrelated mutants such as let-2(mn114). (4) Overexpression of POP-1 gave similar spicule phenotype as disruption of son-1 function. Taken together, son-1 is involved in several Wnt-mediated developmental processes, including vulva formation, somatic gonad development, and male spicule development. These results provide the first evidence for the specific functions of the HMG1/2 class proteins in vivo (Jiang, 1999 and references).
The TCF/LEF class sequence-specific HMG proteins have been implicated in Wnt signaling. They can either positively or negatively regulate Wnt target gene expression depending on their transcription cofactors, with beta-Catenin acting as a co-activator and CBP acting as a co-repressor. The C. elegans TCF/LEF homolog, pop-1, appears to negatively regulate Wnt signaling because loss-of-function in pop-1 causes an opposite phenotype as inactivation of the Wnt signaling during C. elegans embryogenesis. In the specification of SPD neuron fate versus SPDsh fate during male spicule development, overexpression of POP-1 results in the same defect as loss-of-function mutation in lin-17 and son-1. Therefore, a POP-1-like activity also negatively regulates Wnt signaling during C. elegans postembryonic development. Both the sequence-specific TCF/LEF and sequence-nonspecific HMG1/2 class of proteins are involved in lin-17-mediated Wnt signaling and they play opposite roles in regulating Wnt pathway activity (Jiang, 1999 and references).
pop-1 has been shown to act downstream of the Wnt receptor and its expression is negatively regulated by Wnt signaling. Inactivation of the Wnt pathway up-regulates POP-1 protein level. son-1 also acts in the Wnt responding cells. Therefore, both the POP-1-like protein and SON-1 act in the same cell nucleus to regulate gene transcription. The POP-like activity inhibits gene transcription, and SON-1 facilitates gene transcription. Since SON-1 is an HMG-1/2-like protein, it is thought that SON-1 could activate gene transcription in two ways: (1) SON-1 could alter chromatin conformation in favor of gene expression, and (2) SON-1 could physically interact with other transcription factors and help stimulate specific gene expression. Proteins of the HMG1/2 class have been shown to interact with POU domain proteins or HOX proteins in vitro and enhance their DNA-binding and transcriptional activities. The present data suggest that son-1 function may not be rate limiting for Wnt signal transduction. Moreover, son-1::GFP expression does not seem to be altered in a lin-17 mutant background. Therefore, it is possible that SON-1 acts to activate gene transcription after the inhibitory effect of POP-1 is relieved by activation of the Wnt pathway (Jiang, 1999 and references).
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).
In flies and vertebrates, Armadillo/beta-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signaling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative beta-catenin homologs have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homolog POP-1 mediate Wnt signaling by a mechanism that has challenged current views of the Wnt pathway. BAR-1 is the only beta-catenin homolog that interacts directly with POP-1. BAR-1 mediates Wnt signaling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only beta-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. It is concluded that a canonical Wnt pathway exists in C. elegans. Furthermore, this analysis shows that the functions of C. elegans beta-catenins in adhesion and in signaling are performed by separate proteins (Korswagen, 2000).
It is proposed that the signaling and adhesion functions of Armadillo/beta-catenin have been distributed between separate beta-catenin homologs in C. elegans. Two beta-catenins function in Wnt signaling. WRM-1 is part of a divergent Wnt pathway that, in collaboration with a mitogen-activated protein (MAP) kinase pathway, mediates the asymmetric distribution of POP-1 between daughter cells of many anterior/posterior cells. POP-1 probably acts as a repressor in this pathway and differences in POP-1 expression levels between daughter cells may allow the specification of different fates. BAR-1 is part of a Wnt pathway that is similar to that in flies and vertebrates. BAR-1 can directly associate with POP-1 to activate a Tcf reporter. BAR-1 and POP-1 are required for the expression of the endogenous Wnt target gene mab-5. Of the three C. elegans beta-catenins, only BAR-1 contains a set of four conserved GSK3 phosphorylation sites. These sites are essential for the regulation of beta-catenin by the Wnt pathway and are frequently mutated in cancers. C. elegans contains a single putative APC homolog, APR-11. In vertebrates, APC forms a complex with GSK3, Axin and beta-catenin to downregulate beta-catenin levels in the absence of Wnt signaling. None of the C. elegans beta-catenins bind APR-1 in a yeast two-hybrid assay. Together with the observation that the C. elegans genome does not contain a clear Axin homolog, this indicates that BAR-1 may be regulated differently (Korswagen, 2000).
The endoderm of higher organisms is extensively patterned along the anterior/posterior axis. Although the endoderm (gut or E lineage) of the nematode Caenorhabditis elegans appears to be a simple uniform tube, cells in the anterior gut show several molecular and anatomical differences from cells in the posterior gut. In particular, the gut esterase ges-1 gene, which is normally expressed in all cells of the endoderm, is expressed only in the anterior-most gut cells when certain sequences in the ges-1 promoter are deleted. Using such a deleted ges-1 transgene as a biochemical marker of differentiation, the basis of anterior-posterior gut patterning in C. elegans has been investigated. Although homeotic genes are involved in endoderm patterning in other organisms, anterior gut markers are expressed normally in C. elegans embryos lacking genes of the homeotic cluster. Although signaling from the mesoderm is involved in endoderm patterning in other organisms, ablation of all non-gut blastomeres from the C. elegans embryo does not affect anterior gut marker expression; furthermore, ectopic guts produced by genetic transformation express anterior gut markers generally in the expected location and in the expected number of cells. It is concluded that anterior gut fate requires no specific cell-cell contact but rather is produced autonomously within the E lineage. Cytochalasin D blocking experiments fully support this conclusion. The HMG protein POP-1, a downstream component of the Wnt signaling pathway, is important in many anterior/posterior fate decisions during C. elegans embryogenesis. When RNA-mediated interference is used to eliminate pop-1 function from the embryo, gut is still produced but anterior gut marker expression is abolished. Thus the establishment of E lineage polarity is independent of pop-1. It is suggested that the C. elegans endoderm is patterned by elements of the Wnt/pop-1 signaling pathway acting autonomously within the E lineage and that anterior gut marker expression will depend on elements of a Wnt signaling pathway (Schroeder, 1998).
