Table of contents

The Armadillo Family

Proteins of the armadillo family are involved in diverse cellular processes in higher eukaryotes. Some of them, like armadillo, beta-catenin and plakoglobins have dual functions in intercellular junctions and signaling cascades. Others, belonging to the importin-alpha-subfamily are involved in NLS recognition and nuclear transport, while some members of the armadillo family have as yet unknown functions. The Saccharomyces cerevisiae protein Yel013p is a novel armadillo (arm) repeat protein. The ORF Yel013w was identified by the genome project on chromosome V (EMBL: U18530) and codes for an acidic protein of 578 residues with 8 central arm-repeats, which are closely related to the central repeat-domain of Xenopus laevis plakoglobin. Yel013p (Vac8p) is constitutively expressed in diploid and haploid yeasts and is not essential for viability and growth. However, the vacuoles of mutant cells are multilobular or even fragmented into small vesicles and the processing of aminopeptidase I, representing the cytoplasm-to-vacuole transport pathway, is strongly impaired. Consistent with these observations, subcellular fractionation experiments, immunolocalization and expression of green fluorescent protein (GFP) fusion proteins reveal that Yel013p (Vac8p) is associated with the vacuolar membrane. These data provide evidence for the involvement of an arm-family member in vacuolar morphology and protein targeting to the vacuole (Fleckenstein, 1998).

ß-catenin homologs in other insects

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).

Wnt/β-catenin pathway and asymmetric cell divisions in C. elegans embryos

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).

ß-Catenin can promote adhesion at the cell cortex and mediate Wnt signaling in the nucleus. In Caenorhabditis elegans, both WRM-1/ß-catenin and LIT-1 kinase localize to the anterior cell cortex during asymmetric cell division but to the nucleus of the posterior daughter afterward. Both the cortical and nuclear localizations are regulated by Wnts and are apparently coupled. The daughters show different nuclear export rates for LIT-1. These results indicate that Wnt signals release cortical WRM-1 from the posterior cortex to generate cortical asymmetry that may control WRM-1 asymmetric nuclear localization by regulating cell polarity (Takeshita, 2005).

After the four-cell stage of C. elegans development, the polarity of many cells, including the EMS blastomere and the T hypodermal cell, is regulated by the Wnt signaling pathway. Unlike the tissue-polarity Wnt pathway, which regulates cell polarity in Drosophila and mammals independent of ß-catenin, the Wnt pathway that controls the EMS polarity involves WRM-1/ß-catenin and the POP-1/TCF transcription factor and hence is related to the canonical Wnt pathway. Unlike ß-catenin in other organisms, WRM-1 does not bind to cadherins and functions in Wnt signaling only, but not in cell adhesion. In the canonical Wnt pathway, the Wnt signal regulates the stability and nuclear localization of ß-catenin. However, it is not known how the Wnt signal regulates WRM-1, especially because WRM-1 does not have the conserved phosphorylation sites of GSK3ß. Furthermore, the subcellular localization of WRM-1 has not been determined. Therefore, the function of WRM-1 in the regulation of cell polarity has remained obscure (Takeshita, 2005).

In addition to the components of the Wnt pathway, LIT-1/MAP kinase and MOM-4/MAPKKK are involved in the EMS division. MOM-4 activates the LIT-1 kinase, while LIT-1 binds to WRM-1 to phosphorylate POP-1. Activation of this Wnt/MAPK pathway results in the asymmetric distribution of POP-1 between the nuclei of the daughter cells (POP-1 asymmetry). Unlike the Numb and Prospero proteins in Drosophila, however, POP-1 does not localize to the cell cortex during division. Instead, POP-1 asymmetry is regulated by nuclear export. Although the nuclear export of POP-1 is regulated by phosphorylation by the LIT-1-WRM-1 complex, it is not clear how the Wnt signaling pathway determines the difference in the rate of nuclear export between the daughter cells (Takeshita, 2005).

This study shows that WRM-1 and LIT-1 localized asymmetrically to the anterior cell cortex before and during the division of post-embryonic cells. Surprisingly, after division, WRM-1 and LIT-1 localized preferentially to the nucleus of the posterior rather than anterior daughters. These results suggest a role for cortical ß-catenin in the regulation of cell polarity, and provide a novel link between cortical and nuclear ß-catenin (Takeshita, 2005).

