Table of contents

Wnts in worms: asymmetric cell division and cell fate determination

A polarizing signal induces endoderm production by a 4-cell stage blastomere in C. elegans called EMS. Mutations were identified in five genes, mom-1 through mom-5, required for EMS to produce endoderm. mom-1, mom-2, and mom-3 are required in the signaling cell, P2, while mom-4 is required in EMS. P2 signaling downregulates an HMG domain protein, POP-1, in one EMS daughter. The sequence of mom-2 predicts that it encodes a member of the Wnt family of secreted glycoproteins, which in other systems activate HMG domain proteins. Defective mitotic spindle orientations in mom mutant embryos indicate that Wnt signaling influences cytoskeletal polarity in blastomeres throughout the early embryo (Thorpe, 1997).

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).

Mutations in the gene lin-17 result in the disruption of a variety of asymmetric cell divisions in Caenorhabditis elegans. lin-17 mutations affect the divisions of ectodermal, gonadal and neural cells that are not related by their lineage histories, by their positions, or by the developmental stages at which they divide. For example, in lin-17 mutants an abnormal division of the cell P7.p in the mid-body, causes the hermaphrodite to have an ectopic vulva-like protrusion (the multivulva phenotype), while defects in the divisions of the B, T, P10.p, P11.p cells in the male tail result in abnormal tail structures. In most cases, the affected cell divisions are asymmetric in wild-type animals but symmetric in lin-17 animals, producing sister cells with similar cell fates. lin-17 mutations cause divisions that would normally produce sister cells of unequal size to instead generate cells of equal size. For this reason, it has been suggested that lin-17 functions prior to or during cell division to establish the polarity of mother cells and that this polarity determines the asymmetry of these divisions. lin-17 encodes a protein with seven putative transmembrane domains. The LIN-17 protein is most similar to the Drosophila Frizzled protein and its vertebrate homologs. Studies using a lin-17-green fluorescent protein translational fusion indicate that lin-17 is expressed in mother cells before asymmetric cell divisions and in both daughter cells after the divisions. These results suggest that lin-17 encodes a receptor that regulates the polarities of cells undergoing asymmetric cell divisions and raise the possibility that the LIN-17 protein acts as a receptor for the Wnt protein LIN-44, which also controls asymmetric cell divisions (Sawa, 1996).

Mutations in the C. elegans gene lin-44 lead to reversals in the polarity of certain asymmetric cell divisions. lin-44 is a member of the Wnt family of genes that encode secretory glycoproteins implicated in intercellular signaling. Both in situ hybridization experiments using lin-44 transcripts and experiments using reporter constructs designed to mimic patterns of lin-44 expression indicate that lin-44 is expressed in hypodermal cells at the tip of the tail and posterior to the cells with polarities affected by lin-44 mutations. Mosaic analysis indicates that lin-44 acts cell nonautonomously. It is proposed that LIN-44 protein is secreted by tail hypodermal cells and affects the polarity of asymmetric cell divisions that occur more anteriorly in the tail (Herman, 1995).

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 early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. These findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell (Bei, 2002).

The mes-1 gene encodes a probable transmembrane protein with overall structural similarity to receptor tyrosine kinase and is a factor required for proper asymmetry and cell fate specification in embryonic germlineage. Null mutations in mes-1 cause a maternal-effect sterile phenotype in which the progeny of homozygous mothers are viable but mature without germcells. Most cell types are specified properly in mes-1 sterile animals, but the germline cell named P4 adopts the fate of its sister cell, a muscle precursor, called D and produces ectopic muscle at the expense of the germline. Interestingly, MES-1 protein is localized intensely at the contact site between the germline blastomere and intestinal precursor at each early developmental stage, starting from the four-cell stage where MES-1 is localized at the contact site between P2 and EMS. An intense phosphotyrosine signal that depends on mes-1(+) activity is correlated with MES-1 protein localization. MES-1 is required in both P2 and EMS and appears to act through a second gene, src-1, a homolog of the vertebrate protooncogene c-Srcpp60. A probable null mutant of src-1 is described that exhibits a fully penetrant maternal-effect embryonic lethal phenotype. The src-1 and mes-1 mutants exhibit similar germline defects and have a nearly identical set of genetic interactions with Wnt/Wg pathway components (Bei, 2002).

Double mutants between mes-1 or src-1 and each of several Wnt/Wg signaling components exhibit a complete loss of P2/EMS signaling, including a loss of the A/P division orientation in the EMS cell. Synergy was observed between mes-1 or src-1 mutants and each of the following previously described mutants: mom-1 (Porcupine), mom-2 (Wnt/Wg), mom-5 (Frizzled), sgg-1 (GSK-3), and mom-3 (uncloned). In addition, identical synergies were observed in the phenotypes of embryos produced by mes-1 or src-1 homozygotes after injection with a mixture of two double-stranded RNAs targeting the C. elegans Disheveled homologs dsh-2 and mig-5. RNAi targeting these Disheveled homologs individually does not induce visible defects in P2/EMS signaling. mes-1 functions in both EMS and P2 to direct MES-1 protein localization at EMS/P2 junction and to specify A/P cleavage orientation in the EMS cell, while src-1 is required cell autonomously in EMS for the induction of the EMS A/P division axis. These findings suggest that a homotypic interaction between MES-1-expressing cells, P2 and EMS, induces a SRC-1-mediated phosphotyrosine signaling pathway that functions in parallel with Wnt/Wg signaling to specify endoderm and to orient the division axis of EMS in early C. elegans embryos (Bei, 2002).

Recent work on dorsal closure in Drosophila has identified a possible convergence between Src and Wnt signaling at the level of regulation of the Jun N-terminal kinase (JNK). Dorsal closure is the process in which epithelial sheets spread over and enclose the dorsal region of the Drosophila embryo during morphogenesis. JNK signaling is essential for dorsal closure and mutants lacking JNK exhibit a dorsal-open phenotype and also exhibit loss of expression of a TGF-ß homolog decapentaplegic in the epithelial cells that lead the closure process. Recent genetic studies have implicated both Src-like kinases and Wnt signaling components in the dorsal closure process and in regulating the expression of dpp. Mutations in Src42A and Wnt signaling factors produce dorsal closure phenotypes similar to JNK mutants and activation of JNK signaling can partially suppress defects caused by these mutants. These findings suggest that Wnt and Src may converge to regulate JNK activity and dorsal closure in Drosophila and thus provide evidence from another system for interactions between these pathways in a developmental process (Bei, 2002).

How do temporal and spatial interactions between multiple intercellular and intracellular factors specify the fate of a single cell in Caenorhabditis elegans? P12, which is a ventral cord neuroectoblast, divides postembryonically to generate neurons and a unique epidermal cell. Three classes of proteins are involved in the specification of P12 fate: the LIN-3/LET-23 epidermal growth factor signaling pathway; a Wnt protein LIN-44 and its candidate receptor LIN-17, and a homeotic gene product EGL-5. lin-3 encodes a membrane-spanning protein with a single extracellular EGF domain that is similar in structure to members of the EGF family of growth factors. LIN-3 is an inductive signal sufficient to promote the P12 fate, and the conserved EGF signaling pathway is utilized for P12 fate specification: egl-5, an AbdominalB homolog, is a downstream target of the lin-3/let-23 pathway in specifying P12 fate, and LIN-44 and LIN-17 act synergistically with lin-3 in the specification of the P12 fate. The Wnt pathway may function early in development to regulate the competence of the cells to respond to the LIN-3 inductive signal (L. I. Jiang, 1998).

In C. elegans there are twelve ventral cord precursor cells, P1-P12, numbered from anterior to posterior along the body axis. These cells divide postembryonically to generate cells of the ventral nervous system, as well as the vulva. P11/P12 are the most posterior pair of the ventral cord precursors. At hatching, the cells AB.plapappa (left side) and AB.prapappa (right side) are disposed laterally. In hermaphrodites, they start to migrate ventrally several hours after hatching and enter the ventral cord about 8-9 hours after hatching. The left cell migrates to the anterior and becomes P11; the right cell migrates to the posterior and becomes P12. Two hours later they each divide once. The anterior daughters, P11.a and P12.a are neuroblasts that will divide for three more rounds to generate several ventral cord neurons. These neurons are morphologically indistinguishable under Nomarski optics. The posterior daughter of P11, P11.p, does not divide but rather fuses with the large epidermal syncytium hyp7. P12.p divides once more about 1 hour prior to L1 molt to generate two cells:, which becomes a unique epidermal cell (hyp12) and P12.pp, which undergoes cell death. P11.p and can be distinguished by their different nuclear morphologies and positions observed with Nomarski optics. Prior to migration, both P11 and P12 cells are able to express the P12 fate: if only a single cell is present, it will adopt the P12- like fate. Therefore, P12 represents a primary fate, while P11 is a secondary fate (L. I. Jiang, 1998).

Interactions between lin-3 and lin-44 were tested by examining the P11/P12 defect in a strain defective in both genes. The strong synergy observed between lin-3 and lin-44 is consistent with the two signals acting in parallel. To confirm the interaction between lin-3 and lin-44, a test of synergy between lin-3 and lin-17 mutations was performed. lin-17, which encodes a putative seven-transmembrane protein similar to the Drosophila Frizzled protein, has been suggested to be a receptor for the LIN-44 protein. Similar synergistic interactions are found between lin-3 and lin-17 as have been found between lin-3 and lin-44. A synergistic interaction is also found between mutations of let-23, the EGF receptor for LIN-3 signal, and the Wnt signal LIN-44. These data support the hypothesis that both of the two signaling pathways, lin-3 and lin-44, are required for the P12 fate specification. How do these two signaling pathways function in concert to specify P12 fate? The favored model is that both pathways are required for proper P12 fate specification and that they act at different developmental times. Three sources of evidence support this model: (1) LIN-3 overexpression experiments indicate that LIN-3 signal is required in early L1 before P11/P12 enter the ventral cord to induce P12 fate. (2) lin-44 expression is turned on during embryogenesis, much earlier than the time of P11/P12 induction. (3) The unique effect of overexpression of LIN-3EGF during late embryogenesis in lin-44 mutants suggests that LIN-44 function may be important in the early phase of P12 fate specification. It is possible that the Wnt pathway regulates the competence of the cells to respond to the LIN-3 inductive signal. But the Wnt signal alone is not sufficient to promote P12 fate, since overexpression of LIN-44 in wild-type animals has no effect on P11/P12 cell fate specification (L. I. Jiang, 1998).

The following is a model for P12 neuroectoblast fate specification: in newly hatched larvae, LIN-44 signal acts via receptor LIN-17 to set up the competence of P11 and P12 cells to respond to the inductive signal and be able to express P12 fate. egl-5 expression is kept off in both P11 and P12 cells. Later, an inductive signal LIN-3 coming from the posterior region activates LET-23 receptor activity in the posterior cell of the P11/P12 pair. Activation of the lin-3/let-23 pathway turns on egl-5 expression, which specifies the posterior cell to take on P12 fate. lin-15 negatively regulates let-23 activity and prevents the anterior cell from becoming P12. Information from the Wnt signaling pathway may be integrated into the lin-3/let-23 EGF signaling pathway either at the level of LIN-3 signal, LET-23 receptor or egl-5 transcription. Thus the temporal and spatial co-ordination and interactions between the Wnt signal, EGF signal and HOM-C transcription factor are important for P12 fate specification (L. I. Jiang, 1998).

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).

The specification of body pattern along the anteroposterior (A/P) body axis is achieved largely by the actions of conserved clusters of Hox genes. Precise control of the hox gene mab-5 expression is crucial for achieving wild-type development of at least two cell types, the V cells and the migratory Q neuroblasts. In the V5 lineage, mab-5 is switched on and off multiple times: each change in expression regulates a different type of cell-fate decision. The two Q cells are neuroblasts born in identical A/P postions but on opposite sides of the animal: QL on the left and QR on the right. In wild-type animals, after a short poserior migration, QL switches on mab-5, which, in turn, causes descendants of QL to migrate to the posterior. In contrast, mab-5 remains off in QR and its descendants, and as a result the descendants of QR continue to migrate toward the anterior. Limiting expression of Hox genes to localized regional domains and controlling the precise patterns of expression within those domains is critically important for normal patterning. egl-20, a C. elegans gene required to activate expression of the Hox gene mab-5 in the migratory neuroblast QL, encodes a member of the Wnt family of secreted glycoproteins. A second Wnt pathway gene, bar-1, which encodes a beta-catenin/Armadillo-like protein, is also required for activation of mab-5 expression in QL. In addition, the gene pry-1, phenotypically resembling Drosophila Polycomb-group mutants, is required to limit expression of the Hox genes lin-39, mab-5 and egl-5 to their correct local domains. egl-20, pry-1 and bar-1 all function in a linear genetic pathway with conserved Wnt signaling components, suggesting that a conserved Wnt pathway activates expression of mab-5 in the migratory neuroblast QL. Members of this Wnt signaling system are found to play a major role in both the general and fine-scale control of Hox gene expression in other cell types along the A/P axis. Although a similar global role for Wingless in regulating Hox gene expression in Drosophila has not been found, in at least one instance, the expression of Ultrabithorax in the midgut, a Drosophila Hox gene acts downstream of a Wnt gene (Maloof, 1999).

In Caenorhabditis elegans males, a row of epidermal precursor cells called seam cells generates a pattern of cuticular alae in anterior body regions and neural sensilla (called rays) in the posterior. The Abdominal-B homolog mab-5 is required for two posterior seam cells, V5 and V6, to generate rays. The V5 lineage generates one ray and the V6 lineage generates five rays. In mab-5 mutant males, V5 and V6 do not generate sensory ray lineages but instead generate lineages that lead to alae, cuticular ridges that extend along the two sides of the animal. Alae are normally generated by the V1-V4 cells only. Two independent regulatory pathways can activate mab-5 expression in the V cells. (1) The caudal homolog pal-1 turns on mab-5 in V6 during embryogenesis. (2) A Wnt signaling pathway is capable of activating mab-5 in the V cells during postembryonic development, however, during normal development Wnt signaling is inhibited by signals from neighboring V cells. The inhibition of this Wnt signaling pathway by lateral signals between the V cells limits the number of rays in the animal and also determines the position of the boundary between alae and rays (Hunter, 1999).

Wnt signaling systems play important roles in the generation of cell and tissue polarity during development. A Wnt signaling system is described that acts in a new way to orient the polarity of an epidermal cell division in C. elegans. In this system, the EGL-20/Wnt signal acts in a permissive fashion to polarize the asymmetric division of a cell called V5. EGL-20 regulates this polarization by counteracting lateral signals from neighboring cells that would otherwise reverse the polarity of the V5 cell division. These findings indicate that this lateral signaling pathway also involves Wnt pathway components. Overexpression of EGL-20 disrupts both the asymmetry and polarity of lateral epidermal cell divisions all along the anteroposterior (A/P) body axis. Together these findings suggest that multiple, inter-related Wnt signaling systems may act together to polarize asymmetric cell divisions in this tissue (Whangbo, 2000).