In C. 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. This is the case for 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. A novel RNA-mediated interference technique has been used to interfere specifically with pop-1 zygotic function and it has been determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans beta-catenin homologs appears 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 mimic the defects in egl-20/Wnt mutants and block 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 (Herman, 2001).
A model for the generation of T cell polarity is presented. In wild-type hermaphrodites, the T.a cell divides to generate a hypodermal cell and a blast cell that give rise to primarily hypodermal cell fates, whereas the T.p cell divides to generate neural cell fates and a cell that undergoes apoptosis. Based upon the analysis of lin-44 and lin-17 (frizzled/WNT receptor) mutants, the polarity of the T cell appears to be determined before it divides. Thus, it seems that there is an asymmetric segregation of cell fates at the T cell division: hypodermal cell fate is segregated to T.a and neural cell fate is segregated to T.p. The segregation of cell fate is correlated with a particular level of POP-1 protein: a higher level of POP-1 is correlated with hypodermal cell fates, while a lower level of POP-1 is correlated with neural cell fates. The distributions of both cell fate and POP-1 are dependent upon lin-44 and lin-17. However, reducing POP-1 function even further, by RNAi or expression of DN-POP-1, leads to hypodermal cell fates. The model suggests that the LIN-44/Wnt signal, acting through LIT-1 kinase (a homolog of Drosophila Nemo), functions to modify POP-1, which results in decreased POP-1 levels and the activation of neural-specific genes in T.p. The high levels of POP-1 in T.a may be nonfunctional. Specifically, LIN-44/Wnt binds to LIN-17/FZ on the posterior portion of the T cell before it divides (and on T.p and its descendants). Without LIN-44 signal, the T.a cell accumulates a high level of POP-1 and expresses hypodermal-specific genes. Surprisingly, the interference with pop-1 function also causes T.a to take on a hypodermal fate, suggesting that such a fate does not depend upon POP-1 function and may even represent the default state, perhaps achieved by the constitutive expression of hypodermal-specific genes in T.a. In the presence of LIN-44 signal, transduction through LIN-17 and unknown factors, that may not be components of the canonical WNT pathway, leads to the activation of LIT-1, which might lead to the phosphorylation of POP-1, resulting in the reduction of POP-1 levels in T.p by degradation as may occur in the E blastomere. This may occur by LIT-1 combining with an unidentified factor that performs a function similar to WRM-1 in the embryo. The interference with pop-1 function also leads to the T.p descendants taking on hypodermal cell fates, suggesting that some pop-1 function is required for specification of neural cell fates. One possibility is that a low level of a modified, perhaps phosphorylated, form of POP-1 is required for the activation of neural-specific genes, one or more of which might function to repress hypodermal-specific genes in T.p. The observation that overexpression of DN-POP-1 also causes the loss of neural cell fates suggests that the N-terminal domain of POP-1 may be necessary for activation of neural-specific genes, perhaps because it becomes modified or it interacts with an unknown factor. The isolation and characterization of additional genes that function in the control of T cell polarity will help to elucidate how this novel Wnt signaling pathway can function through POP-1/Tcf to control cell polarity (Herman, 2001).
In C. elegans, histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein, POP-1, through an unknown mechanism. CBP-1, HDA-1 and POP-1 converge on end-1, a Serpent-related GATA factor that acts as an initial endoderm-determining gene. In the E lineage, an essential function of CBP-1 appears to be the activation of end-1 transcription. A molecular mechanism has been identified for the endoderm-suppressive effect of POP-1 in the MS lineage by demonstrating that POP-1 functions as a transcriptional repressor that inhibits inappropriate end-1 transcription. Evidence is provided that POP-1 represses transcription via the recruitment of HDA-1 and UNC-37, the C. elegans homolog of the co-repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate-determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co-repressors ensure maximal repression of inappropriate cell type-specific gene transcription (Calvo, 2001).
The shape and polarity of the C. elegans gonad is defined during early gonadogenesis by two somatic gonadal precursor cells, Z1 and Z4, and their descendants. Z1 and Z4 divide asymmetrically to establish the proximal-distal axes of the gonad and to generate regulatory leader cells that control organ shape. pop-1, the C. elegans TCF/LEF-1 transcription factor, controls the first Z1/Z4 asymmetric division and hence controls proximal-distal axis formation. Two pop-1(Sys) alleles (for symmetrical sisters) have been identified that render the Z1/Z4 divisions symmetrical. The pop-1(q645) allele is fully penetrant for the Sys gonadogenesis defect in hermaphrodites, but affects male gonads weakly; pop-1(q645) alters a conserved amino acid in the ß-catenin binding domain. The pop-1(q624) allele is weakly penetrant for multiple defects and appears to be a partial loss-of-function mutation; pop-1(q624) alters a conserved amino acid in the HMG-box DNA binding domain. Zygotic pop-1(RNAi) confirms the role of pop-1 in Z1/Z4 asymmetry and reveals additional roles of pop-1, including one in leader cell migration. Two other Wnt pathway regulators, wrm-1 and lit-1, have the same effect as pop-1 on Z1/Z4 asymmetry. Therefore, wrm-1 and lit-1 are required for pop-1 function, rather than opposing it as observed in the early embryo. It is concluded that POP-1 controls the Z1/Z4 asymmetric division and thereby establishes the proximal-distal axes of the gonad. This control over proximal-distal polarity extends the view of Wnt signaling in C. elegans, which had previously been known to control anterior-posterior polarities (Siegfried, 2002).
This work introduces the sys-2 locus. sys-2 is allelic with pop-1. Therefore the sys-2 gene name has been dropped, but the Sys name has been retained to describe Z1/Z4 division defects typical of sys-1, lin-17 and pop-1(Sys) mutants. lin-17 gene encodes a frizzled-like receptor, implicating Wnt signaling in control of Z1/Z4 asymmetric divisions. The phenotypes of these two pop-1(Sys) alleles are distinct, both from each other and from those of pop-1(RNAi) progeny. To explore the pathway controlling Z1/Z4 asymmetry, components of the canonical Wnt signaling pathway and components of a Map kinase pathway that regulate POP-1 function were analyzed. Animals lacking either the ß-catenin homolog wrm-1 or the nemo-like kinase (NLK) homolog lit-1 have typical Sys gonadal defects. Similarly, loss of lit-1 in a post-embryonic cell, called the T cell, also results in the same phenotype as loss of pop-1 (Siegfried, 2002).