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).

Wnt signaling regulates many aspects of metazoan development, including stem cells. In C. elegans, Wnt/MAPK signaling controls asymmetric divisions. A recent model proposed that the POP-1/TCF DNA binding protein works together with SYS-1/β-catenin to activate transcription of target genes in response to Wnt/MAPK signaling. The somatic gonadal precursor (SGP) divides asymmetrically to generate distal and proximal daughters of distinct fates: only its distal daughter generates a distal tip cell (DTC), which is required for stem cell maintenance. No DTCs are produced in the absence of POP-1/TCF or SYS-1/β-catenin, and extra DTCs are made upon overexpression of SYS-1/β-catenin. This study reports that POP-1/TCF and SYS-1/β-catenin directly activate transcription of ceh-22/nkx2.5 isoforms in SGP distal daughters, a finding that confirms the proposed model of Wnt/MAPK signaling. In addition, it is demonstrated that the CEH-22/Nkx2.5 homeodomain transcription factor is a key regulator of DTC specification. It is speculated that these conserved molecular regulators of the DTC niche in nematodes may provide insight into specification of stem cell niches more broadly (Lam, 2006).

Thus Wnt signaling and ceh-22/nkx2.5 work together to specify the DTC fate. The common function of DTCs in hermaphrodites and males is that of a stem cell niche. Wnt signaling has emerged as a key regulator of stem cells in many tissues and in many organisms, and that role relies on transcriptional activation by TCF/LEF and β-catenin transcription factors. The current work suggests that one role of Wnt signaling may be to control the stem cell niche. A similar suggestion was recently put forward with respect to osteoblasts, which provide a niche for hematopoietic stem cells. CEH-22/Nkx2.5 and its homologs have not previously been implicated in the control of stem cells. Indeed, the fly and vertebrate homologs, termed Tinman and Nkx2.5, respectively, are best known for their roles in heart specification and differentiation. Nematodes have no heart, but CEH-22 controls development of the rhythmically contracting musculature of the pharynx, and zebrafish Nkx2.5 can functionally replace CEH-22. Therefore, the CEH-22/Nkx2.5 class of homeodomain transcription factors has broadly conserved functions in animal development (Lam, 2006).

A remaining question is whether CEH-22 control of the DTC fate reflects a conserved role for this class of homeodomain transcription factors in regulating stem cell niches. Mouse mutants deleted for Nkx2.5 die with a broad spectrum of defects, including severe defects in vasculogenesis and angiogenesis as well as hematopoiesis in the yolk sac. Intriguingly, endothelial cells appear to function as stem cell niches. It is tempting to speculate that the severe vasculature defects in Nkx2.5 mutants may reflect some role of this conserved regulator in control of a vertebrate niche, much as CEH-22 controls the DTC. Two important challenges for the future are to learn how CEH-22 specifies the DTC niche in C. elegans and to learn whether its homologs specify an analogous stem cell niche in flies and vertebrates (Lam, 2006).

In C. elegans, Wnt signaling regulates a number of asymmetric cell divisions. During telophase, WRM-1/beta-catenin localizes asymmetrically to the anterior cortex and the posterior daughter's nucleus. However, cortical WRM-1's functions are not known. This study used a membrane-targeted form of WRM-1 to show that cortical WRM-1 inhibits Wnt signaling and the nuclear localization of WRM-1. These functions are mediated by APR-1/APC, which regulates WRM-1 nuclear export. APR-1 as well as PRY-1/Axin and Dishevelled homologs localize asymmetrically to the cortex. These results suggest a model in which cortical WRM-1 recruits APR-1 to the anterior cortex before and during division, and the cortical APR-1 stimulates WRM-1 export from the anterior nucleus at telophase. Because beta-catenin and APC are localized to the cortex in many cell types in different species, these results suggest that these cortical proteins may regulate asymmetric divisions or Wnt signaling in other organisms as well (Mizumoto, 2007).