In Caenorhabditis elegans, Ras/ERK and Wnt/ß-catenin signaling pathways cooperate to induce P12 and vulval cell fates in a Hox-dependent manner. eor-1 and eor-2, two new positively acting nuclear components of the Ras and Wnt pathways, are described. eor-1 and eor-2 act downstream or in parallel to ERK and function redundantly with the Mediator complex gene sur-2 and the functionally related gene lin-25, such that removal of both eor-1/eor-2 and sur-2/lin-25 mimics the removal of a main Ras pathway component. Furthermore, the eor-1 and eor-2 mutant backgrounds reveal an essential role for the Elk1-related gene lin-1. eor-1 and eor-2 also act downstream or in parallel to pry-1 Axin and therefore act at the convergence of the Ras and Wnt pathways. eor-1 encodes the ortholog of Drosophila Tramtrack and human PLZF, a BTB/zinc-finger transcription factor that is fused to RARalpha in acute promyelocytic leukemia. eor-2 encodes a novel protein. EOR-1/PLZF and EOR-2 appear to function closely together and cooperate with Hox genes to promote the expression of Ras- and Wnt-responsive genes. Further studies of eor-1 and eor-2 may provide insight into the roles of PLZF in normal development and leukemogenesis (Howard, 2002).

In the leech Helobdella robusta, an annelid worm, the early pattern of cell divisions is stereotyped. The unequal first cleavage yields cells AB and CD, which differ in size, cytoplasmic inheritance, normal fate, and developmental potential. A dynamic and transcription-independent pattern of WNT signaling is reported in the two-cell stage of H. robusta. Surprisingly, HRO-WNT-A is first expressed in a stochastic manner, such that either AB or CD secretes the protein in each embryo. This stochastic phase is followed by a deterministic phase during which first AB, then CD expresses HRO-WNT-A. When contact between the cells is reduced or eliminated, both AB and CD express HRO-WNT-A simultaneously. Finally, bathing embryos in anti-HRO-WNT-A antibody during first cleavage reduces the adhesion between cells AB and CD. These findings show that the stochastic phase of HRO-WNT-A signaling in the two-cell stage of Helobdella is negatively regulated by cell-cell contact and that this early signaling affects cell adhesion without affecting cell fate. It is speculated that the primordial function of wnt class genes may have been to regulate cell-cell adhesion and that the nuclear signaling components of the wnt pathway arose later in association with the evolution of diverse cell types (Huang, 2001).

Wnt proteins are intercellular signals that regulate various aspects of animal development. In C. elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva (Inoue, 2004).

Since lin-44(null) enhances lin-18(null) but not lin-17(null), lin-44 must function in parallel to lin-18. Similarly, since mom-2(null) enhances lin-17(null), mom-2 must function in parallel to lin-17. Based on these results, it is proposed that LIN-44 preferentially functions as the ligand for LIN-17/Frizzled and MOM-2 preferentially functions as the ligand for LIN-18/Ryk. Since lin-44 and mom-2 single mutant phenotypes are weaker than those of lin-17 and lin-18, each receptor likely transduces additional signals (including LIN-44/LIN-18 and MOM-2/LIN-17 combinations as well as CWN-2). A weak enhancement of lin-18(e620) by mom-2(RNAi) supports this possibility. The results do not rule out the possibility that LIN-44 or MOM-2 signals through a third pathway. However, the complete reversal of the P7.p orientation observed in the lin-17; lin-18 double mutant suggests that the two receptors account for most of the P7.p orienting activity. LIN-17 and LIN-44 are also required for other fate specifications in C. elegans, suggesting that LIN-17 acts as a LIN-44 receptor in multiple tissues. Sequence analysis suggests that CWN-2 is the ortholog of Wnt5, the ligand for Derailed in Drosophila. Therefore, the involvement of CWN-2 is consistent with it functioning as a LIN-18 ligand, although it was not possible to resolve the receptor specificity for this ligand. The orthology relationship of MOM-2 is not clear. MOM-2/Wnt and MOM-5/Frizzled are required for endoderm induction. However, no evidence of MOM-5 involvement in P7.p orientation was found, and LIN-18 is not required for endoderm induction (Inoue, 2004).

How cells integrate the input of multiple polarizing signals during division is poorly understood. Two distinct C. elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/β-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. CKI (KIN-19) plays a role not only in the Wnt/β-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, a model is established for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event (Walston, 2004).

During development, certain cell divisions must occur with a specific orientation to form complex structures and body plans. In many cases, the polarizing input for oriented divisions involves Wnt signaling. One example of such division involves neuroblasts in Drosophila, in which the first division of the pI sensory organ precursor cell is under the control of Frizzled (Fz) and Dishevelled (Dsh). The orientation of blastomere divisions in the early C. elegans embryo has also been shown to require Wnt signaling. In the 4-cell embryo, the EMS blastomere is induced by its posterior neighbor, the P2 blastomere. This induction has two consequences: it specifies the fates of EMS daughter cells and properly positions the mitotic spindle of EMS. Although both processes are under the control of Wnt signaling, they are controlled through divergent pathways. When EMS divides, the anterior daughter, MS, gives rise to progeny that are primarily mesodermal, and the posterior daughter, E, produces all of the endoderm. The fates of MS and E are controlled in part by a Wnt signaling pathway that regulates the activity of the Tcf/Lef transcription factor, POP-1, in conjunction with the β-catenin WRM-1. WRM-1 interacts with POP-1 through a cofactor, LIT-1, a NEMO-like kinase that is activated through a parallel mitogen-activated protein kinase (MAPK) pathway. Pathways that utilize a β-catenin to alter transcription are referred to as Wnt/β-catenin pathways. Removal of some components of the Wnt/β-catenin pathway alters the fates of the two EMS daughters. Although the fate of the EMS daughters is controlled by a Wnt/β-catenin pathway, the orientation of the EMS division is controlled by a different Wnt pathway (Walston, 2004).

In wild-type embryos, the EMS spindle initially aligns along the left/right (L/R) axis and rotates to adopt an anterior/posterior (A/P) orientation during the initial stages of mitosis. In embryos that lack the function of certain Wnt signaling components, the EMS spindle often sets up in the proper orientation but fails to rotate along the A/P axis until the onset of anaphase. In some cases, the delayed spindle rotates dorsoventrally (D/V) before it adopts the proper A/P alignment. The Wnt spindle orientation pathway that controls EMS orientation involves a Wnt (MOM-2), Porcupine (Porc; MOM-1), and Fz (MOM-5). GSK-3, the C. elegans GSK-3β homolog, has been reported to act positively downstream of the Fz receptor to regulate EMS spindle positioning, rather than as a downregulator of β-catenin accumulation as observed with Wnt/β-catenin signaling. Indeed, Wnt/β-catenin signaling components downstream of GSK-3 are not involved in controlling EMS spindle alignment, and EMS spindle alignment occurs independently of gene transcription. Pathways such as the one that positions the spindle in EMS, which utilize GSK-3 but are independent of transcription, are referred to as Wnt spindle orientation pathways (Walston, 2004).

Although many Wnt signaling components have been identified that participate in spindle orientation, the role of the Dsh family has not been clearly characterized. The Dsh family proteins transmit Wnt signals received from Fz receptors. The Dshs use three domains (DIX, PDZ, and DEP) to interact with different downstream proteins and activate multiple Wnt pathways specifically. The C. elegans genome contains three Dsh family genes that possess the three conserved domains: dsh-1, dsh-2, and mig-5. Transcripts of dsh-2 and mig-5 are at similar, enriched levels in the 4- and 8-cell embryo based on microarray analysis, while dsh-1 levels are low (Walston, 2004).

Another molecule involved in Wnt signaling is Casein Kinase I (CKI). CKI has been shown to prime β-catenin for degradation by phosphorylating it at a specific serine residue. Once primed, the β-catenin can be further phosphorylated and targeted for destruction by GSK-3β. CKI has also been shown to bind and phosphorylate Dsh and may assist in inhibiting GSK-3β when Wnt signaling is active. Loss of function of the CKIα homolog, kin-19, causes defects in the fate of EMS daughter cells. Although the role of CKI in spindle alignment has not been examined, CKIα localizes to centrosomes and mitotic spindles in vertebrate systems (Walston, 2004).

A pathway involving MES-1, a receptor tyrosine kinase, and SRC-1, a Src family tyrosine kinase, acts redundantly with Wnt signaling with respect to the fate of EMS daughters and the orientation of the EMS spindle. When a Src pathway member and a member of the Wnt spindle orientation pathway are removed simultaneously, the EMS spindle fails to rotate into the proper A/P position prior to division and remains misaligned throughout division. Removal of Src pathway members also enhances endoderm fate specification defects observed following removal of Wnt/β-catenin pathway members. Spindle orientation defects in dsh-2(RNAi);mig-5(RNAi) embryos have not been reported unless the Src pathway is also removed; however, only defects in cell division orientation have been reported, as opposed to abnormalities in initial spindle positioning (Walston, 2004).

In addition to regulating the orientation of the EMS division, four of the mom (more mesoderm) genes, mom-1 (Porc), mom-2 (Wnt), mom-5 (Fz), and mom-3 (uncloned), cause spindle alignment defects in the ABar blastomere of the 8-cell embryo. Three of the four AB granddaughters, ABal, ABpl, and ABpr, divide with spindle orientations that are parallel to one another. ABar divides in an orientation that is roughly perpendicular to the other three, an event best viewed from the right side of the embryo, placing anterior to the right. When the function of one of the above mom genes is removed, ABar divides parallel to the other AB granddaughters, resulting in mispositioning of its daughter cells, such that ABarp, the wild-type posterior daughter cell, adopts a position that is anterior to its sister, ABara. The source of the polarizing cue(s) that orients the division of ABar is unclear. However, using blastomere isolations, it has been demonstrated that C, MS, and E are all competent to align the spindle and generate asymmetric expression of POP-1 within unidentified, dividing AB granddaughters, suggesting that one or more of these cells could produce signals that orient the division of ABar in vitro (Walston, 2004).

In this study, the roles of two Wnt signaling pathways involved in regulating the mitotic spindle are demonstrated. (1) The nontranscriptional Wnt spindle alignment pathway requires contact from the C blastomere to align the spindle of ABar. The three Dshs differentially participate in aligning the spindles of EMS and ABar and vary with respect to their interaction with the Src signaling pathway during spindle orientation. Moreover, while KIN-19 participates in endoderm induction through the Wnt/β-catenin pathway, it also acts in the Wnt spindle orientation pathway. (2) A Wnt/β-catenin pathway regulates the timing of spindle rotation in ABar, presumably by specifying the fate of neighboring blastomeres. Taken together, these studies indicate that spindle orientation during early development is a tightly regulated event, influenced by multiple cues transmitted via redundant pathways (Walston, 2004).

Wnt signals in the early embryo are transmitted from P2 to EMS to orient its spindle and to specify the fate of the EMS daughters. The orientation of the spindle relies on Wnt ligands, including MOM-2, that are secreted from P2 and activate MOM-5/Fz on the surface of EMS. This ultimately activates GSK-3, resulting in spindle alignment irrespective of gene transcription or other downstream Wnt/β-catenin components. The current analysis suggests that all three Dsh proteins are upstream of GSK-3 activation. Removal of the function of any of the dshs results in an incorrectly positioned EMS spindle, with varying penetrance. The strongest effect is seen in offspring of dsh-2 mutant mothers, suggesting that DSH-2 is primarily responsible for transducing the signal from MOM-5 to GSK-3 in EMS. Antibody staining shows an enrichment of DSH-2 at the area of cell-cell contact between EMS and P2, consistent with a MOM-2/Wnt signal activating DSH-2 at the cell cortex through the MOM-5/Fz receptor (Walston, 2004).

This analysis also shows that kin-19 contributes to the Wnt spindle orientation pathway in both EMS and ABar. Although KIN-19 participates in EMS fate specification, it has not been demonstrated to influence the orientation of the EMS spindle. Depletion of KIN-19 results in spindle misalignment in EMS and ABar. Additionally, KIN-19 localizes to centrosomes during mitosis: this has been shown to be important in establishing the initial polarization axis in the 1-cell embryo. How kin-19 operates within the pathway remains unclear. Because CKI family members have the ability to prime β-catenin for further phosphorylation by GSK-3, KIN-19 may act as a priming kinase for GSK-3-mediated phosphorylation of other unidentified target proteins. Based on the localization of KIN-19, these targets may be linked to the cytoskeleton, thereby affecting the physical alignment of the spindles of EMS and ABar (Walston, 2004).

This analysis shows that the same Wnt spindle orientation pathway that orients the EMS blastomere also aligns the spindle of the ABar blastomere. The results indicate that, as in EMS, this pathway does not require gene transcription to align the ABar spindle and that GSK-3 could be interacting directly or indirectly with the cytoskeleton (Walston, 2004).

All three dsh genes also act redundantly during ABar spindle orientation as well. Surprisingly, the data show that MIG-5 is the Dsh that is most important during ABar spindle orientation, contrary to the case for EMS spindle alignment, where DSH-2 is most important. The ABar spindle defects seen in dsh-2(or302) embryos suggest that DSH-2 also contributes significantly to ABar spindle orientation. DSH-1 seems to play only a minor role, since dsh-1(RNAi) does not result in ABar spindle defects unless performed along with mig-5(RNAi). This combination may remove enough total Dsh protein to prevent ABar from dividing correctly. In contrast, when dsh-1 function is removed in combination with that of dsh-2, the amount of MIG-5 present may be sufficient to maintain the total Dsh protein at a high enough level that the removal of dsh-1 function has no effect. Alternatively, the Dshs may have slightly different functions in regulating spindle orientation (Walston, 2004).

In Wnt signaling mutants, defective EMS spindle orientation is eventually corrected to the proper orientation, which is presumably due to the activity of the parallel src-1 pathway. In contrast, the Src pathway does not rescue spindle defects in ABar, although the src-1 pathway does influence ABar division. At this time, targets of SRC-1 in spindle orientation are unknown. It is possible that one or more of the Dshs are SRC-1 targets; however, the more severe phenotype of src-1 mutants in EMS suggests that other targets are also affected. Interestingly, in EMS and ABar, removal of src-1 function along with the function of either dsh-1 or mig-5 has very little additional effect on spindle polarity; however, when src-1 function is removed in dsh-2(or302) mutants, spindle misalignment is enhanced to nearly complete penetrance in EMS and ABar. Thus, while the three Dsh proteins act partially redundantly, there may be differences in how they impinge on other pathways (Walston, 2004).