How might POP-1 govern Z1/Z4 asymmetry? Two simple models are suggested, although they are not mutually exclusive and others certainly exist. Both models take advantage of the fact that Z1 and Z4 contact each other ventrally beneath the two germline precursor cells, Z2 and Z3. In the first model, a Wnt signal polarizes Z1 and Z4, which leads to enhanced POP-1 activity in the distal daughter. Polarization may result from an inhibitory signal in the underlying hypodermis or from a decreased access of the Wnt to proximal regions of Z1/Z4. In the second model, no Wnt signal is invoked. Instead, contact between Z1 and Z4 (or contact between Z1/Z4 and the hypodermis) polarizes the cell (for example, excluding LIN-17 from the proximal region), which leads to enhanced POP-1 activity in the distal daughters (Siegfried, 2002).
Wnt signaling controls primary axis formation in vertebrates and limb axis formation in vertebrates and flies. In C. elegans, POP-1 controls axis formation during early organogenesis by controlling a key cell polarity. In vertebrate primary axis formation, Wnt signaling is restricted to a dorsal developmental field by opposing ventral signals, leading to dorsal-ventral (D-V) axis specification; similar processes control limb DV axis specification. Polarization of Z1 and Z4 in C. elegans gonad development may act in a similar fashion: the proximal-distal axis may be determined by restriction of Wnt signaling to the distal Z1 and Z4 daughter cells (Siegfried, 2002).
POP-1, a Tcf/Lef factor, functions throughout Caenorhabditis elegans development as a Wnt-dependent reiterative switch to generate nonequivalent sister cells that are born by anterior-posterior cell divisions. The interaction between POP-1 and a target gene that it represses has been observed as it responds to Wnt signaling. Dynamic observations in living embryos reveal that POP-1 undergoes Wnt-dependent nucleocytoplasmic redistribution immediately following cytokinesis, explaining the differential nuclear POP-1 levels in nonequivalent sister cells. In unsignaled (anterior) but not Wnt-signaled (posterior) sister cells, POP-1 progressively coalesces into subnuclear domains during interphase, coincident with its action as a repressor. While the asymmetric distribution of POP-1 in nonequivalent sisters apparently requires a 124-amino-acid internal domain, neither the HMG box nor ß-catenin interaction domains are required. A transcriptional activator, MED-1, a GATA type protein, associates in vivo with the end-1 and end-3 target genes in the mesoderm (anterior sister) and in the endoderm (posterior sister) following the asymmetric cell division that subdivides the mesendoderm. However, in the anterior sister, binding of POP-1 to the end-1 and end-3 genes blocks their expression. In vivo, binding of POP-1 to the end-1 and end-3 targets (in the posterior sister) is blocked by Wnt/MAPK signaling. Thus, a Tcf/Lef factor represses transactivation of genes in an unsignaled daughter cell by abrogating the function of a bound activator (Maduro, 2002).
C. elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, (but unlike in flies, not all of them) are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes has been performed; the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. Two enhancer fragments have been identified that are involved in different aspects of the embryonic ceh-13 expression pattern. Important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. The data show that the molecular nature of Hox1 class gene autoregulation has been conserved among worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos (Streit, 2002).
An enhancer element that has been identified in the ceh-13 promoter region is a 740-bp fragment (enh740) located at the downstream end of enh3.4. enh740 drives early embryonic expression of a reporter gene in C. elegans in a pattern indistinguishable from ceh-13. This fragment does not contain candidate sequences for the kind of auto-regulatory element discussed above. Nevertheless, it is able to induce LacZ expression in groups of cells of all germ layers in the Drosophila embryo around stage 15. Interestingly, enh740 is activated by and dependent on Wg signaling and its nuclear effector Pan in all three germ layers and therefore acts as a strong Wg sensor that shows little or no tissue specificity. This is in contrast to the Wg-dependent Drosophila enhancer elements characterized so far that are all specific for particular tissues (Streit, 2002).
Expression of lab also depends on the Wg signaling pathway. In the embryonic midgut of Drosophila, low Wg levels stimulate its transcription and that of another Hox gene (Ubx), whereas high levels repress both genes. Analysis of the Ubx midgut enhancer reveals that activation and repression are mediated by different enhancer elements. Whereas Wg-mediated transcriptional activation is conferred directly by binding of Pangolin (the Drosophila LEF-1/TCF homolog) and its coactivator Armadillo, the Drosophila ß-catenin equivalent, it appears that repression in response to high Wg levels is indirect and based on transcriptional activation of the Teashirt (Tsh) repressor. enh740 may contain only Wg activator elements but lacks Tsh repressor sequences and therefore acts as an activator even at high Wg concentrations. This may also explain why the expression domain of enh740 was shifted toward the posterior compared to endogenous Lab. Further biochemical and genetic analyses are required to determine whether Pangolin binds directly to the 740-bp element and how it activates reporter gene expression (Streit, 2002).
The finding that enh740 acts as a strong Wg response element in Drosophila raises the challenging question of whether expression of enh740 in C. elegans also depends on Wnt/Wg signaling. Indeed, genetic studies in C. elegans have revealed that Wnt/Wg signaling acts very early during nematode development. In four-cell-stage embryos, for example, MOM-2/Wnt/Wg signaling specifies the production of endoderm. Furthermore, several C. elegans Wnt/Wg pathway members are involved in a embryo-wide polarity system that generates differences in cell fate between many sister cells generated by a/p divisions. The ß-catenin/Armadillo-related protein WRM-1 and LIT-1, a serine/threonine protein kinase homologous to the Drosophila tissue polarity protein Nemo, are effectors of this signaling pathway. WRM-1 binds and activates LIT-1, which in turn phosphorylates POP-1 and causes a reduction of its apparent nuclear levels in the posterior daughters of a/p dividing cells. In the AB lineage, for example, POP-1 asymmetry is present at each of several sequential a/p divisions and is first established during the a/p divisions of the ABxx cells (Streit, 2002).