In the 8-cell embryo, ABar contacts the C and MS blastomeres. Blastomere isolations have been used to demonstrate that C and MS can orient the spindle of unidentified AB granddaughters. They also demonstrate that AB granddaughters have random spindle orientation when presented with a mom-2 mutant C blastomere, but not with a mom-2 mutant MS blastomere. Using pal-1(RNAi) to alter the fate of C and laser killing of blastomeres to create steric hindrance within the embryo, ABar has been unambiguously identified. These results show that a loss of contact between C and ABar results in misalignment of its spindle in virtually all cases. Thus, contact with C is not only sufficient to align the spindle of an AB granddaughter but is also necessary to properly orient the ABar spindle through the Wnt spindle alignment pathway. These results further suggest that the polarizing activity of C is mediated by MOM-2/Wnt (Walston, 2004).

The orientation of the EMS spindle is not affected when Wnt/β-catenin signaling is abrogated through disruption of transcription or removal of WRM-1/β-catenin or POP-1/Tcf/Lef. In contrast, when wrm-1, lit-1, pop-1, or ama-1 function is removed, the ABar spindle is delayed in rotating into position. All of these treatments are known to affect the differentiation of the progeny of EMS. Moreover, MS has been shown to be capable of orienting the spindle of AB granddaughters in isolated blastomeres independent of MOM-2 function. Given the physical proximity of the blastomeres to ABar in the wild-type embryo, MS may produce a MOM-2-independent signal that ultimately affects positioning of the ABar centrosome further from C. The data further suggest that abnormalities in the fate of EMS daughters result in rotation defects. In wrm-1(RNAi) embryos, both EMS daughters become MS-like, and β-tubulin::GFP analysis reveals that the centrosomes of ABar do not rotate properly in many cases. If a signal that aids orientation of the spindle of ABar is normally secreted by MS, the two MS-like daughter cells specified in wrm-1(RNAi) embryos could produce competing signals that result in spindle rotation defects in ABar. Similarly, when both of the EMS daughters adopt an E-like fate, as in pop-1(RNAi), altered signaling from EMS daughters could again lead to a similar phenotype. In these cases, the centrosomal positioning presumably relies solely on the Wnt signal from C to eventually position the spindle in the correct orientation (Walston, 2004).

In conclusion, spindle orientation in the early C. elegans embryo is regulated through a Wnt spindle alignment pathway involving the Dshs and KIN-19 but independent of gene transcription. In addition, in ABar, the Wnt/β-catenin pathway regulates the timing of spindle rotation in a transcription-dependent manner, presumably indirectly by altering the fates of E and MS. The components of the Wnt spindle orientation pathway downstream of KIN-19 and GSK-3 are unknown; future work should be aimed at identifying these components and determining which Wnts are involved in specific inductive events (Walston, 2004).

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands, were examined. During regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-catenin1 RNAi animals. In addition, regeneration defects of the nervous system were observed that are not found after Smed-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, Smed-WntP-1 and Smed-Wnt11-2 were identifed as the putative posterior organizers; Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, this study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through β-catenin-dependent and -independent pathways (Adell, 2009).

Wnt signals orient mitotic spindles in development, but it remains unclear how Wnt signaling is spatially controlled to achieve precise spindle orientation. This study shows that C. elegans syndecan (SDN-1; see Drosophila Syndecan) is required for precise orientation of a mitotic spindle in response to a Wnt cue. SDN-1 is the predominant heparan sulfate (HS) proteoglycan in the early C. elegans embryo, and that loss of HS biosynthesis or of the SDN-1 core protein results in misorientation of the spindle of the ABar blastomere. The ABar and EMS spindles both reorient in response to Wnt signals, but only ABar spindle reorientation is dependent on a new cell contact and on HS and SDN-1. SDN-1 transiently accumulates on the ABar surface as it contacts C, and is required for local concentration of Dishevelled (MIG-5; see Drosophila Dishevelled) in the ABar cortex adjacent to C. These findings establish a new role for syndecan in Wnt-dependent spindle orientation (Dejima, 2014).

FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. This study shows that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/beta-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, it was shown that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans (Minor, 2013).

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. This study demonstrates that Wnt (see Drosophila Wingless), the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the β-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease (Shen, 2021).

Opposing Wnt pathways orient cell polarity during organogenesis

The orientation of asymmetric cell division contributes to the organization of cells within a tissue or organ. For example, mirror-image symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. This study characterized the molecular mechanisms contributing to this division pattern. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs toward the center. These Wnts act via Fz/LIN-17 and Ryk/LIN-18, which control beta-catenin localization and activate gene transcription. In addition, VPCs on both sides of the axis of symmetry possess a uniform underlying 'ground' polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving the Ror receptor tyrosine kinase CAM-1 and the planar cell polarity component Van Gogh/VANG-1. Thus, tissue polarity is determined by the integration of multiple Wnt pathways (Green, 2008).

These results describe the contributions of multiple Wnt pathways to the orientation of cell polarity in the C. elegans vulval epithelium. Because no factor required for the posterior orientation of P5.p or P7.p had previously been identified, this orientation was thought to be signaling independent or 'default'. However, when a new approach was used to reduce Wnt levels in a spatiotemporally controlled manner (overexpression of Ror/CAM-1, a Wnt sink), the VPCs displayed instead a randomized orientation, which is likely to be the true default. The posterior orientation seen in the absence of Fz/lin-17 and Ryk/lin-18 depends on the instructive activity of Wnt/EGL-20. This polarity is referred to as 'ground' polarity. In response to centrally located Wnt/MOM-2 (and possibly Wnt/LIN-44), the receptors Fz/LIN-17 and Ryk/LIN-18 orient P5.p and P7.p toward the center. This reorientation of P7.p, 'refined' polarity, provides the mirror-image symmetry required for a functional organ (Green, 2008).

That P7.p is oriented toward the center in wild-type worms suggests that Wnts LIN-44 and MOM-2 have a greater ability to affect P7.p orientation than does EGL-20. Although the posterior-anterior EGL-20 gradient reaches the VPCs, EGL-20 levels may be much lower here than the levels of Wnts secreted from the nearby AC. Indeed, it was found that local expression of egl-20 in the AC can overcome the effects of distally expressed egl-20. lin-44 is expressed in the tail in addition to the AC but has not been shown to have long-range activity. It is thus possible that this posterior source of lin-44 does not affect P7.p orientation and that LIN-44, in addition to MOM-2, acts as a central cue (Green, 2008).

LIN-17 and LIN-18 were previously reported to reorient P7.p and to reverse the AP pattern of nuclear TCF/POP-1 levels in P7.p daughters. This study extended knowledge of the signaling downstream of Fz/LIN-17 and Ryk/LIN-18 by showing that these receptors control the asymmetric localization of two β-catenins, SYS-1 and BAR-1, the first evidence that Ryk proteins regulate β-catenin. Although asymmetric localization of SYS-1 suggests involvement of the Wnt/β-catenin asymmetry pathway, disruption of pathway components either did not cause a P-Rvl phenotype (lit-1(rf)) or caused only a weakly penetrant P-Rvl phenotype [pop-1(RNAi), sys-1(rf), and wrm-1(rf)], making the function of the Wnt/β-catenin asymmetry pathway in refined polarity unclear. LIN-17 and LIN-18 were also shown to activate transcription in the proximal VPC daughters. Yet, this transcription is not required for P7.p reorientation, since transcriptional states observed by POPTOP, a reporter of Wnt target genes, do not always correspond with the morphological phenotype. Therefore, refined polarity may be largely independent of BAR-1 or the Wnt/β-catenin asymmetry pathway and instead be analagous to the spindle reorientation of the EMS cell during C. elegans embryogenesis, in which Wnt signaling affects the cytoskeleton independent of Wnt's effect on gene expression (Green, 2008).

What then, is the purpose of the Wnt/β-catenin asymmetry pathway in the VPCs? The weakly penetrant A-Rvl phenotype seen in wrm-1(rf) and lin-17(lf); lit-1(lf) worms, combined with the observation that EGL-20 regulates SYS-1 asymmetry, suggests that the Wnt/β-catenin asymmetry pathway functions in ground polarity. Therefore, both ground and refined polarity may converge on regulation of these components, although they are not absolutely required for refined polarity. Because the localization of Wnt/β-catenin asymmetry pathway components in ground polarity matches the reiterative pattern seen in most other asymmetric cell divisions in C. elegans, it is hypothesized that localization of these components is initially established as part of a global anterior-posterior polarity. It is likely that LIN-17 and LIN-18 overcome ground polarity by inhibiting the Wnt/β-catenin asymmetry pathway, a scenario consistent with the ability of lit-1(rf) to suppress lin-17(lf) and lin-18(lf) mutations (Green, 2008).

Remarkably, it is only by peeling back the layer of refined polarity that ground polarity can be observed and manipulated. By doing so, it was found that Wnt/EGL-20, expressed from a distant posterior source, imparts uniform AP polarity to the field of VPCs via a new pathway involving Van Gogh/vang-1, a core PCP pathway component. It is noteworthy that Fz is also a core PCP pathway component, yet it does not seem to be involved in EGL-20 signaling via VANG-1. This is not incompatible with other descriptions of PCP. For example, in the Drosophila wing, Van Gogh and Fz antagonize each other and cause wing hairs to orient in opposite directions. The molecular mechanism by which VANG-1 functions in ground polarity is unknown; however, regulation of SYS-1 by VANG-1 provides evidence that the pathway involving egl-20 and vang-1is associated with the Wnt/β-catenin asymmetry pathway (Green, 2008).

A major difference between VPC orientation in C. elegans and PCP in Drosophila is that no Wnt has been directly implicated in Drosophila PCP. Therefore, VPC orientation may be more similar to some forms of PCP in vertebrates. For example, Wnts are believed to act as permissive polarizing factors during vertebrate convergent extension. Also, VPC orientation is strikingly similar to hair cell orientation in the utricular epithelia of the mammalian inner ear, wherein hair cells flanking the axis of symmetry are oriented in opposite directions. In this system, both medial and lateral hair cells possess a uniform underlying polarity as evidenced by asymmetric localization of Prickle, a core PCP pathway component, to the medial side of cells in both populations. Van Gogh is required for proper Prickle asymmetry, perhaps similarly to the role of vang-1 in ground polarity of the VPCs. It is not understood how the position of the utricular axis of symmetry is determined, but the similarities between these two systems suggest that it may represent a local source of Wnt (Green, 2008).

By moving the source of EGL-20 from the posterior to the anterior side of P7.p and thereby reversing P7.p orientation, this study showed that EGL-20 acts as a directional cue. Although it is not presently clear if the pathway involving egl-20 and vang-1 is mechanistically similar to the PCP pathway described in Drosophila and vertebrates, the result nonetheless provides a long-sought example of a Wnt that acts instructively via a PCP pathway component. Detailed description of the subcellular localization of Van Gogh/VANG-1 and other PCP pathway components in the VPCs will be required to make meaningful comparisons between VPC orientation and established models of PCP (Green, 2008).

In addition to vang-1, a role of Ror/cam-1 in ground polarity was identified. The results provide the first evidence that Ror proteins interpret directional Wnt signals, as well as the first evidence that they interact with Van Gogh. Although a Xenopus Ror homolog, Xror2, was previously described to function in PCP during convergent extension, a recent report indicates that the involvement of Xror2 in convergent extension (CE) is actually via a different pathway. In response to Wnt5a, Xror2 activates JNK by a mechanism requiring Xror2 kinase activity. In contrast to Wnt5a/Xror2 signaling, Ror/CAM-1 function in ground polarity does not require JNK. Therefore, the ground polarity pathway involving Wnt/EGL-20, Ror/CAM-1, and Van Gogh/VANG-1 may be a new type of Wnt signaling (Green, 2008).

Using C. elegans vulva development as a model, this study showed that multiple coexisting Wnt pathways with distinct ligand specificities and signaling mechanisms act in concert to regulate the polarity of individual cells during their assembly into complex structures (Green, 2008).

The C. elegans ROR receptor tyrosine kinase, CAM-1, non-autonomously inhibits the Wnt pathway

Inhibitors of Wnt signaling promote normal development and prevent cancer by restraining when and where the Wnt pathway is activated. ROR proteins, a class of Wnt-binding receptor tyrosine kinases, inhibit Wnt signaling by an unknown mechanism. To clarify how RORs inhibit the Wnt pathway, the relationship between Wnts and the sole C. elegans ROR homolog, cam-1, was examined during C. elegans vulval development, a Wnt-regulated process. It was found that loss and overexpression of cam-1 causes reciprocal defects in Wnt-mediated cell-fate specification. The molecular and genetic analyses revealed that the CAM-1 extracellular domain (ECD) is sufficient to non-autonomously antagonize multiple Wnts, suggesting that the CAM-1/ROR ECD sequesters Wnts. A sequestration model is supported by findings that the CAM-1 ECD binds to several Wnts in vitro. These results demonstrate how ROR proteins help to refine the spatial pattern of Wnt activity in a complex multicellular environment (Green, 2007).

Despite studies in several different organisms, the mechanism of ROR action remains elusive. This work characterized the role of CAM-1/ROR as a regulator of Wnt distribution and determined that one function of ROR proteins is to sequester Wnts. It has been hypothesized that CAM-1/ROR could sequester Wnts. Kim (2003) found that expression of the membrane-anchored CAM-1 ECD was sufficient to rescue the cell migration defects of cam-1(lf) worms and that overexpression of the membrane-anchored CAM-1 CRD caused defects in HSN and Q cell migration similar to those caused by mutation of egl-20/Wnt, leading these authors to propose that the CAM-1 CRD might sequester EGL-20/WNT. Indeed, CAM-1 was later shown to inhibit EGL-20 signaling in cell migration independently of the CAM-1 cytoplasmic domain (Forrester, 2004). However, the mechanism of this inhibition was not demonstrated. In particular, since the ROR2 CRD is capable of dimerizing with Fz (Oishi, 2003), the CAM-1 ECD could potentially function cell-autonomously by inhibiting the Wnt receptor (Green, 2007).

Genetic data indicate that CAM-1 antagonizes Wnt signaling during vulval development. It was found that in lin-17 and lin-18 mutant backgrounds, cam-1 mutations cause an 'overinduced' phenotype owing to elevated levels of Wnt activity. Loss of lin-17 or lin-18 might provide a sensitized background if Wnt receptors LIN-17 and LIN-18, like CAM-1, also affect the extracellular distribution of Wnts. According to this hypothesis, mutation of lin-17 or lin-18 would similarly result in elevated extracellular Wnt levels; however, the data do not conclusively support this hypothesis (Green, 2007).