Because of this a/p polarity system, POP-1 is also asymmetrically distributed after the fourth AB division that leads to the formation of the ceh-13 expressing ABxxxp. Therefore, and given the fact that POP-1 has been proposed to function as a transcriptional repressor in this pathway, it is tempting to speculate that high POP-1 levels may negatively regulate ceh-13 expression in the ABxxxa cells, the anterior sisters of the ABxxxp cells. This view is supported by the complete absence of ceh-13:: gfp reporter gene expression in the AB lineage of lit-1 mutant animals, that lack POP-1 asymmetry and show high levels of POP-1 immunostaining in the nuclei of all ABxxxx cells. Surprisingly, however, a complete absence of pMF1 reporter gene expression is found in all ABxxxx cells of pop-1 (RNAi) animals that lack maternal and embryonic POP-1 expression. Thus, it is possible that POP-1, at low nuclear concentrations or upon phosphorylation by LIT-1, may act as a transcriptional activator of ceh-13. However, since mutations in pop-1 and lit-1 result in cell fate changes and start to act earlier in development than ceh-13 is first expressed, other explanations for these results can not be excluded. An argument for a direct involvement of POP-1 in control of ceh-13 is the fact that enh740 does contain several putative POP-1 binding sites and at least one of them does bind POP-1 in vitro. Altogether, it is thought that WNT signaling, or more specifically POP-1, is very likely to be one of the spatiotemporal cues involved in control of ceh-13 expression in the early embryo. However, early embryonic ceh-13/enh740 expression cannot depend on Wg mediated polarity cues only: POP-1 asymmetry in the AB lineage is established already in the daughters of the ABxx cells, but onset of ceh-13 expression occurs only one cell division later. Regardless of the POP-1 distribution, ceh-13 continues to be expressed in both the anterior and posterior daughters of almost all of the ABxxxp cells. Furthermore, ceh-13 is not expressed in the cells of the early C lineage. Thus, it is proposed that, in addition to Wnt/Wg-mediated polarity cues, other yet unidentified factors participate in the regulation of ceh-13/ enh740 early expression and provide the cells with temporal and lineage-specific inputs. Further work will be required to confirm the involvement of the worm Wnt/Wg pathway and its nuclear effector POP-1 on enh740 and to identify other regulatory proteins that bind to it (Streit, 2002).
Earlier experiments have suggested that the pattern of expression of the worm Hox genes are determined, at least in part, by mechanisms independent of the global position of the cells along the a/p axis. This is in contrast to Drosophila and vertebrates, where the Hox genes are controlled by global positional mechanisms. Based on these observations, it has been suggested that Hox gene regulation in C. elegans may rely on different strategies than those previously characterized in Drosophila and mammalians. Nevertheless, it has been show in this study that the molecular nature of the mechanisms that control Hox 1 class genes may be more conserved between flies and worms than previously assumed (Streit, 2002).
In C. elegans embryos, the nuclei of sister cells that are born from anterior/posterior divisions show an invariant high/low asymmetry, respectively, in their level of the transcription factor POP-1. Previous studies have shown that POP-1 asymmetry between the daughters of an embryonic cell called EMS results in part from a Wnt-like signal provided by a neighboring cell, called P2. This study identifies additional signaling cells that play a role in POP-1 asymmetry for other early embryonic cells. Some of these cells have signaling properties similar to P2, whereas other cells use apparently distinct signaling pathways. Although cell signaling plays a critical role in POP-1 asymmetry during the first few cell divisions, later embryonic cells have an ability to generate POP-1 asymmetry that appears to be independent of prior Wnt signaling (Park, 2003).
The pattern of cell cleavage and differentiation in C. elegans is largely invariant. The spindles of most cells are oriented such that mitosis generates an anterior daughter and a posterior daughter, and most of the early anterior/posterior (a/p) cell divisions result in sister cells with different fates. The transcription factor POP-1 is present in the nuclei of all early embryonic, and several postembryonic, cells. After an embryonic cell divides, its anterior daughter invariably shows a higher level of nuclear POP-1 than its posterior daughter. Thus each a/p pair of sister cells exhibits POP-1 asymmetry with a reproducible high/low polarity. Conditions that result in high POP-1 in the posterior sister cause an anterior transformation in fate, whereas the absence of POP-1 in the anterior sister causes a posterior transformation in fate. POP-1 functions in a variety of cell-type decisions, including mesodermal/endodermal choices and epidermal/neuronal choices. These observations suggest that POP-1 asymmetry provides the a/p coordinate system that collaborates with more widely expressed transcription factors to diversify sister cells (Park, 2003).
POP-1 is related to TCF/Pangolin, a transcriptional effector of the canonical Wnt signaling pathway, and POP-1 has been shown to function in canonical Wnt signaling during larval development. However, POP-1 asymmetry in the early embryo is regulated by a non-canonical Wnt pathway (see Eisenmann's Wnt Signaling), with parallel input from a mitogen-activated protein kinase (MAPK) pathway. Components of these pathways include MOM-2/Wnt, MOM-5/Frizzled, WRM-1/beta-catenin, MOM-4/MAPKKK/TAK1 and LIT-1/Nemo. Sister cells show POP-1 asymmetry because they differ in their nucleo/cytoplasmic distributions of POP-1. Studies with cultured vertebrate cells suggest that WRM-1/beta-catenin can activate LIT-1/Nemo, resulting in phosphorylated POP-1 that accumulates in the cytoplasm (Park, 2003).
How is the a/p polarity system established? The most detailed experimental studies to date have focused on the development of the EMS cell. EMS divides into an anterior, mesodermal precursor and a posterior, endodermal precursor. This a/p polarity is induced during the 4-cell stage of embryogenesis by a neighboring cell called P2. For example, removing P2 causes both EMS daughters to have anterior fates (high POP-1) and repositioning P2 on the opposite surface of EMS reverses the polarity of the division (Park, 2003).