Using vulval development as a model, this study shows conclusively that CAM-1/ROR can act non-autonomously. The source of the Wnts required for vulval induction is unknown and a sequestration model would require that Pmyo-3::CAM-1::GFP (muscle expression) and Psnb-1::CAM-1::GFP (neuronal expression) are expressed in positions that enable them to restrict diffusion or transport of the Wnts to the VPCs. EGL-20/WNT forms a gradient of decreasing concentration from its site of expression in the tail extending anteriorly past the VPCs (Coudreuse, 2006). The distance between the source of EGL-20 and the VPCs provides ample opportunity for CAM-1 expressed in nervous or muscle tissue to prevent EGL-20 from reaching the VPCs. CWN-1/WNT is expressed in ventral cord neurons (VCNs) and posterior body wall muscle. Endogenous CAM-1 expression in body wall muscle and VCNs, which are in close proximity to the VPCs, could place CAM-1 between the source of cwn-1 expression and the VPCs, allowing CAM-1 to act as a barrier and limit the amount of Wnt signal received by the VPCs. CAM-1 could also function at the Wnt source to limit secretion. Consistent with inhibition by sequestration, CAM-1 overexpression antagonizes Wnt signaling independently of the cytoplasmic domain. Also, phenotypes of cam-1 mutants indicate that the membrane-anchored ECD is sufficient to inhibit Wnt signaling (Green, 2007).

A sequestration model also predicts that CAM-1 specifically binds to those Wnts that it antagonizes. In agreement with genetic data, it was found that the CAM-1 CRD can bind to Wnts CWN-1, EGL-20 and MOM-2 in vitro. The initial experimental design included measuring binding at various concentrations of CRD-AP that would allow calculation of the binding affinity of each receptor-ligand pair. However, preliminary results showed high background binding to untransfected Drosophila S2 cells. The concentration of CRD-AP was chosen at which the greatest difference existed between binding to Nrt-Wnt-expressing and to untransfected cells, and all of the combinations were tested at this concentration. This study never observed a difference greater than 2-fold. Weak binding could be caused by a species barrier, whereby the Drosophila cells do not express a necessary cofactor or do not process Wnts in a manner conducive to high-affinity binding to C. elegans receptors. Although the binding that was detected is not as robust as that observed for Drosophila Wnts and Fzs, it might still be informative (Green, 2007).

Although sequestration through Wnt-CRD binding can account for many functions of CAM-1/ROR, there are examples in which CAM-1 might function by a different mechanism. The membrane-anchored ECD, but not the membrane-anchored CRD alone, has been shown to be sufficient to rescue all cell migration defects of cam-1(lf) worms. In cases where the CRD is not sufficient, ligand binding might require additional CAM-1 ECD(s) - e.g. the kringle or Ig domain - or these might be cases in which CAM-1 functions by a non-sequestration mechanism. Other examples of CAM-1 function that are probably not due to sequestration include cell-autonomous roles in CAN migration and development of the ASI sensory neuron. Also, CAM-1 function in Pn.aap division orientation in males requires CAM-1 kinase activity. Although this study has furthered the understanding of ROR function, the role of the cytoplasmic domains remains elusive. CAM-1 shares 44% identity in the kinase domain to human ROR1 and ROR2 and none of the 21 invariant amino acids is altered. Although ROR proteins have demonstrated kinase activity, the precise function of this activity has not been identified (Green, 2007).

Genetic and biochemical observations that CAM-1 interacts not only with EGL-20, but also with other Wnts, suggest that CAM-1 is an important general regulator of Wnt activity, rather than a specific EGL-20 antagonist. As a system in which neighboring cells reproducibly adopt distinct fates, vulva induction has enabled this study of how CAM-1 affects the precision of Wnt distribution. The subtle effects observed upon cam-1 manipulation suggest that CAM-1 serves to buffer Wnt levels rather than to dramatically affect Wnt localization. Such buffering mechanisms might provide robustness to the Wnt morphogen gradient. The high degree of similarity between CAM-1 and vertebrate ROR proteins, in addition to the ability of ROR proteins to inhibit Wnt signaling in a kinase-independent manner, suggest a conserved function of ROR proteins to fine-tune the spatial profile of Wnt activity and to help create regions of distinct cell fate in complex multicellular organisms (Green, 2007).

Wnt gradient formation in C. elegans requires retromer function in Wnt-producing cells

Wnt proteins function as morphogens that can form long-range concentration gradients to pattern developing tissues. This study shows that the retromer, a multiprotein complex involved in intracellular protein trafficking, is required for long-range signaling of the C. elegans Wnt ortholog EGL-20. In a genome-wide RNA-mediated interference (RNAi)-based screen, it was found that an ortholog of the yeast retromer complex subunit Vps35p (see Drosophila Vps35) is required for posterior localization of the Q neuroblast progeny QL.d. In yeast, the retromer directs endosome-to-Golgi retrieval of proteins such as the carboxypeptidase Y receptor Vps10p. In vertebrate epithelial cells, it physically interacts with the immunoglobulin receptor (IgR) and mediates basal-to-apical transcytosis of the IgR-IgA complex. The retromer functions in EGL-20-producing cells to allow the formation of an EGL-20 gradient along the anteroposterior axis. This function is evolutionarily conserved, because Wnt target gene expression is also impaired in the absence of the retromer complex in vertebrates. These results demonstrate that the ability of Wnt to regulate long-range patterning events is dependent on a critical and conserved function of the retromer complex within Wnt-producing cells (Coudreuse, 2006).

Retromer function may be required for the formation of an active Wnt protein, for secretion, or for other processes within the expressing cells that enable long-range Wnt signaling. Several lines of evidence suggest that retromer function is not required for the formation and secretion of an active form of Wnt. (1) The specificity of the retromer mutant phenotype argues against a general role in active Wnt formation. Thus, whereas mutation of the Porcupine ortholog MOM-1 (which is required for active Wnt formation) leads to embryonic lethality, mutation of the retromer complex primarily disrupts EGL-20 function. (2) The observation that loss of retromer function mainly affects long-range EGL-20 signaling indicates that short-range-acting forms of EGL-20 and other Wnts are still produced and secreted. (3) Mutation of vps-35 did not affect EGL-20 levels within the EGL-20-producing cells, indicating that EGL-20 is normally produced and secreted. (4) Knockdown of Vps35 in transfected HEK cells did not affect Wnt3A secretion (Coudreuse, 2006).

It has recently been shown that binding of Wnt to lipoprotein particles is important for long-range gradient formation (Panakova, 2005). The association of Wnt with these particles may take place within Wnt-producing cells, in an endosomal compartment that contains both Wnt and internalized lipoprotein particles. This mechanism would require Wnt to be sorted out of the default secretory pathway by a specific cargo receptor and to be transported to endosomes before secretion. It is speculated that, similar to its function in Vps10p trafficking, the retromer complex may recycle the Wnt cargo receptor from the endosome to the Golgi network. In the absence of retromer, a lack of Wnt cargo receptors in the Golgi would lead to Wnt secretion via the default pathway. This would impair Wnt association with lipoprotein particles and limit its range of signaling (Coudreuse, 2006).

Wnt gradient formation is a complex and tightly regulated process. Factors expressed at the surface of cells along the gradient domain control its spatial extension and determine the range of its effects. However, these results show that the establishment of a Wnt concentration gradient is not dependent only on the capacity of neighboring cells to attract and spread Wnt proteins. An early permissive mechanism within the Wnt-producing cells, which requires retromer function, is an essential step that allows secreted Wnt molecules to respond to the different cues that will shape the gradient (Coudreuse, 2006).

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. This study demonstrates that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. Retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. It is proposed that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue (Yang, 2008).

Two Wnts instruct topographic synaptic innervation in C. elegans

Gradients of topographic cues play essential roles in the organization of sensory systems by guiding axonal growth cones. Little is known about whether there are additional mechanisms for precise topographic mapping of synaptic connections. Whereas the C. elegans DA8 and DA9 neurons have similar axonal trajectories, their synapses are positioned in distinct but adjacent domains in the anterior-posterior axis. This study found that two Wnts, LIN-44 and EGL-20, are responsible for this spatial organization of synapses. Both Wnts form putative posterior-high, anterior-low gradients. The posteriorly expressed LIN-44 inhibits synapse formation in both DA9 and DA8, and creates a synapse-free domain on both axons via LIN-17 /Frizzled. EGL-20, a more anteriorly expressed Wnt, inhibits synapse formation through MIG-1/Frizzled, which is expressed in DA8 but not in DA9. The Wnt-Frizzled specificity and selective Frizzled expression dictate the stereotyped, topographic positioning of synapses between these two neurons (Hizumoto, 2013).

Wnt signaling positions neuromuscular connectivity by inhibiting synapse formation in C. elegans

Nervous system function is mediated by a precisely patterned network of synaptic connections. While several cell-adhesion and secreted molecules promote the assembly of synapses, the contribution of signals that negatively regulate synaptogenesis is not well understood. This study examined synapse formation in the Caenorhabditis elegans motor neuron DA9, whose presynapses are restricted to a specific segment of its axon. The Wntlin-44 localizes the Wnt receptor lin-17/Frizzled (Fz) to a subdomain of the DA9 axon that is devoid of presynaptic specializations. When this signaling pathway, composed of the Wnts lin-44 and egl-20, lin-17/Frizzled and dsh-1/Dishevelled, is compromised, synapses develop ectopically in this subdomain. Conversely, overexpression of LIN-44 in cells adjacent to DA9 is sufficient to expand LIN-17 localization within the DA9 axon, thereby inhibiting presynaptic assembly. These results suggest that morphogenetic signals can spatially regulate the patterning of synaptic connections by subdividing an axon into discrete domains (Klassen, 2007).

In the canonical pathway, Wnt signaling results in the inhibition of the destruction complex, thereby allowing the accumulation of cytosolic β-catenin, which can translocate to the nucleus and regulate transcription through the TCF/Lef family of transcription factors. Interestingly, previous research has implicated components of this pathway in the stabilization of postsynaptic glutamate receptors in C. elegans (Dreier, 2005). Mutations in the β-catenins bar-1(ga80) and wrm-1(ne1982ts), or in the TCF/LEF pop-1(q645), did not result in defects in DA9 presynaptic positioning. No DA9 synaptic defects were observed in the mutants for the effectors lrp-1(ku156)/Arrow, lit-1(or131ts)/NLK, pry-1(mu38cs)/Axin, and tap-1(gk202)/TAB-1. As with the canonical pathway, mutations in fmi-1(gm122)/Flamingo in the planar cell polarity pathway and unc-43(n498n1186)/CaMKinase in the calcium-dependent pathway do not phenocopy lin-44, lin-17, or dsh-1 mutants. Therefore, a positive identification of downstream signaling mechanisms may require the discovery of novel effectors (Klassen, 2007).

Reciprocal signaling by Wnt and Notch specifies a muscle precursor in the C. elegans embryo

The MS blastomere produces one third of the body-wall muscles (BWMs) in the C. elegans embryo. MS-derived BWMs require two distinct cell-cell interactions, the first inhibitory and the second, two cell cycles later, required to overcome this inhibition. The inductive interaction is not required if the inhibitory signal is absent. Although the Notch receptor GLP-1 (see Drosophila Notch) was implicated in both interactions, the molecular nature of the two signals was unknown. This study now shows that zygotically-expressed MOM-2 (Wnt; see Drosophila Wingless) is responsible for both interactions. Both the inhibiting and the activating interactions require precise spatiotemporal expression of zygotic MOM-2, which is dependent upon two distinct Notch signals. In a Notch mutant defective only in the inductive interaction, MS-derived BWMs can be restored by preventing zygotic MOM-2 expression, which removes the inhibitory signal. These results suggest that the inhibitory interaction ensures the differential lineage specification of MS and its sister blastomere, whereas the inductive interaction promotes the expression of muscle-specifying genes by modulating TCF (see Drosophila Pangolin) and β-catenin (see Drosophila Armadillo) levels. These results highlight the complexity of cell fate specification by cell-cell interactions in a rapidly dividing embryo (Robertson, 2017).

C. elegans AP-2 and Retromer control Wnt signaling by regulating MIG-14/Wntless

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. This study reports that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on these observations, it is proposed that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion (Pan, 2008).

Three groups have found a new molecule known as Wntless, Evi, or Sprinter, that is necessary for Wnt function (Banziger, 2006; Bartscherer, 2006; Goodman, 2006). Wntless interacts physically with Wingless, targeting it to the cell surface for secretion (Banziger, 2006). C. elegans MIG-14, which is also known as MOM-3, is the homolog of Wntless (Banziger, 2006). The mig-14 alleles were originally identified in screens for mutants with defects in QL migration, which the Wnt EGL-20 regulates. A screen for mutants with defects in the asymmetric division of the EMS blast cell identified the original mom-3 allele. MOM-3 acts in P2, the cell that secretes the Wnt MOM-2 and signals to EMS, causing it to divide asymmetrically (Pan, 2008).

This paper establishes a connection between endocytosis, retromer function, and MIG-14. The C. elegans gene dpy-23 encodes the mu subunit of the AP-2 clathrin adaptor complex that is necessary for receptor-mediated endocytosis and functions in several Wnt-related processes. The observations indicate that efficient Wnt secretion requires endocytosis and trafficking of MIG-14 by retromer (Pan, 2008).

Wnt function requires the C. elegans AP-2 μ subunit DPY-23. Together with retromer, DPY-23 regulates the intracellular distribution of MIG-14, a Wnt-binding factor required for Wnt secretion. It is speculated that newly synthesized EGL-20/Wnt binds to MIG-14 in the Golgi, targeting the Wnt to the cell membrane for secretion. In this model, AP-2-mediated endocytosis and retromer retrieval at the sorting endosome would recycle MIG-14 to the Golgi, where it can bind to EGL-20/Wnt for next cycle of secretion (Pan, 2008).

Studies in Drosophila demonstrated a role for endocytosis in the formation of a Wingless gradient. Models based on a nonautonomous requirement for dynamin in Wnt function implicated endocytosis as part of a relay that transferred Wingless from one cell to the next. Other studies have proposed that the Wingless gradient was generated by diffusion. It was proposed that the effects of dynamin loss on Wnt function reflected a lack of Wingless secretion from cells expressing the morphogen. While the current results do not directly resolve this controversy, the requirement for DPY-23 in MIG-14 endocytosis supports the hypothesis that endocytosis is necessary for Wnt secretion and provides a mechanism for how endocytosis regulates Wnt secretion (Pan, 2008).

A previous study argued that retromer was not necessary for Wnt secretion, but instead was necessary for production of a functional Wnt (Coudreuse, 2006). In this study it was also proposed that retromer was necessary for long-range Wnt signaling, but only played a minor role in short-range signaling. It was argued that retromer mutants produced Wnt molecules that could only act on nearby cells but failed to act on more distant cells. The current findings that retromer is required for MIG-14 trafficking and the previous discovery that Wntless, the Drosophila MIG-14 homolog, is necessary for Wingless secretion are at odds with the interpretation that retromer plays a specific role in production of a Wnt that acts in long-range signaling (Banziger, 2006; Bartscherer, 2006; Pan, 2008 and references therein).

An argument for retromer playing a specific role in long-range signaling was based on the observations that retromer mutants have little effect on processes that require MOM-2 and LIN-44, Wnts that are produced near responding cells (Coudreuse, 2006). Further support for the long-range hypothesis was based on the higher frequency of V5 defects in egl-20 mutants compared to retromer mutants (Coudreuse, 2006). This difference contrasted with the high frequency of QL migration defects in both egl-20 and retromer mutants. The discrepancy between the V5 and QL defects in the two types of mutants was explained by the closer proximity of the V5 cell to the EGL-20 source. The model that retromer plays a specific role in long-range Wnt signaling has led to speculation that the trafficking events regulated by this complex might control the production of a specifically modified form of Wnt (Coudreuse, 2007; Coudreuse, 2006; Hausmann, 2007), for example, a Wnt that could associate with lipoprotein particles (Pan, 2008).

A simpler hypothesis is favored where retromer is required for MIG-14 recycling and where blocked recycling leads to defects in Wnt secretion. The observation that excess MIG-14 can ameliorate the Wnt phenotypes of dpy-23 and vps-35 mutants is consistent with the notion that low levels of functional Wnts are still secreted in these mutants. It is proposed that the phenotypic differences observed between retromer and egl-20 mutants may result from differential sensitivities of various responding cells to lowered Wnt levels, and a similar explanation could account for the phenotypic differences between dpy-23 and Wnt mutants (Pan, 2008).

While the phenotypes of mig-14 mutants have most of the defects displayed by either single Wnt mutants or Wnt mutant combinations, dpy-23 mutants do not exhibit certain Wnt mutant phenotypes. They do not have the severe ALM polarity defects that are exhibited by cwn-1; egl-20 or cwn-1; cwn-2 double mutants and completely lack the PLM polarity defects of lin-44 mutant. Yet the dpy-23 defects in HSN and QL migration are extremely severe. One explanation for these differences between dpy-23 and mig-14 mutants, as well as the differences between retromer and mig-14 mutants, is that different Wnt-producing cells vary in their dependence on AP-2 or retromer to secrete Wnts. It is speculated that endocytosis and retomer recycle MIG-14 for multiple rounds of Wnt secretion. If this hypothesis is correct, phenotypic differences could reflect the ability of some cells to synthesize sufficient MIG-14 resulting in less dependence on recycling. Alternatively, independent mechanisms for trafficking MIG-14 could operate in different Wnt-secreting cells (Pan, 2008).

Neuroblast migration along the anteroposterior axis of C. elegans is controlled by opposing gradients of Wnts and a secreted Frizzled-related protein

The migration of neuroblasts along the anteroposterior body axis of C. elegans is controlled by multiple Wnts that act partially redundantly to guide cells to their precisely defined final destinations. How positional information is specified by this system is, however, still largely unknown. This study used a novel fluorescent in situ hybridization methods to generate a quantitative spatiotemporal expression map of the C. elegans Wnt genes. The five Wnt genes were found to be expressed in a series of partially overlapping domains along the anteroposterior axis, with a predominant expression in the posterior half of the body. Furthermore, a secreted Frizzled-related protein is expressed at the anterior end of the body axis, where it inhibits Wnt signaling to control neuroblast migration. These findings reveal that a system of regionalized Wnt gene expression and anterior Wnt inhibition guides the highly stereotypic migration of neuroblasts in C. elegans. Opposing expression of Wnts and Wnt inhibitors has been observed in basal metazoans and in the vertebrate neurectoderm. These results in C. elegans support the notion that a system of posterior Wnt signaling and anterior Wnt inhibition is an evolutionarily conserved principle of primary body axis specification (Harterink, 2011).

Wingless and insect segmentation

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects [like Schistocerca (grasshopper)] anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of orthopteran orthologs of the Drosophila segmentation genes may be involved in orthopteran segmentation, little is known about the role of these genes. Segmentation processes of Gryllus bimaculatus have been investigated, focusing on its orthologs of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, RNA interference (RNAi) was used. No significant effects of RNAi for Gbwg and Gbhh on segmentation were observed, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. These results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila (Miyawaki, 2004).

Gbwg is expressed continuously in the posterior-most region of G. bimaculatus embryos. This expression pattern is a common feature in the intermediate- and short-germ insects. The posterior elongation takes place between the T3 segment and the posterior-most region of embryos. Judged from the phenotypes of the GbarmRNAi embryos, it is reasonable to propose that the canonical Wnt/Wg pathway is involved in the posterior sequential elongation and segmentation. Since intermediate phenotypes, for example, lack of the T3-A5 segments, were not observed even in mild RNAi conditions, GbWg in the posterior growth zone should be responsible for the phenotypes of the GbarmRNAi embryos, and is likely to act as an organizing signal in posterior sequential segmentation (Miyawaki, 2004).

There are several lines of evidence supporting the idea that Wg/Wnt may play essential roles in pattern formation through a process of cell-cell interaction. In Hydra, Wnt expressed in the putative head organizer may be involved in axis formation. Furthermore, a recent advance in the study of vertebrate somitogenesis reveals that the somites appear by the progressive anterior conversion of a temporally periodic pattern into a spatially periodic pattern, requiring the expression of homologs of the Drosophila pair-rule gene hairy and segment-polarity gene wingless. Recently, Wnt3a was reported to play major roles in the segmentation clock controlling somitogenesis in vertebrates. In this case, Wnt/ß-catenin signaling pathway is known to link to the segmentation clock through negative-feedback loop with Axin2. The existence of this mechanism in vertebrates makes the clock model for short and intermediate-germ insects plausible. Since the posterior segments are formed sequentially with about 2 h periodicity in cricket embryos, it is probable therefore that a segmentation clock operates in the insect embryos (Miyawaki, 2004).

Although the molecular mechanisms directing anteroposterior patterning of the Drosophila embryo (long-germband mode) are well understood, how these mechanisms were evolved from an ancestral mode of insect embryogenesis remains largely unknown. In order to gain insight into mechanisms of evolution in insect embryogenesis, the expression and function of the orthologue of Drosophila caudal (cad) was examined in the intermediate-germband cricket Gryllus bimaculatus. A posterior (high) to anterior (low) gradient in the levels of Gryllus bimaculatus cad (Gbcad) transcript is formed in the early-stage embryo, and then Gb' cad is expressed in the posterior growth zone until the posterior segmentation is completed. Reduction of Gb' cad expression level by RNA interference results in deletion of the gnathum, thorax, and abdomen in embryos, remaining only anterior head. The gnathal and thoracic segments are formed by Gb' cad probably through the transcriptional regulation of gap genes including Gb' hunchback and Gb' Krüppel. Furthermore, Gb'cad is found to be involved in the posterior elongation, acting as a downstream gene in the Wingless/Armadillo signalling pathways. These findings indicate that Gb'cad does not function as it does in Drosophila, suggesting that regulatory and functional changes of cad occurred during insect evolution. The Wg/Cad pathway in the posterior pattern formation may be common in short- and intermediate-germband embryogenesis. During the evolutionary transition from short- or intermediate- to long-germband embryogenesis, an ancestral cell-cell signalling system including Wg/Arm signalling may have been replaced by a diffusion system of transcription factors as found in Drosophila. Since Wnt/Cdx pathways are involved in the posterior patterning of vertebrates, such mechanisms may be conserved in animals that undergo sequential segmentation from the posterior growth zone (Shinmyo, 2005).

In the long germ insect Drosophila, all body segments are determined almost simultaneously at the blastoderm stage under the control of the anterior, the posterior, and the terminal genetic system. Most other arthropods (and similarly also vertebrates) develop more slowly as short germ embryos, where only the anterior body segments are specified early in embryogenesis. The body axis extends later by the sequential addition of new segments from the growth zone or the tail bud. The mechanisms that initiate or maintain the elongation of the body axis (axial growth) are poorly understood. The terminal system in the short germ insect Tribolium was functionally analyzed. Unexpectedly, Torso signaling is required for setting up or maintaining a functional growth zone and at the anterior for the extraembryonic serosa. Thus, as in Drosophila, fates at both poles of the blastoderm embryo depend on terminal genes, but different tissues are patterned in Tribolium. Short germ development as seen in Tribolium likely represents the ancestral mode of how the primary body axis is set up during embryogenesis. It is therefore concluded that the ancient function of the terminal system mainly was to define a growth zone and that in phylogenetically derived insects like Drosophila, Torso signaling became restricted to the determination of terminal body structures (Schoppmeier, 2005).

In Drosophila, the anterior- and posterior-most terminal body regions of the embryo depend on the maternal terminal-group genes. One of them, the torso-like (tsl) gene is expressed in somatic follicle cells located at the anterior and posterior pole of the oocyte. In the embryo, tsl contributes to the local activation of the receptor tyrosine kinase Torso at the egg poles. The signal is transduced to the nucleus via a Ras-Raf-MAP-K/Erk phosphorylation cascade, and leads to the expression of the zygotic target genes tailless (tll) and huckebein (hkb) at the posterior terminus of the embryo. Failure to activate Torso signaling results in defects in the head skeleton and loss of all segments posterior to abdominal segment 7, in addition to loss of the hindgut and posterior midgut anlagen (Schoppmeier, 2005 and references therein).

Whether an anteriorly acting terminal system is a general feature of all insects has been challenged because under certain conditions, Torso function at the anterior is dispensable for head development in Drosophila. This hypothesis is supported by the expression of the Tribolium ortholog of tll at the posterior, but not at the anterior pole of blastoderm stage embryos. Thus, in Tribolium, posterior terminal cells appear to be determined before the onset of abdomen formation. It is unknown, however, whether these cells specify posterior fate after axis elongation and abdomen formation is completed or whether they also contribute to earlier steps of segmentation (Schoppmeier, 2005).

The orthologs of the key components of the Torso pathway have been isolated in the short germ beetle Tribolium torso (Tc-tor) and torso-like (Tc-tsl). As in Drosophila, Tc-torso mRNA is maternally inherited by the embryo and expressed ubiquitously in freshly laid eggs, and Tc-tsl is expressed during oogenesis anteriorly and posteriorly in the follicle cells of the oocyte (Schoppmeier, 2005).

Knocking down the function of Tc-torso or Tc-tsl using parental RNA interference leads to identical embryonic phenotypes. Whereas the head and the anterior thorax are unaffected, unexpectedly the most extreme Tc-torsoRNAi and Tc-tslRNAi embryos lack all structures that develop during postblastodermal abdominal growth. Thus, the head and thoracic segments that form in torso or tsl RNAi embryos likely represent the structures, which are determined already during the Tribolium blastoderm stage. Less strongly affected embryos fail to form the full number of abdominal segments (Schoppmeier, 2005).

To determine whether the Tc-torso RNAi phenotype does not reflect a late function of maintaining abdominal fate prior to cuticularization, the expression of Engrailed protein was examined in Tc-torsoRNAi embryos at a stage when abdominal segments should already have developed. Indeed, in strongly affected embryos, Engrailed stripes corresponding to the head and thorax, but not to abdominal segments, are present (Schoppmeier, 2005).

The emergence of segments was visualized in embryos with impaired Torso signaling by analyzing the Tc-even-skipped (Tc-eve) expression pattern. In wild-type embryos, Tc-eve is initially expressed in a double segmental pattern that later resolves into secondary segmental stripes. Tc-tsl RNAi does not interfere with the formation of the first two primary Tc-eve stripes that give rise to the gnathal and the first thoracic (T1) segments. However, although the third primary Tc-eve expression domain (Tc-eve stripe 3) forms normally, this domain does not resolve into segmental stripes, and no additional primary eve-stripes form. In the wild-type, Tc-eve stripe 3 covers the region where the second (T2) and third thoracic (T3) segment will develop. Although Tc-eve stripe 3 does not split in Tc-tsl RNAi embryos, this domain gives rise to the second thoracic segment. Thus, Torso signaling is required for the initiation of axial growth or maintaining the segmentation process (Schoppmeier, 2005).

As revealed by DAPI staining and by morphology, posterior invagination of cells is abolished in both Torso- and tsl RNAi embryos, and as a consequence, no posterior pit forms. To understand how Torso signaling is propagated at the posterior pole and to test whether downstream gene activity is affected in the growth zone in Tc-torsoRNAi embryos, the activity of the Map-kinase and the expression of Tc-wingless (Tc-wg), Tc-tailless (Tc-tll), Tc-caudal (Tc-cad), and Tc-forkhead (Tc-fkh) RNA was analyzed in early embryos (Schoppmeier, 2005).

The active state of the Torso receptor is transduced to the nucleus via the Ras-Raf signal transduction pathway and leads to the activation of zygotic target genes. The activity of this pathway can be visualized with an antibody that recognizes ErkPP but does not discriminate between the different pathways that involve ErkPP signaling. In nontreated embryos, ErkPP can be detected in a subpopulation of the serosa, a single row of cells at the border of the serosa and the embryonic anlage; at the rims of the mesoderm; and at the posterior pole. In torsoRNAi embryos posterior ErkPP expression is lost, further indicating that ErkPP is involved in propagating terminal signaling. ErkPP expression in the serosa is mildly affected whereas the other sites where ErkPP activity is detected in the wild-type are normal. ErkPP activity in the amnion appears not to be reduced; however, the amnion itself does also not form properly. Whether this is a direct or indirect consequence of Torso reduction is unclear (Schoppmeier, 2005).

In addition to segmental stripes, a terminal wingless (wg) expression domain first seen at the blastoderm stage is present throughout the phase of body elongation in the growth zone of the wild-type. In Tc-torsoRNAi embryos, the posterior terminal Tc-wg domain is missing at the blastoderm stage, as well as in older embryos corresponding in age to wild-type embryos undergoing body axis extension. Drosophila-torso mutant embryos also lack the posterior terminal wg expression domain, indicating, that the dependence of wg on torso is conserved. The segmental wg stripes that were built prior to the growth process, form close to the posterior end. This shows that a presegmented region (PSR) normally separating the last segment formed at the posterior end of the embryo is strongly reduced or absent in torsoRNAi embryos. The absence of Tc-cad and Tc-tll in torsoRNAi embryos establishes these genes as potential targets of terminal signaling also in Tribolium (Schoppmeier, 2005).