Relatively little is known about the cellular events that establish a/p polarity in other embryonic cells. At the 2-cell stage, the posterior cell is called P1 (the parent of P2 and EMS) and the anterior cell is called AB. Within the AB lineage, POP-1 asymmetry is first detectable after the third division of AB, when there are four a/p sister pairs of AB descendants; for convenience this stage is referred to as the AB8 stage. POP-1 function is essential for a/p differences in cell fate within each of the four sister pairs of AB8 cells. Previous studies on how a/p differences are generated in the AB lineage have reached contradictory conclusions. In one set of experiments, AB was separated from P1 and allowed to develop to the AB16 stage. As many as eight of the AB16 cells expressed a transgenic marker that normally is expressed in the eight posterior AB16 cells, suggesting that AB has an inherent a/p polarity. In a different study, videomicroscopy was used to follow AB development after killing P1 or P1 descendants. Several AB8 cells showed posterior to anterior transformations in fate after killing P1, but not P1 descendants, suggesting that P1 induces an a/p polarity in AB that is maintained in a latent form until the AB8 stage (Park, 2003).
To further analyze the cellular basis for a/p polarity in the AB lineage, POP-1 levels were analyzed directly by immunostaining isolated and cultured embryonic cells. The results indicate that POP-1 asymmetry at the AB8 stage results from interactions with specific P1 descendants, rather than with P1. These interactions are mediated in part by MOM-2/Wnt signaling. Surprisingly, by the AB16 stage embryonic cells have acquired an ability to generate POP-1 asymmetry that appears to be independent of MOM-2/Wnt signaling or prior interactions with other cells, but that requires MOM-5/Frizzled (Park, 2003).
MOM-5/Frizzled is essential for POP-1 asymmetry in isolated cells that have not been exposed to MOM-2/Wnt signaling. Therefore MOM-5/Frizzled may be a component of the signaling pathway that generates low/high POP-1 polarity independent of MOM-2/Wnt. Drosophila Frizzled is an essential component of the planar cell polarity pathway, however the role of Wnt proteins has not been determined. It will be of interest to determine whether other genes involved in Drosophila planar cell polarity have functions in low/high signaling in C. elegans. MOM-4/MAPKKK and proteins such as LIT-1/Nemo and WRM-1/Beta-catenin are essential for POP-1 asymmetry in AB descendants, and thus appear to be core components of the asymmetry-generating machinery (Park, 2003 and references therein).
Mutations in tcl-2, a gene encoding a novel protein, cause defects in the specification of the fates of the descendants of the TL and TR blast cells, whose polarity is regulated by lin-44/Wnt and lin-17/frizzled, during Caenorhabditis elegans development. In wild-type animals, POP-1/TCF/LEF, is asymmetrically distributed to the T cell daughters, resulting in a higher level of POP-1 in the nucleus of the anterior daughter. The POP-1 asymmetric distribution is controlled by lin-44 and lin-17. However, in tcl-2 mutants, POP-1 is equally distributed to T cell daughters as is observed in lin-17 mutants, indicating that, like lin-17, tcl-2 functions upstream of pop-1. In addition, tcl-2 mutations cause defects in the development of the gonad and the specification of fate of the posterior daughter of the P12 cell, both of which are controlled by the Wnt pathway. Double mutant analyses indicate that tcl-2 can act synergistically with the Wnt pathway to control gonad development as well as P12 descendant cell fate specification. A functional tcl-2::gfp construct is weakly expressed in the nuclei of the T cell and its descendants. These results suggest that tcl-2 functions with Wnt pathways to control T cell fate specification, gonad development, and P12 cell fate specification (Zhao, 2003).
The functions of the Wnt signaling pathway seem to be the activation of the responsive gene(s), usually transcription factors, in the nucleus. In C. elegans, mab-5 and lin-39 encode homeodomain proteins, apparent targets of the Wnt signaling pathway that function in the control of QL migration and vulva induction, respectively. tlp-1, which encodes a C2H2 type zinc finger protein, is a potential target of lin-44-mediated Wnt signaling pathway in the control of the asymmetric T cell division. Furthermore, although tlp-1 mutations could cause the T.p cell to adopt hypodermal fates, the POP-1 distribution pattern is not altered, suggesting that tlp-1 functions downstream of pop-1. tcl-2 mutations also affect tlp-1 expression. Although these experiments do not define the exact point where TCL-2 might function, it must be before POP-1 and the end point might be to regulate tlp-1 to promote neural cell fates through the Wnt pathway (Zhao, 2003).
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 C. elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. Most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk/Derailed family) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva (Deshpande, 2005).
Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, since neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. The C. elegans dsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. It is suggested that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ (Chang, 2005).
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).
The C. elegans somatic gonadal precursor cell (SGP) divides asymmetrically to establish gonad-specific coordinates in both sexes. In addition, the SGP division is sexually dimorphic and initiates sex-specific programs of gonadogenesis. Wnt/MAPK signaling determines the gonadal axes, and the FKH-6 transcription factor specifies the male mode of SGP division. This paper demonstrated that C. elegans cyclin D controls POP-1/TCF asymmetry in the SGP daughters as well as fkh-6 and rnr expression in the SGPs. Although cyclin D mutants have delayed SGP divisions, the cyclin D defects are not mimicked by other methods of retarding the SGP division. EFL-1/E2F has an antagonistic effect on fkh-6 expression and gonadogenesis, which is relieved by cyclin D activity. It is proposed that cyclin D and other canonical regulators of the G1/S transition coordinate key regulators of axis formation and sex determination with cell cycle progression to achieve the sexually dimorphic SGP asymmetric division (Tilmann, 2005).
Asymmetric cell divisions are a widespread mechanism for generating diverse cell types during animal development. Model asymmetric divisions include those of the C. elegans zygote, the C. elegans EMS blastomere, and the Drosophila neuroblast and sensory organ precursor. This study has embarked on an in depth analysis of a different asymmetric division, that of the somatic gonadal precursor cell (SGP) in C. elegans. This division establishes the proximal-distal axis of the gonad of both sexes, and it is sexually dimorphic. By teasing apart the molecular regulators of the SGP division, it wll be learned how precursor cells establish an organ coordinate system and how asymmetric divisions can be modulated during development to generate distinct organs (Tilmann, 2005).
The C. elegans embryo generates a four-celled gonadal primordium that appears the same in XX hermaphrodites and XO males. Within the primordium, one SGP resides at each of the two opposite poles, and two germline precursors lie between. During the first larval stage (L1), the SGP divides asymmetrically in both sexes to generate proximal and distal daughters that establish gonadal axes. The SGP division is also sexually dimorphic with respect to both size and fate of its daughters. The hermaphrodite SGP makes distal and proximal daughters of roughly equal size, but the male SGP produces a smaller distal daughter and a larger proximal daughter. In addition, the SGP daughters exhibit sex-specific behaviors (e.g., migration) and generate sex-specific regulatory cells that control gonad elongation and germline proliferation. Therefore, the SGP asymmetric division initiates sex-specific programs of gonadogenesis that generate a double-armed ovotestis in hermaphrodites and a single-armed testis in males (Tilmann, 2005).