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, Tc-prd and Tc-slp were analyzed to determine the extent to which this paradigm is conserved in Tribolium. It was found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning (Choe, 2007; full text of article).

wingless/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved. Wnt signaling is also implicated in posterior patterning and germband elongation, but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions. Tribolium contains additional Wnt family genes that are also expressed in the growth zone. After depleting Tc-WntD/8, a small percentage of embryos were found lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods and vertebrates (Bolognesi, 2008).

Wingless and insect wing patterns

The morphological and functional evolution of appendages has played a critical role in animal evolution, but the developmental genetic mechanisms underlying appendage diversity are not understood. Given that homologous appendage development is controlled by the same Hox gene in different organisms, and that Hox genes are transcription factors, diversity may evolve from changes in the regulation of Hox target genes. Two impediments to understanding the role of Hox genes in morphological evolution have been the limited number of organisms in which Hox gene function can be studied and the paucity of known Hox-regulated target genes. An analysis was carried out of Hindsight, a butterfly homeotic mutant in which portions of the ventral hindwing pattern are transformed to ventral forewing identity, and the regulation of target genes by the Ultrabithorax (Ubx) gene product was compared in Lepidopteran and Dipteran hindwings. Ubx gene expression is lost from patches of cells in developing Hindsight hindwings, which correlates with changes in wing pigmentation, color pattern elements, and scale morphology. This mutant was used to study how regulation of target genes by Ubx protein differs between species. Drosophila Serum response factor (blistered), Achaete-Scute Complex, and wingless are repressed in Drosophila halteres. Portions of the expression pattern of Lepidopteran homologs of these genes are not repressed in butterfly hindwings. Unlike the expression patterns of the homologous genes in halteres, butterfly wg is not repressed along the posterior margin in the hindwing, nor is butterfly SRF repressed in intervein regions, and the AS-C homologs are not repressed in cells flanking the dorsal-ventral boundary. These differences in the regulation of wg, SRF and AS-C between Drosophila halteres and butterfly hindwings suggest that these genes became repressed by Ubx when an ancestral hindwing evolved into a haltere in the dipteran lineage, with a concomitant reduction of appendage size, loss of margin bristles, and changes in shape. Two additional exampes of Ubx-regulated differences in gene expression between fly and butterfly flight appendages were found. (1) wg is expressed in two stripes in butterfly forewings that roughly correspond to the future location of the proximal band elements. This protein of the wg pattern is absent from butterfly hindwings and has not counterpart in flies and represents a novel feature regulated by Ubx in butterflies. (2) Dll is expressed along the margin of both butterfly wings and the Drosophila forewing, but this expression is modified in halteres and may be regulated by Ubx. Changes in Hox-regulated target gene sets are, in general, likely to underlie the morphological divergence of homologous structures between animals (Weatherbee, 1999).

Wingless and insect leg development

Insects can be grouped into two main categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), has been studied focusing on expression patterns of the three key signaling molecules, hedgehog, wingless and decapentaplegic, which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anterior/posterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, expression pattern of dpp is significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence is found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology (Niwa, 2000).

In the allocation phase of Drosophila 5h embryos, wg and hh are expressed in a stripe along the A/P compartment boundary and in the posterior region of each segment, respectively. However, dpp is expressed throughout the dorsal region and then in the dorsal side of the wg stripe. Later, the expression changes to give two thin stripes running anteroposteriorly along the length of the embryo. Wg, but not Dpp, is responsible for initial specification of the limb primordia with a distal identity and for induction of Dll. A model for the allocation of the limb primordium (the G-H model) is presented. A stripe of Wg induces the limb primordium expressing Dll. Repression of Dll by Dpp from the dorsal side and by Spitz (Drosophila EGF) from the ventral side limits the limb formation only in the lateral position. Then, Dpp specifies proximal cell identity in the primordium in a concentration-dependent manner. In Gryllus and Schistocerca embryos, expression of wg is detected in a stripe along the A/P compartment boundary of the body segment. In Gryllus embryos, expression of dpp is first detected along the periphery of the germ band. Similar expression patterns have been observed in Tribolium. Although the cricket and grasshopper belong to the same Orthoptera, the expression patterns of Sadpp are more complicated than those of Gbdpp. In Schistocerca embryos at early stages, Sadpp is expressed in two partial stripes in each hemisegment, intrasegmentally and intersegmentally, paralleling the D/V axis. The intrasegmental stripes extend along both dorsal and ventral sides of the presumptive leg field. Early expression patterns of Gbdpp resemble those of Dmdpp or Tcdpp more closely than those of Sadpp. Thus, the wg expression pattern appears conserved in the allocation phase, while early expression patterns of dpp seems divergent even in the Orthoptera. Thus, more data are necessary to judge whether the G-H model is also applicable as a model for initiation of limb formation in other insects (Niwa, 2000 and references therein).

In Phase 2, in the Drosophila leg imaginal disc, hh is expressed in the posterior compartment of the disc, determining the A/P pattern, and induces dpp and wg expression in the dorsal and ventral side of the A/P boundary, respectively. They act cooperatively in a concentration-dependent manner to organize the P/D axis and induce expression of Dll at the center of the disc. In Gryllus and Schistocerca limb buds, since hh and wg are expressed in the posterior and the ventral side of the A/P boundary, respectively, their functions during limb development should be conserved among the fly, cricket, beetle and grasshopper. However, expression patterns of Gbdpp are considerably different from those of Drosophila dpp: Gbdpp expression is limited to a dorsal stripe, transiently around the time of limb bud emerging, at stage 6-7. At this time, expression of Dll was found in the distal tip of the limb bud. This transient expression pattern also occurs in Schistocerca embryos. In Drosophila, removal of Dpp signaling prior to the second larval instar results in loss of Dll expression, while later removal of Dpp does not affect Dll expression, indicating that Dpp is required for the initiation but not maintenance of Dll transcription. Thus, it is reasonable to consider that transient dpp expression is enough to induce expression of Dll, which is required for the P/D leg pattern formation (Niwa, 2000 and references therein).

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. Patterns of hedgehog, wingless, and decapentaplegic expression were examined during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns are essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, it is concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM (Mito, 2002).

In the cricket leg, the single layer of surface epidermal cells forms precise patterns of structures, including bristles and spines, in the overlying cuticle. The regional specialization of the leg epidermal cells is evident along the three major axes of the leg, which include the anteroposterior (A/P), dorsoventral (D/V), and proximodistal (P/D) axes. The P/D axis relates to the distance from the body trunk, while the A/P and D/V axes unite to form the single circumferential axis. When a metathoracic leg of a Gryllus nymph in the third instar is amputated at the tibia, the distal missing part is completely recovered after about 30-35 days through four molts subsequent to the amputation. Just after the amputation, a trachea running along the P/D axis, reticulate fat bodies, and muscles are observed in sagittal sections. By 6 h after amputation, wounded muscles already start to degenerate, while hemocytes aggregate in the wound to form a scab. By day 2, epidermal cells migrate over the wound surface, and epidermal continuity is restored underneath the scab. Cell proliferation can be detected in epidermis lining the scab during this process. By day 5, the wound epidermis thickens to form a regeneration bud, or blastema, and cell proliferation is greatly activated in the blastema. Cells in the blastema lose their differentiated character and start to grow. By day 7, the blastema becomes the primordia of the tibia and tarsus concomitant with muscle recovery. By day 10, the boundary of the tibia-tarsus is visible in the blastema. Finally, all of the structures that normally lie distal to the point of amputation are restored (Mito, 2002).

In normally developing cricket leg buds, hh i expressed in the posterior (P) compartment, while wg and dpp are expressed in the ventral (V) side and dorsal (D) side of the anteroposterior (A/P) boundary, respectively. In a normal leg at the stage corresponding to the regeneration samples, hybridization signals for hh are weakly detected in epidermal cells located in the posterior region, whereas the expressions of wg and dpp are not observed. In contrast, the induced expressions of hh, wg, and dpp are observed in the blastemata of regenerating legs. The expression signals of hh are localized on the posterior side of the leg epidermis. The localization of the En protein was examined in cryosections with the monoclonal antibody mAb4D9. Signals were detected in both sagittal and transverse sections, indicating that En is localized in the posterior half of epidermis and supporting the results for hh. In the transverse sections, the En expression domain looks slightly broader than that of hh (Mito, 2002).

The expression pattern of wg is clearly observed in the ventral region of the blastema with a distal-to-proximal gradient in the signal intensity. The signals of the dpp expression are much weaker than the wg signals. Furthermore, there was variation in the expression patterns. Since such variation was not observed in the wg expression pattern, it is considered that the expression pattern of dpp is dynamically changed, as observed during leg development. The observed expression patterns of dpp were classified mainly into three types: Type I, with signals restricted in dorsodistal epithelial cells of the blastema, where intense non-specific signals appear in the trachea due to longer staining reactions; Type II, with signals observed in dorsal and distal epithelial cells, and weakly in ventral cells; and Type III, with signals so weak that no pattern is discernible (n=24). Type I expression patterns are observed in the early stages, while Type II patterns are observed even in the later stages (~4 days). Therefore, it is reasonable to consider that the expression pattern of dpp changes from Type I to Type II as the regeneration proceeds (Mito, 2002).

The expression patterns of wg and dpp in the blastema are comparable to those in the leg bud of the cricket embryo. In particular, the discrete expression of dpp (Type I) observed in the blastema is also observed in the dorsal side along the A/P boundary in the cricket leg bud, which differs from the expression of dpp in the leg imaginal disc. However, a major difference between the leg bud and blastemata is the size of the wg/dpp expression boundary: the boundary becomes a line in the blastema, similar to the apical ectodermal ridge of vertebrate limb buds, rather than a point in the insect leg bud. After wound healing, the restoration of the epidermal continuity results in the formation of a D/V boundary where dpp-expressing epidermal cells abut wg-expressing cells, which possibly initiates the formation of the P/D axis in the regeneration blastema (Mito, 2002).

The Drosophila genes wingless and decapentaplegic comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda. Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g., endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative. All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions (Prpic, 2004).

ß-Catenin regulates cell adhesion and cellular differentiation during development, and misregulation of ß-catenin contributes to numerous forms of cancer in humans. This study describes C. elegans conditional alleles of mom-2/Wnt, mom-4/Tak1, and wrm-1/ß-catenin. These reagents were used to examine the regulation of WRM-1/ß-catenin during a Wnt-signaling-induced asymmetric cell division. While WRM-1 protein initially accumulates in the nuclei of all cells, signaling promotes the retention of WRM-1 in nuclei of responding cells. Both PRY-1/Axin and the nuclear exportin homolog IMB-4/CRM-1 antagonize signaling. These findings reveal how Wnt signals direct the asymmetric localization of ß-catenin during polarized cell division (Nakamura, 2005).

A possible insight into the connection between cortical and nuclear signaling events comes from preliminary findings on the cortical localization of WRM-1. In the course of these studies, a faint localization of WRM-1::GFP to the cell cortex was seen during each mitosis. Interestingly, in the EMS cell (the 4-cell stage blastomere in C. elegans ), WRM-1::GFP is lost along the posterior cortex proximal to the signaling cell P2, while staining is maintained along the anterior cortex of the dividing EMS cell. This cortical localization is also visible at later stages and in developing larvae. These preliminary studies suggest that MOM-5/Frizzled is required for cortical association, while cortical release correlates with signaling via MOM-2/Wnt. Although these observations require further investigation, they suggest an interesting model that could explain how signaling at the cortex could drive nuclear WRM-1 asymmetries. Importantly, this model could also explain the difference between the penetrance of the endoderm defects seen in mom-2/Wnt mutants (~60% gutless) and mom-5/Fz mutants (only 5% gutless), and the surprising finding that the lower penetrance gutless phenotype of mom-5 is epistatic to mom-2 (Nakamura, 2005).

According to this model, P2/EMS signaling alters the affinity of WRM-1 for the posterior cortex of EMS and simultaneously activates WRM-1 for downstream signaling. This activation could be direct (e.g., by phosphorylation of WRM-1) or indirect (e.g., by modification of a WRM-1-interacting protein such as LIT-1). For simplicity in this discussion, the direct activation model will be considered. At steady state, only a small percentage of WRM-1 protein localizes at the cortex and this level drops during signaling, suggesting that cortical association may reflect a dynamic process that is modulated by signaling. Cortical signaling events also ensure that the mitotic apparatus of the cell is oriented such that division produces one nucleus that is more proximal to the posterior cortex and thus exposed to higher concentrations of an activated and less cortically associated form of WRM-1. At the beginning of telophase, WRM-1 accumulates in both nascent nuclei via a mechanism that depends on the kinases MOM-4 and LIT-1. During late telophase, and shortly after cytokinesis, IMB-4/CRM-1-dependent export begins to reduce WRM-1 nuclear levels in MS. However, in E, the signal-dependent release of an activated form of WRM-1 from the cortex induces a net nuclear retention of WRM-1. Finally, retention of WRM-1 in the nascent E (endoderm-restricted precursor) nucleus correlates with a simultaneous CRM-1-dependent nuclear export of POP-1 (Nakamura, 2005).

This model explains the phenotypic differences between mom-2 and mom-5 mutants. In mom-2 mutants, MOM-5 sequesters WRM-1 at the posterior cortex, reducing WRM-1 nuclear retention in E, and resulting in the higher penetrance of the mom-2 endoderm defect. In mom-5 mutants or in mom-2; mom-5 double mutants, signaling from P2 via the parallel SRC-1 tyrosine kinase pathway can activate WRM-1, which is then free to enter the nucleus and promote POP-1 nuclear export. Since SRC-1 has little effect on WRM-1 localization, these findings suggest that SRC-1 may instead alter WRM-1 activity (Nakamura, 2005).

The details of the mechanism that drives the reciprocal nuclear accumulation of WRM-1 and POP-1 are still not clear. The finding that the nuclear accumulation of WRM-1 partially depends on POP-1 suggests that WRM-1 and POP-1 may directly compete for nuclear export factors or nuclear/cytoplasmic retention sites. For example, WRM-1-dependent phosphorylation of POP-1 might increase the affinity of POP-1 for CRM-1, perhaps by promoting the interaction of POP-1 with PAR-5/14-3-3. This could lead to a direct competition that displaces WRM-1 from the export machinery in responding cells. Alternatively, signaling may alter the relative affinity of WRM-1 and/or POP-1 for binding to mutually exclusive partners in the nucleus or in the cytoplasm, causing a simultaneous and codependent shift in the net balance of their nuclear/cytoplasmic retention (Nakamura, 2005).

In summary, this study has analyzed the regulation of a ß-catenin homolog, WRM-1, during a polarized cell division in C. elegans. The findings suggest that WRM-1 is subject to regulation at multiple levels, and begin to place the surprising genetic complexity of P2/EMS signaling into a cell-biological context. Furthermore, the findings suggest that Wnt signaling can control the nuclear accumulation of ß-catenin and may also influence its cortical distribution. These modes of regulation may be of particular importance when Wnt signaling induces a polarized, asymmetric cell division (Nakamura, 2005).