The Wnt/MAPK pathway specifies the distal SGP daughter fate in both sexes. The terminal regulators of this pathway are POP-1/TCF and SYS-1/β-catenin, a DNA binding protein and its transcriptional coactivator, respectively. In the early embryo, activated Wnt/MAPK signaling promotes nuclear export of POP-1 so that the daughter cell receiving the Wnt signal has less nuclear POP-1 than its sister, a phenomenon called POP-1 asymmetry. A similar situation is observed after the SGP division: the distal daughter is specified by Wnt/MAPK activation and contains less nuclear POP-1 than its proximal sister. In mutants lacking POP-1, SYS-1, or upstream components of the Wnt/MAPK pathway, distal-specific cells are not made, and extra proximal-specific cells are sometimes seen; by contrast, gonads with excess SYS-1 produce extra distal cells and lack proximal cells. Therefore, the Wnt/MAPK pathway establishes the proximal-distal axes of both hermaphrodite and male gonads (Tilmann, 2005).
Wnt signaling regulates many aspects of metazoan development, including stem cells. In C. elegans, Wnt/MAPK signaling controls asymmetric divisions. A recent model proposed that the POP-1/TCF DNA binding protein works together with SYS-1/β-catenin to activate transcription of target genes in response to Wnt/MAPK signaling. The somatic gonadal precursor (SGP) divides asymmetrically to generate distal and proximal daughters of distinct fates: only its distal daughter generates a distal tip cell (DTC), which is required for stem cell maintenance. No DTCs are produced in the absence of POP-1/TCF or SYS-1/β-catenin, and extra DTCs are made upon overexpression of SYS-1/β-catenin. This study reports that POP-1/TCF and SYS-1/β-catenin directly activate transcription of ceh-22/nkx2.5 isoforms in SGP distal daughters, a finding that confirms the proposed model of Wnt/MAPK signaling. In addition, it is demonstrated that the CEH-22/Nkx2.5 homeodomain transcription factor is a key regulator of DTC specification. It is speculated that these conserved molecular regulators of the DTC niche in nematodes may provide insight into specification of stem cell niches more broadly (Lam, 2006).
Thus Wnt signaling and ceh-22/nkx2.5 work together to specify the DTC fate. The common function of DTCs in hermaphrodites and males is that of a stem cell niche. Wnt signaling has emerged as a key regulator of stem cells in many tissues and in many organisms, and that role relies on transcriptional activation by TCF/LEF and β-catenin transcription factors. The current work suggests that one role of Wnt signaling may be to control the stem cell niche. A similar suggestion was recently put forward with respect to osteoblasts, which provide a niche for hematopoietic stem cells. CEH-22/Nkx2.5 and its homologs have not previously been implicated in the control of stem cells. Indeed, the fly and vertebrate homologs, termed Tinman and Nkx2.5, respectively, are best known for their roles in heart specification and differentiation. Nematodes have no heart, but CEH-22 controls development of the rhythmically contracting musculature of the pharynx, and zebrafish Nkx2.5 can functionally replace CEH-22. Therefore, the CEH-22/Nkx2.5 class of homeodomain transcription factors has broadly conserved functions in animal development (Lam, 2006).
A remaining question is whether CEH-22 control of the DTC fate reflects a conserved role for this class of homeodomain transcription factors in regulating stem cell niches. Mouse mutants deleted for Nkx2.5 die with a broad spectrum of defects, including severe defects in vasculogenesis and angiogenesis as well as hematopoiesis in the yolk sac. Intriguingly, endothelial cells appear to function as stem cell niches. It is tempting to speculate that the severe vasculature defects in Nkx2.5 mutants may reflect some role of this conserved regulator in control of a vertebrate niche, much as CEH-22 controls the DTC. Two important challenges for the future are to learn how CEH-22 specifies the DTC niche in C. elegans and to learn whether its homologs specify an analogous stem cell niche in flies and vertebrates (Lam, 2006).
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. The β-catenin SYS-1 also exhibits reiterated asymmetry throughout multiple A-P divisions, and this asymmetry is reciprocal to that of POP-1. Furthermore, SYS-1 functions as a coactivator for POP-1, and 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. The SYS-1 and POP-1 asymmetries are regulated independently, each by a subset of genes in the Wnt/MAP kinase pathways. It is proposed 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 (Huang, 2007).
How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. This study establishes a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/β-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. This study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/β-catenin pathway is widely used to control the identity of neuronal lineages (Bertrand, 2009).
Several examples have by now well illustrated that the differentiation of individual neuron types is governed by terminal selector genes that encode transcription factors which directly activate large batteries of terminal differentiation genes. However, how these terminal selector genes are regulated by earlier specification processes, in particular asymmetric divisions, remains poorly understood. This study has uncovered a direct regulatory cascade that links the asymmetric division machinery to the activation of the terminal selector genes ttx-3 and ceh-10 during embryogenesis in C. elegans. These results will first be discussed in the context of the broad concept of progressive regulatory states before analyzing two other general implications of these studies, namely, a common theme of Zic gene function in neural precursors and a potentially broadly conserved role of Wnt signaling in neuronal specification (Bertrand, 2009).
The Zic transcription factor REF-2 is transiently expressed in the SMDD/AIY mother, where it directly activates the expression of the ttx-3 LIM homeobox gene in cooperation with the bHLH transcription factor HLH-2. Following division of the mother cell, TTX-3 is inherited in both SMDD and AIY and activates ceh-10 expression in AIY, but not in SMDD. The difference in ttx-3 activity between AIY and SMDD is due to the Wnt/β-catenin asymmetry pathway. The transcriptional mediators of this pathway, the TCF transcription factor POP-1 and its coactivator the β-catenin SYS-1, are asymmetrically localized after division of the SMDD/AIY mother. In AIY, the POP-1 nuclear concentration is low and SYS-1 concentration is high. This may allow most of the POP-1 proteins to be associated with the coactivator SYS-1 and to activate the transcription of ceh-10 via the predicted POP-1 binding sites present in its promoter. In SMDD, where the POP-1 nuclear concentration is high and SYS-1 concentration is low, most of the POP-1 proteins may not be associated with SYS-1 and therefore repress ceh-10 transcription. Finally, once coexpressed in postmitotic AIY, TTX-3 and CEH-10 directly activate a large battery of terminal differentiation genes responsible for AIY differentiation and specific function. TTX-3 and CEH-10 also maintain their own expression so that the system is locked during larval and adult stages (Bertrand, 2009).