Wingless and parasegmental organization in invertebrates

Spiders belong to the chelicerates, which is a basal arthropod group. To shed more light on the evolution of the segmentation process, orthologs of the Drosophila segment polarity genes engrailed, wingless/Wnt and cubitus interruptus have been recovered from the spider Cupiennius salei. The spider has two engrailed genes. The expression of Cs-engrailed-1 is reminiscent of engrailed expression in insects and crustaceans, suggesting that this gene is regulated in a similar way. This is different for the second spider engrailed gene, Cs-engrailed-2, which is expressed at the posterior cap of the embryo from which stripes split off, suggesting a different mode of regulation. Nevertheless, the Cs-engrailed-2 stripes eventually define the same border as the Cs-engrailed-1 stripes. The spider wingless/Wnt genes are expressed in different patterns from their orthologs in insects and crustaceans. The Cs-wingless gene is expressed in iterated stripes just anterior to the engrailed stripes, but is not expressed in the most ventral region of the germ band. However, Cs-Wnt5-1 appears to act in this ventral region. Cs-wingless and Cs-Wnt5-1 together seem to perform the role of insect wingless. Although there are differences, the wingless/Wnt-expressing cells and en-expressing cells seem to define an important boundary that is conserved among arthropods. This boundary may match the parasegmental compartment boundary and is even visible morphologically in the spider embryo. An additional piece of evidence for a parasegmental organization comes from the expression domains of the Hox genes that are confined to the boundaries, as molecularly defined by the engrailed and wingless/Wnt genes. Parasegments, therefore, are presumably important functional units and conserved entities in arthropod development and form an ancestral character of arthropods. The lack of engrailed and wingless/Wnt-defined boundaries in other segmented phyla does not support a common origin of segmentation (Damen, 2002).

There is an ongoing discussion of whether segmentation in different phyla has a common origin. The presumably conserved segment-polarity network and the organization into parasegments can be seen as an ancestral character for arthropods. In the closely related onychophorans, engrailed expression points to a comparable organization. However, segment polarity gene orthologs are apparently not involved in body segmentation in other segmented phyla. In annelids, engrailed is expressed in segmentally iterated spots in the CNS and in mesodermal cells, but is probably not involved in body segmentation as in arthropods. The establishment of segment polarity in leeches is independent of cell interactions along the anteroposterior axis; this is in contrast to the situation in arthropods, where anterior and posterior fates of the segments are specified by intercellular signaling between wg- and en-expressing cells. Furthermore, there are no indications that the annelid embryo is constructed from units like the parasegment. In the leech, progeny of particular teloblasts overlap with respect to segmental boundaries and do not form genealogical units as in crustaceans. Some key aspects of arthropod segmentation are thus not present in annelids. The segmentation of annelids and arthropods, therefore, seems to be brought about by different mechanisms. This is an important argument against a common origin of segmentation in annelids and arthropods. In chordates it is also doubtful whether engrailed plays a role in somitogenesis. engrailed but not wingless is expressed in reiterated pattern in the somites of the cephalochordate amphioxus, which suggests that the segment polarity gene network as present in arthropods is not conserved. Furthermore, vertebrate engrailed orthologs do not play a role in somite formation or maintenance of the somite boundaries. This points to a different mode of segmentation in vertebrates and arthropods, and does not support a common origin of segmentation. However additional evidence is required to prove this (Damen, 2002).

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. The expression patterns of the genes even-skipped, engrailed, and wingless in a centipede, Lithobius atkinsoni, were examined. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods (Hughes, 2002).

Wnts in other invertebrate species

During Caenorhabditis elegans development, the HSN neurons and the right Q neuroblast and its descendants undergo long-range anteriorly directed migrations. Both of these migrations require EGL-20, a C. elegans Wnt homolog. Through a canonical Wnt signaling pathway, EGL-20/Wnt transcriptionally activates the Hox gene mab-5 in the left Q neuroblast and its descendants, causing the cells to migrate posteriorly. CAM-1, a Ror receptor tyrosine kinase (RTK) family member, inhibits EGL-20 signaling. Excess EGL-20, like loss of cam-1, causes the HSNs to migrate too far anteriorly. Excess CAM-1, like loss of egl-20, shifts the final positions of the HSNs posteriorly and causes the left Q neuroblast descendants to migrate anteriorly. The reversal in the migration of the left Q neuroblast and its descendants results from a failure to express mab-5, an egl-20 mutant phenotype. These data suggest that CAM-1 negatively regulates EGL-20 (Forrester, 2004).

Arthropods are the most diverse and speciose group of organisms on earth. A key feature in their successful radiation is the ease with which various appendages become readily adapted to new functions in novel environments. Arthropod limbs differ radically in form and function, from unbranched walking legs to multi-branched swimming paddles. To uncover the developmental and genetic mechanisms underlying this diversification in form, it was asked whether a three-signal model of limb growth based on Drosophila experiments applies to the development of arthropod limbs with variant shape. A Wnt-1 ortholog (Tlwnt-1) was cloned from Triops longicaudatus, a basal crustacean with a multibranched limb. The mRNA in situ hybridization pattern during larval development was examined to determine whether changes in wg expression are correlated with innovation in limb form. During larval growth and segmentation Tlwnt-1 is expressed in a segmentally reiterated pattern in the trunk. Unexpectedly, this pattern is restricted to the ventral portion of the epidermis. During early limb formation the single continuous stripe of Tlwnt-1 expression in each segment becomes ventrolaterally restricted into a series of shorter stripes. Some but not all of these shorter stripes correspond to what becomes the ventral side of a developing limb branch. It is concluded that the Drosophila model of limb development cannot explain all types of arthropod proximodistal outgrowths, and that the multi-branched limb of Triops develops from an early reorganization of the ventral body wall. In Triops, Tlwnt-1 plays a semiconservative role similar to that played by Drosophila wingless in segmentation and limb formation: morphological innovation in limb form arises in part through an early modulation in the expression of the Tlwnt-1 gene (Nulsen, 1999).

At hatching, all newly formed Triops segments express Tlwnt-1 transcripts in the anterior of each segment and En in a single row of cells in the posterior of each segment. However, Tlwnt-1 transcripts are expressed only ventrally during segmentation, whereas in Drosophila, wg transcripts are detected encircling the embryo, albeit slightly weaker dorsally. The absence of dorsal Tlwnt-1 transcripts was unexpected and suggests that there is a difference in the way in which ventral versus dorsal tissue is patterned in Triops. The phenotype of Drosophila wg mutation has not suggested a difference in wg function dorsally. However, the maintenance of wg activity in dorsal and ventral epidermis requires separate regulatory mechanisms. The lack of Tlwnt-1 transcripts in the dorsal half of each Triops segment is unlikely to be an artifact, because no dorsal staining is seen in both the carapace and the posterior ring. Nor is it proposed that this difference is due to a shift in timing, because this pattern is observed in all newly forming segments as well. Is there any other evidence for the dissociation of dorsal and ventral segmentation in arthropods? Most arthropods bear one pair of limbs per segment, yet it has been observed that the posterior abdominal segments in Triops are variable in this regard. They can bear from one to seven limbs with the number increasing posteriorly. A discrepancy in the number of dorsal and ventral En stripes is observed in these posterior segments, suggesting a mechanistic separation of dorsal from ventral segmentation. Fossil evidence for decoupled dorsal and ventral segmentation has also been reported for euthycarcinoids and Fuxianhuia. However, in all these cases only the posterior-most segments are not congruent in pattern from ventral to dorsal sides, whereas Tlwnt-1 transcripts are never observed dorsally, even during the formation of the more anterior segments (Nulsen, 1999).

Millipedes provide another possible example of dorsal/ventral dissociation, since these animals bear two pairs of limbs ventrally for each dorsal segment. However, in this case the variance between dorsal and ventral segmentation is thought to arise as a later fusion event of two segment primordia dorsally. The lack of Tlwnt-1 expression dorsally suggests that it does not play a role in either the delineation or direction of dorsal cell fates in Triops. The dorsal cells may employ a different development mechanism in order to grow and/or maintain segment polarity. In this regard, it will be interesting to determine whether other Triops wnt family members have dorsal expression patterns (Nulsen, 1999).

Does Tlwnt-1 regulate segment formation? Another interesting feature of the Triops Tlwnt-1 expression pattern is the terminal ring. A terminal ring of wg expression has now been observed in Drosophila, Tribolium and Triops. The Drosophila terminal ring expression has always been attributed to the precursor cells to the proctodeum, which also stain with wg later. However, the proctodeal staining can be clearly distinguished from the terminal ring in Triops. wg is a growth factor known to play a role in proliferation in the notum of the wing and the formation of Malphigian tubules in Drosophila. It is reasonable to expect that it plays a similar role in the proliferation of segments from posterior in Triops. Triops segments become delineated from the posterior of the larva in the region of the posterior ring. Similar to the 'progress zone' model in the chick limb bud, cells may be actively proliferating from the posterior. The posterior Tlwnt-1 expressing cells could be the source of a morphogen necessary for the function of the growth zone. High levels of Tlwnt-1 posteriorly could function either as a signal for mitosis, or as an inhibitor for differentiation. Evidence for the existence of posterior morphogens has also been suggested from experimental manipulations on many insect embryos. Once the segments are found, the more anterior, segmentally reiterated ventral stripes of Tlwnt-1 may then play a role in the maintenance and polarity of each newly formed segment (Nulsen, 1999 and references therein).

Tlwnt-1 expression during 'late' appendage development exhibits parallels to the Drosophila uniramous paradigm. Expression patterns of several genes are conserved in the development of branches of the multibranched limb of Triops and the uniramous limb of Drosophila. In Drosophila limb development, wg is required in an anterior ventral sector of the leg imaginal disc, en in the posterior of the disc and Dll at the center of the disc, where it promotes proximodistal outgrowth. The interaction between these genes has led to a model of limb development in Drosophila termed the uniramous paradigm. During the development of the multibranched limbs of Triops, Tlwnt-1 is restricted to an anteroventral portion of most of the ventral branches: En is posterior and Dll is detected in each of the developing limb branches. Thus, Triops Tlwnt-1, En and Dll expression patterns show striking molecular parallels between the patterning of an individual branch in a multi-branched limb and the patterning of a uniramous Drosophila limb. This suggests that Triops limb branches are patterned individually, each branch consisting of its own set of orthogonal axes and supports the hypothesis that multiple branches in a multibranched limb are patterned as a molecular reiteration of the key elements utilized to pattern a Drosophila limb (Nulsen, 1999 and references therein).

However, drawing a strict parallel is problematic: Tlwnt-1 is not restricted to the anteroventral sector of all limb branches. Notably, in the most dorsal branch, the epipod, Tlwnt-1 transcripts are detected in a one-cell-wide row encircling the entire branch. Another variation in the Tlwnt-1 expression pattern during limb outgrowth occurs in the exopod and gnathobase of the developing trunk limbs. These two branches exhibit Tlwnt-1 transcripts not only in a ventral sector of the branch but also in a dorsal one. A similar expression pattern is observed in the developing mandibles and maxillae. Interestingly, the mandible is branched and the maxilla unbranched, yet both exhibit similar expression patterns. It is clear from these data that the three-way intersection model of limb development generated from Drosophila experiments is not the only mechanism for elaboration of a proximodistal axis. Proximodistal outgrowths can develop in the absence of a restriction of wg to the ventral sector. The variants of Tlwnt-1 expression seen in different limb branches provide molecular evidence that individual multibranched limb branches are not all formed by a simple iterative process. Rather, in this crustacean, individual limb branches have unique characteristics to their patterning, apparent from the earliest stages of development. It appears that each limb modification, whether it be a flattened, lobate dorsal epipod or a chewing appendage such as the mandible, is correlated with a change in Tlwnt-1 expression (Nulsen, 1999 and references therein).

Evidence has been provided that crustacean dorsal epipods and insect wings are homologous structures, based on the expression of the Drosophila wing patterning genes apterous and nubbin in one of the two dorsal epipods of the branchiopod crustacean Artemia franciscana and the malacostracan Pacifastacus leniusculus. The Tlwnt-1 expression pattern in the Triops epipod appears to provide additional support for this hypothesis. The Tlwnt-1 expression in the epipod is similar to the wg expression pattern in the middle stages of Drosophila wing development, where wg transcripts are detected along the entire wing margin. An alternative interpretation, however, would be that this pattern of Tlwnt-1 expression functions to generate a branch shape that is more flattened and lobate and on this basis has been selected for independently in both crustacean and insect lineages. An examination of the wnt expression pattern in the rod-shaped dorsal epipod of the malacostracan crustacean would be informative (Nulsen, 1999 and references therein).

The Tlwnt-1 pattern during 'early' limb development deviates from the uniramous paradigm The most striking deviation from the Drosophila uniramous paradigm concerns the initial specification of the limb primordia from the ventral body wall. In Drosophila the developing limb primordia occupies at most one-fifth of the dorso-ventral extent of the body wall. By contrast, the limb field of the Triops larva consists of nearly the entire ventral body wall. It is interesting to speculate, based on the Wnt and Dll expression patterns, that Triops develops eight separate legs on each hemisegment, which later in development fuse to form one swimming appendage. However, the interpretation that there is one large limb primordia that subsequently divides into eight parts is preferred. Morphologically, the ventral body wall first protrudes as a single epithelial ridge. From this ridge the eight limb branches subsequently protrude. At the first signs of limb differentiation, when the limbs protrude from the ventral body wall, Tlwnt-1 transcripts are still observed in a nearly continuous stripe. P/D elongation of branches occurs before Tlwnt-1 is restricted to the ventral portion of each branch. The disruption of the continuous Tlwnt-1 stripe does not occur until the lobes are clearly distinguishable. Similarly, Dll protein can only be detected in the Triops limb lobes after P/D elongation of the branches. In contrast, in the Drosophila imaginal disc, P/D elongation requires that wg be restricted to a discrete ventral domain of expression before Dll expression proceeds, and this temporal and spatial expression is required for P/D elongation. These data suggest an additional mechanism for the P/D axis formation in Triops, as compared to Drosophila (Nulsen, 1999 and references therein).

The dynamic expression of Tlwnt-1 argues that innovation in limb form found in Triops is likely due to an early change in D/V patterning. The Drosophila paradigm relies on an initial specification of the imaginal primordia via an early interaction between D/V and A/P patterning genes. How does Triops establish multiple branches from one set of A/P and D/V coordinates? It is proposed that limbs with many branches, such as those seen in the trunk swimming appendages of the branchiopod crustacean Triops longicaudatus, result from an increase in the size of the limb primordia allocated during dorsal-ventral axis formation in the earliest stages of development. A change in how the D/V axis is patterned results in the production of a much larger limb primordia. Tlwnt-1 transcripts are subsequently repressed in particular groups of cells, within this enlarged primordia. An expression analysis of conserved D/V patterning genes, as well as the expression of wg and Dll in arthropods with biramous limbs, will help test this hypothesis (Nulsen, 1999).