It has been proposed that during development a cell progresses through a succession of 'regulatory states' each characterized by a combination of specific gene regulatory factors. In the case of the AIY terminal division, two regulatory states are observed. The first one (state 1) is characterized by the transient expression of REF-2 and HLH-2 in the SMDD/AIY mother. The second (state 2p) corresponds to the terminal differentiation state defined by the expression of the terminal complex TTX-3/CEH-10 and the battery of terminal differentiation genes. The transition between those two states is driven by a binary decision system based on the Wnt/β-catenin asymmetry pathway (Bertrand, 2009).
These findings provide explicit support for a theoretical model initially proposed by Priess and coworkers (Lin, 1998). In this model a transcription factor 'B' expressed in both daughter cells following the division cooperates with a high POP-1 level in the anterior cell to specify state 2a and cooperates with a low POP-1 level in the posterior cell to specify state 2p. In the case of AIY, this lineage-specific factor 'B' corresponds to the transcription factor TTX-3 (Bertrand, 2009).
Before discussing general principles of Wnt/β-catenin signaling in neuronal specification, ref-2, one specific member of the regulatory network studied here, will be discussed. ref-2 is expressed in several neuronal precursors in the embryo; in contrast, there is no detectable expression of ref-2 in postmitotic neurons at larval and adult stages. Similarly, in Hydra and vertebrates, Zic transcription factors are also expressed in several neural progenitors, while expression in adult postmitotic neurons is only rarely seen. This indicates that Zic transcription factors may have a conserved function in neural precursor development. While in vertebrates Zic transcription factors have been shown to play a role in promoting the proliferation of the progenitors, it is conceivable that they also function as transient initiators of the terminal differentiation program of specific neurons, as observed in the case of AIY. For example, an intriguing parallel can be drawn between the development of the AIY interneurons and the cholinergic projection neurons/interneurons of the vertebrate basal forebrain. These vertebrate cholinergic neurons have an important function in memory formation, as is the case for the cholinergic interneuron AIY. In vertebrates, these postmitotic neurons and their progenitors express the TTX-3-related LIM-homeodomain transcription factor Lhx7/8, which is required for their differentiation. It has been recently reported that the Zic transcription factors Zic1 and Zic3 are also expressed in these progenitors and that inactivation of both genes reduces the number of cholinergic neurons. While these Zic factors seem to regulate primarily the proliferation of the precursors, it would be interesting to test whether, in analogy to ttx-3 initiation by REF-2, they also initiate the expression of Lhx7/8 and endow the progenitors with the ability to generate cholinergic neurons (Bertrand, 2009).
A particular Wnt pathway, the Wnt/β-catenin asymmetry pathway, is involved in many asymmetric blastomere divisions in the early embryo as well as some asymmetric divisions during larval development in C. elegans. Analysis of temperature-sensitive mutants of the upstream kinase gene lit-1(t1512) has shown that this pathway is involved in six successive asymmetric division rounds in the early embryo. However, this pathway has not been shown so far to be implicated in the terminal division of embryonic neuroblasts. This study has observed that the three terminal neuroblast divisions analyzed (giving rise to AIY, AIN, and ASER, respectively) are affected by disrupting this Wnt pathway. Moreover, lit-1(t1512); mom-4(ne1539) embryos shifted at restrictive temperature just before most embryonic neuroblasts undergo their last division give rise to larvae showing strongly uncoordinated movements, suggesting additional defects in motor neuron lineages. These observations predict that the Wnt/β-catenin asymmetry pathway is widely used in terminal neuroblast division in the C. elegans embryo (Bertrand, 2009).
While it was shown that the transcriptional mediators of this pathway, POP-1/TCF and SYS-1/β-catenin, are asymmetrically localized after the terminal division of embryonic neuroblasts, how the asymmetry in this pathway is initially established remains obscure. Both POP-1 and SYS-1 are regulated by this pathway at a posttranslational level (Mizumoto, 2007). In the early embryo POP-1 asymmetry in the AB lineage requires an initial MOM-2/Wnt signal coming from the P1 lineage that may be transmitted among AB blastomeres by a relay mechanism, but POP-1 asymmetry becomes later independent of MOM-2/Wnt. MOM-5/Frizzled is enriched in the posterior pole of early AB derivatives, and in analogy to the planar cell polarity in Drosophila, a Wnt-independent asymmetric Frizzled localization could be responsible for generating asymmetric cell divisions. Additional studies on Wnt requirement and Frizzled localization are required to assess their mode of function in the context of the terminal division of embryonic neuroblasts (Bertrand, 2009).
Neurons are also generated via asymmetric divisions in Drosophila and vertebrates. Recent results suggest a possible role for β-catenin in the asymmetric division of neural progenitors in the mouse brain. For example, it has been proposed that β-catenin may regulate the asymmetric division generating intermediate progenitors from radial glial cells during corticogenesis. A Wnt/β-catenin system, similar to the one shown in this study to operate in terminal neuroblast divisions in C. elegans, may therefore be used in binary cell fate decisions during the development of the nervous system in other organisms (Bertrand, 2009).
The Wnt signaling pathway regulates multiple aspects of the development of stem cell-like epithelial seam cells in C. elegans, including cell fate specification and symmetric/asymmetric division. This study demonstrates that lit-1, encoding the Nemo-like kinase in the Wnt/β-catenin asymmetry pathway, plays a role in specifying temporal identities of seam cells. Loss of function of lit-1 suppresses defects in retarded heterochronic mutants and enhances defects in precocious heterochronic mutants. Overexpressing lit-1 causes heterochronic defects opposite to those in lit-1(lf) mutants. LIT-1 exhibits a periodic expression pattern in seam cells within each larval stage. The kinase activity of LIT-1 is essential for its role in the heterochronic pathway. lit-1 specifies the temporal fate of seam cells likely by modulating miRNA-mediated silencing of target heterochronic genes. It was further shown that loss of function of other components of Wnt signaling, including mom-4, wrm-1, apr-1, and pop-1, also causes heterochronic defects in sensitized genetic backgrounds. This study reveals a novel function of Wnt signaling in controlling the timing of seam cell development in C. elegans (Ren, 2010).
Wnt target gene activation in C. elegans requires simultaneous elevation of β-catenin/SYS-1 and reduction of TCF/POP-1 nuclear levels within the same signal-responsive cell. SYS-1 binds to the conserved N-terminal β-catenin-binding domain (CBD) of POP-1 and functions as a transcriptional co-activator. Phosphorylation of POP-1 by LIT-1, the C. elegans Nemo-like kinase homolog, promotes POP-1 nuclear export and is the main mechanism by which POP-1 nuclear levels are lowered. A mechanism is described whereby SYS-1 and POP-1 nuclear levels are regulated in opposite directions, despite the fact that the two proteins physically interact. The C terminus of POP-1 is essential for LIT-1 phosphorylation and is specifically bound by the diverged β-catenin WRM-1. WRM-1 does not bind to the CBD of POP-1, nor does SYS-1 bind to the C-terminal domain. Furthermore, binding of WRM-1 to the POP-1 C terminus is mutually inhibitory with SYS-1 binding at the CBD. Computer modeling provides a structural explanation for the specificity in WRM-1 and SYS-1 binding to POP-1. Finally, WRM-1 exhibits two independent and distinct molecular functions that are novel for β-catenins: WRM-1 serves both as the substrate-binding subunit and an obligate regulatory subunit for the LIT-1 kinase. Mutual inhibitory binding would result in two populations of POP-1: one bound by WRM-1 that is LIT-1 phosphorylated and exported from the nucleus, and another, bound by SYS-1, that remains in the nucleus and transcriptionally activates Wnt target genes. These studies could provide novel insights into cancers arising from aberrant Wnt activation (Yang, 2011).
Other invertebrate LEF-1 homologs
Members of the Wnt/wingless family of secreted proteins act as short-range inducers and long-range organizers during axis formation, organogenesis and tumorigenesis in many developing tissues. Wnt signaling pathways are conserved in nematodes, insects and vertebrates. Despite its developmental significance, the evolutionary origin of Wnt signaling is unclear. Described here is the molecular characterization of members of the Wnt signaling pathway (Wnt, Dishevelled, GSK3, beta-Catenin and Tcf/Lef) in Hydra, a member of the evolutionarily old metazoan phylum Cnidaria. Wnt and Tcf are expressed in the putative Hydra head organizer, the upper part of the hypostome. Wnt, beta-Catenin and Tcf are transcriptionally upregulated when head organizers are established early in bud formation and head regeneration. Wnt and Tcf expression domains also define head organizers created by de novo pattern formation in aggregates. These results indicate that Wnt signaling may be involved in axis formation in Hydra and support the idea that it was central in the evolution of axial differentiation in early multicellular animals (Hobmayer, 2000).
The mechanism of animal-vegetal (AV) axis formation in the sea urchin embryo is incompletely understood. Specification of the axis is thought to involve a combination of cell-cell signals and as yet unidentified maternal determinants. In Xenopus the Wnt pathway plays a crucial role in defining the embryonic axes. Recent experiments in sea urchins have shown that at least two components of the Wnt signaling pathway, GSK3beta and beta-catenin, are involved in embryonic AV axis patterning. These results support the notion that the developmental network that regulates axial patterning in deuterostomes is evolutionarily conserved. To further test this hypothesis, the role of beta-catenin nuclear binding partners, members of the TCF family of transcriptional regulators, was examined in sea urchin AV axis patterning. To test the role of TCFs in mediating beta-catenin signals in sea urchin AV axis development, the consequences of microinjecting RNAs encoding altered forms of TCF were examined on sea urchin development. Expression of a dominant negative TCF results in a classic 'animalized' embryo. In contrast, microinjected RNA encoding an activated TCF produces a highly 'vegetalized' embryo. Transactivational activity of endogenous sea urchin TCF is potentiated by LiCl treatment, which vegetalizes embryos by inhibiting GSK3, consistent with an in vivo interaction between endogenous beta-catenin and TCF. Evidence is provided indicating that all of beta-catenin's activity in patterning the sea urchin AV axis is mediated by TCF. Using a glucocorticoid-responsive TCF, it has been shown that TCF transcriptional activity affects specification along the AV axis between fertilization and the 60-cell stage (Vonica, 2000).
Patterning of cell fates along the sea urchin animal-vegetal embryonic axis
requires the opposing functions of nuclear ß-catenin/TCF-Lef, which
activates the endomesoderm gene regulatory network, and SoxB1, which
antagonizes ß-catenin and limits its range of function. A crucial aspect
of this interaction is the temporally controlled downregulation of SoxB1,
first in micromeres and then in macromere progeny. SoxB1 is
regulated at the level of protein turnover in these lineages. This mechanism
is dependent on nuclear ß-catenin function. It can be activated by Pmar1,
but not by Krl, both of which function downstream of ß-catenin/TCF-Lef.
At least partially distinct, lineage-specific mechanisms operate, since turnover
in the macromeres depends on entry of SoxB1 into nuclei, and on redundant
destruction signals, neither of which is required in micromeres. Neither of
these turnover mechanisms operates in mesomere progeny, which give rise to
ectoderm. However, in mesomeres, SoxB1 appears to be subject to
negative autoregulation that helps to maintain tight regulation of
SoxB1 mRNA levels in presumptive ectoderm. Between the seventh and
tenth cleavage stages, ß-catenin not only promotes degradation of SoxB1,
but also suppresses accumulation of its message in macromere-derived
blastomeres. Collectively, these different mechanisms work to regulate
precisely the levels of SoxB1 in the progeny of different tiers of blastomeres
arrayed along the animal-vegetal axis (Angerer, 2005).
Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. This study shows that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles (Yoon, 2019)
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