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 current form of a provisional DNA sequence-based regulatory gene network is presented that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see End-mes: Gene Network Update for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the ß-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin, and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated (Davidson, 2002).

Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg2 endomesodermal domain, the blastula-stage separation of the central veg2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg2 endodermal domain, the stabilization of specification state within these domains, and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model that treats regulatory events within the relevant embryonic nuclei at particular stages (Davidson, 2002).

A gene encoding Wnt8 (see Drosophila Wnt8), a ligand that activates the ß-catenin/Tcf system, is expressed in the same prospective endomesodermal cells in which the autonomous maternal system initially causes ß-catenin nuclearization. This observation implies an autoreinforcing Tcf control loop, which is set up within the endomesodermal domain once this is defined. This loop is necessary, for if it is blocked by introduction of a negatively acting form of the Wnt8 ligand, so is endomesoderm specification. The inferred Wnt8 loop conforms to the 'community effect' concept of Gurdon, i.e., a requirement for intercellular signaling within a field of cells in a given state of specification that is necessary for the maintenance and the further developmental progression of that state (Davidson, 2002).

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis has dismissed the Articulata and thus challenged the segmentation homology hypothesis. In Platynereis, engrailed and wingless orthologs are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, these results are interpreted as molecular evidence of a segmented ancestor of protostomes (Prud'homme, 2003).

During posterior growth, both during normal juvenile segment formation and after caudal regeneration, Pdu-en is expressed in ectodermal circular stripes in developing segments. This segmental expression appears in continuous rings of cells immediately after the growth zone has produced them (in younger, posterior-most segments) and persists in differentiating (more anterior) segments. The pattern is more complicated on the ventral face, since, in addition to the continuous segmental expression, Pdu-en is expressed in mesodermal groups of cells and in forming ganglia of the ventral nerve cord. A longitudinal section shows that the segmental stripes of expression occur long before segmental coelomic cavities or segmental boundaries are visible. As segments mature, it becomes apparent that continuous segmental stripes of Pdu-en expression are always restricted to the anterior-most row of epidermal cells within a segment immediately posterior to the forming segmental groove corresponding to the actual segmental boundary. These segmental grooves are the only ones to form and do not seem to shift during segment differentiation, as indicated by the relative position of an appendage marker, distal-less. Hence, this expression pattern suggests that during postlarval growth in Platynereis, engrailed is involved both in the establishment of the segmental boundaries in the ectoderm and in the specification of particular cell types in the mesoderm and the central nervous system (Prud'homme, 2003).

Pdu-wnt1 is also expressed early in ectodermal stripes in each developing segment both during normal juvenile segment formation and after caudal regeneration, although the signal level is much weaker compared to that in Pdu-en. Pdu-wnt1 is expressed in the posterior-most ectodermal cells of each developing trunk segment, immediately anterior to the segmental boundary. In contrast with Pdu-en, the thickness of Pdu-wnt1 stripes increases in proportion with the segment length. Pdu-wnt1 is also expressed in the posterior part and in an anterior-proximal spot of the parapodia, as well as in the proctodaeum (Prud'homme, 2003).

Based on morphological landmarks (i.e., segmental grooves), these results suggest that Pdu-en and Pdu-wnt1 are expressed in adjacent domains on either side of the segmental boundary and play a role in the formation and maintenance of this boundary. According to these observations, Pdu-en and Pdu-wnt1 are most likely expressed in directly neighboring cells. However, due to technical difficulties with double in situ stainings, it has not been been possible to ascertain this point (Prud'homme, 2003).

The primary (animal-vegetal) (AV) and secondary (oral-aboral) (OA) axes of sea urchin embryos are established by distinct regulatory pathways. However, because experimental perturbations of AV patterning also invariably disrupt OA patterning and radialize the embryo, these two axes must be mechanistically linked. This study shows that FoxQ2, which is progressively restricted to the animal plate during cleavage stages, provides this linkage. When AV patterning is prevented by blocking the nuclear function of beta-catenin, the animal plate where FoxQ2 is expressed expands throughout the future ectoderm, and expression of nodal, which initiates OA polarity, is blocked. Surprisingly, nodal transcription and OA differentiation are rescued simply by inhibiting FoxQ2 translation. Therefore, restriction of FoxQ2 to the animal plate is a crucial element of canonical Wnt signaling that coordinates patterning along the AV axis with the initiation of OA specification (Yaguchi, 2008).

Planarians have high regenerative ability, which is dependent on pluripotent adult somatic stem cells called neoblasts. Recently, canonical Wnt/β-catenin signaling was shown to be required for posterior specification, and Hedgehog signaling was shown to control anterior-posterior polarity via activation of the Djwnt1/P-1 gene at the posterior end of planarians. Thus, various signaling molecules play an important role in planarian stem cell regulation. However, the molecular mechanisms directly involved in stem cell differentiation have remained unclear. This study demonstrates that one of the planarian LIM-homeobox genes, Djislet, is required for the differentiation of Djwnt1/P-1-expressing cells from stem cells at the posterior end. RNA interference (RNAi)-treated planarians of Djislet [Djislet(RNAi)] show a tail-less phenotype. Thus, it is speculated that Djislet might be involved in activation of the Wnt signaling pathway in the posterior blastema. When the expression pattern of Djwnt1/P-1 was carefully examined by quantitative real-time PCR during posterior regeneration, two phases of Djwnt1/P-1 expression were found: the first phase was detected in the differentiated cells in the old tissue in the early stage of regeneration and then a second phase was observed in the cells derived from stem cells in the posterior blastema. Interestingly, Djislet is expressed in stem cell-derived DjPiwiA- and Djwnt1/P-1-expressing cells, and Djislet(RNAi) only perturbed the second phase. Thus, it is proposed that Djislet might act to trigger the differentiation of cells expressing Djwnt1/P-1 from stem cells (Hayashi, 2011).

Wnt genes in amphioxus and the evolution of somitogenesis

The amphioxus tail bud is similar to the amphibian tail bud in having an epithelial organization without a mesenchymal component. Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) have been characterized; their early expression around the blastopore can subsequently be traced into the tail bud. In vertebrate embryos, there is a similar progression of expression domains for Wnt3, Wnt5, and Wnt6 genes from the blastopore lip (or its equivalent) to the tail bud. In amphioxus, AmphiWnt3, AmphiWnt5, and AmphiWnt6 are each expressed in a specific subregion of the tail bud, tentatively suggesting that a combinatorial code of developmental gene expression may help generate specific tissues during posterior elongation and somitogenesis. In spite of similarities within their tail buds, vertebrate and amphioxus embryos differ markedly in the relation between the tail bud and the nascent somites: vertebrates have a relatively extensive zone of unsegmented mesenchyme (i.e., presomitic mesoderm) intervening between the tail bud and the forming somites, whereas the amphioxus tail bud gives rise to new somites directly. It is likely that presomitic mesoderm is a vertebrate innovation made possible by developmental interconversions between epithelium and mesenchyme that first became prominent at the dawn of vertebrate evolution (Schubert, 2001).

Wnt-Ror signaling to SIA and SIB neurons directs anterior axon guidance and nerve ring placement in C. elegans

Wnt signaling through Frizzled proteins guides posterior cells and axons in C. elegans into different spatial domains. This study demonstrates an essential role for Wnt signaling through Ror tyrosine kinase homologs in the most prominent anterior neuropil, the nerve ring. A genetic screen uncovered cwn-2, the C. elegans homolog of Wnt5, as a regulator of nerve ring placement. In cwn-2 mutants, all neuronal structures in and around the nerve ring are shifted to an abnormal anterior position. cwn-2 is required at the time of nerve ring formation; it is expressed by cells posterior of the nerve ring, but its precise site of expression is not critical for its function. In nerve ring development, cwn-2 acts primarily through the Wnt receptor CAM-1 (Ror), together with the Frizzled protein MIG-1, with parallel roles for the Frizzled protein CFZ-2. The identification of CAM-1 as a CWN-2 receptor contrasts with CAM-1 action as a non-receptor in other C. elegans Wnt pathways. Cell-specific rescue of cam-1 and cell ablation experiments reveal a crucial role for the SIA and SIB neurons in positioning the nerve ring, linking Wnt signaling to specific cells that organize the anterior nervous system (Kemmerdell, 2009).

CWN-2 has an essential role in nerve ring placement. The results suggest that CWN-2 is a ligand for the CAM-1 (Ror) receptor in the SIA and SIB neurons, perhaps with MIG-1 (Frizzled) as a co-receptor. In the absence of this signaling pathway, many axons and cell bodies in the nerve ring are displaced towards the anterior. The similar effects of Wnt pathway mutations and genetic ablations suggest that SIA and SIB neurons direct normal nerve ring placement. Additional nerve ring guidance genes that act at least partly parallel to cwn-2, cam-1 and mig-1 are the Frizzled gene cfz-2, the Wnt gene cwn-1, and the Robo gene sax-3 (Kemmerdell, 2009).

cwn-2 is required at a discrete time in development, but the site of cwn-2 expression is relatively unimportant. The rescue of cwn-2 mutants by uniform expression or misexpression echoes the rescue of egl-20 and lin-44 Wnt defects by cDNAs expressed from heat-shock promoters, and suggests that C. elegans Wnts can sometimes function as non-spatial cues. For example, CWN-2 could stimulate axon outgrowth of SIA and SIB at a particular time, with spatial information provided by the distribution of receptors or by other guidance cues near the nerve ring, such as UNC-6 and SLT-1. Alternatively, cwn-2 activity could be spatially limited by cell-specific post-translational pathways or by extracellular Wnt-binding proteins. Finally, additional Wnts, such as CWN-1, might contribute spatial information when CWN-2 is misexpressed: disrupting cwn-2 alone may not eliminate the overall posteriorly biased pattern of Wnt expression. Indeed, in the posterior body, overlapping functions of lin-44, egl-20 and cwn-1 can mask the effects of misexpressing a single Wnt (Kemmerdell, 2009).

CAM-1 has been proposed to act as an extracellular inhibitor of Wnts owing to its non-cell-autonomous action in vulval development and the apparent dispensability of its intracellular domain. However, the CAM-1-related protein Ror2 is an established tyrosine kinase receptor for mammalian Wnts, although kinase-independent functions are also known for vertebrate Rors. Nerve ring development initially appeared not to require the intracellular domain of CAM-1, but many double mutants that included the Frizzleds mig-1, cfz-2 and lin-17 and the LRP-like mig-13 uncovered a requirement for the intracellular domain. Together with a specific requirement for cam-1 expression in the SIA and SIB neurons, these results support a receptor function of CAM-1 in nerve ring development. The overlapping expression and rescue of cam-1 and mig-1 in SIA and SIB matches the genetic results suggesting that they act together in a common process, perhaps as co-receptors for CWN-2. In mammalian osteocytes and lung epithelial cells, Frizzled and Ror or Ryk receptors can function together in a signaling complex. The relevant cellular sites of action for cfz-2, lin-17 and mig-13 are unknown, and expression of cfz-2 in SIA and SIB neurons did not rescue cfz-2 mutants, suggesting that cfz-2 has primary functions outside of SIA and SIB. It is too early to determine whether CFZ-2, LIN-17 and MIG-13 might also be CAM-1 co-receptors (Kemmerdell, 2009).

One interesting implication of the use of multiple Wnt receptors is that spatially and temporally restricted receptor expression might be as important in development as restricted ligand expression. Rather than responding passively to an instructive Wnt cue, developing neurons can shape their response to Wnts through their receptor complement. They can also shape the response of more-distant cells by capturing Wnt ligands, as shown for CAM-1 near the vulva (Kemmerdell, 2009).

Cell-type-specific rescue of cam-1, mig-1 and sax-3 and cell ablation experiments revealed an important role for SIA and SIB neurons in nerve ring placement. Several models could explain cwn-2 effects on SIA and SIB. First, cwn-2 could act in a traditional Wnt patterning role to determine SIA and SIB cell fates; SIA and SIB would then organize nerve ring development through other molecular pathways. However, several SIA and SIB markers are expressed normally in cwn-2 mutants, arguing against a cell fate change (Kemmerdell, 2009).

The model that cwn-2 directly affects axon guidance of SIA and SIB neurons, which in turn instruct the positioning of the nerve ring. SIA and SIB neurons occupy a position near the base of the nerve ring, where they might detect CWN-2, as well as the ventral attractant UNC-6 and the anterior repellent SLT-1. In wild-type animals, the nerve ring axon trajectories of SIA and SIB neurons are unusually complex, consistent with a special patterning role. In cwn-2 and cam-1 mutants, the disruption of axon trajectories in SIA and SIB neurons is more complicated than in other cell types: SIA and SIB have guidance defects at many positions, whereas other neurons simply move to an anterior location. It is suggested that the guidance of SIA and SIB neurons is under the direct control of CWN-2, which generates a temporally precise and spatially less precise signal to form a nerve ring at the correct location. Other nerve ring neurons follow SIA and SIB neurons to this location if possible; if SIA and SIB neurons are misguided or absent, the nerve ring shifts to a more anterior position that might be a default position, or one specified by another guidance cue (Kemmerdell, 2009).

Neuroblast migration along the anteroposterior axis of C. elegans is controlled by opposing gradients of Wnts and a secreted Frizzled-related protein

The migration of neuroblasts along the anteroposterior body axis of C. elegans is controlled by multiple Wnts that act partially redundantly to guide cells to their precisely defined final destinations. How positional information is specified by this system is, however, still largely unknown. This study used a novel fluorescent in situ hybridization methods to generate a quantitative spatiotemporal expression map of the C. elegans Wnt genes. The five Wnt genes were found to be expressed in a series of partially overlapping domains along the anteroposterior axis, with a predominant expression in the posterior half of the body. Furthermore, a secreted Frizzled-related protein is expressed at the anterior end of the body axis, where it inhibits Wnt signaling to control neuroblast migration. These findings reveal that a system of regionalized Wnt gene expression and anterior Wnt inhibition guides the highly stereotypic migration of neuroblasts in C. elegans. Opposing expression of Wnts and Wnt inhibitors has been observed in basal metazoans and in the vertebrate neurectoderm. These results in C. elegans support the notion that a system of posterior Wnt signaling and anterior Wnt inhibition is an evolutionarily conserved principle of primary body axis specification (Harterink, 2011).

Table of contents

wingless continued: Biological Overview | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.