The results strongly suggest that cortical WRM-1/β-catenin inhibits its own nuclear localization during asymmetric cell division in C. elegans. This inhibitory role contrasts with that of nuclear WRM-1, which is a positive regulator of the Wnt/MAPK pathway, probably through its promotion of the nuclear export of POP-1/TCF. Thus, WRM-1 possesses dual and antagonistic functions in the Wnt/MAPK pathway. Because, in the apr-1(RNAi) animals, WRM-1 localized to both daughters at telophase with WRM-1 being at the anterior cortex, APR-1 probably mediates cortical WRM-1's effects on the localization of nuclear WRM-1. Thus, the data indicate a novel role for cortical β-catenin as a regulator of its asymmetric nuclear localization, mediated by APC during asymmetric cell divisions (Mizumoto, 2007).

The results show that Wnt proteins regulate the asymmetric cortical localization of components of the Wnt/MAPK pathway during postembryonic asymmetric cell divisions in C. elegans. In the absence of Wnts, LIN-17/Fz, APR-1/APC, PRY-1/Axin, WRM-1/β-catenin, and LIT-1/MAPK were localized symmetrically to the cortex. Wnt signals from the posterior side of the cell provide cues for polarization, leading to the posterior localization of Fz receptors, like LIN-17 in the T cell and MOM-5 in the Q and V5 cells. These localized and activated Fz receptors probably recruit the Dsh proteins to the posterior cortex, as has been shown in Drosophila and Xenopus. Although there is no experimental evidence for the involvement of Dsh, the cortical Dsh proteins may stimulate the disassembly of the complex containing PRY-1, APR-1, LIT-1, and WRM-1, releasing them from the cortex. The delay in the establishment of cortical asymmetry in pry-1 mutants suggests that PRY-1 probably functions redundantly in this process with one or more additional proteins, e.g., APR-1. This model seems consistent with previous observations in mammals and Drosophila that Wnt signals stimulate the dissociation of β-catenin from the destruction complex. In this scenario, the disassembly of the cortical multiprotein complex does not occur at the anterior cortex, which restricts WRM-1 and APR-1 to the anterior cortex prior to cell division. The detailed mechanism by which these proteins are targeted to the cortex is currently unknown. It does not seem to depend on the direct interaction between β-catenin and cadherin, because WRM-1 does not bind cadherin. Consistently, zygotic RNAi of hmr-1, which caused severe defects in embryonic morphogenesis, did not affect the cortical WRM-1 localization in larvae that escaped lethality. Nonetheless, WRM-1 appears to play a central role in this targeting mechanism, because, as was shown in this study, cortical WRM-1 affects the targeting of APR-1 and LIT-1 to the cortex (Mizumoto, 2007).

The results strongly suggest that APR-1 shuttles between the cytoplasm and nucleus and exports WRM-1 from both the anterior and posterior nuclei at telophase, when WRM-1 nuclear asymmetry is established. In addition, WRM-1 regulates the cortical localization of APR-1. Therefore, it is plausible that cortical WRM-1 regulates its own nuclear localization through cortical APR-1 at telophase. In this case, a remaining question is how cortical APR-1 regulates WRM-1 export. One possibility is that APR-1 functions at the cortex to regulate microtubules, given that in Drosophila and mammals APC localizes to the plus ends of microtubules to regulate their stability. APC is also known to move along microtubules toward the plus ends. In the anterior nucleus, the complex containing WRM-1 and APR-1 that exits from the nucleus may be efficiently loaded at the anterior perinuclear region onto microtubules stabilized by cortical WRM-1 and APR-1. This might result in the enhancement of nuclear export from the anterior nucleus at telophase. Further analyses will be necessary to elucidate the roles of microtubules and APR-1 in asymmetric cell divisions. Whatever the functions of cortical APR-1, the results indicate that APR-1 plays a central role in converting the polarity of mother cells to the asymmetries of the daughter nuclei (Mizumoto, 2007).

In contrast to the finding that apr-1 inhibits the Wnt/MAPK pathway in postembryonic cells, apr-1 is a positive regulator of the pathway in the EMS division. Consistent with this, in apr-1(RNAi) embryos, WRM-1 fails to localize to the nuclei of the EMS daughter cells, suggesting that apr-1 is required for the nuclear localization of WRM-1. Therefore, the molecular mechanisms of the Wnt/MAPK pathway may be different between the EMS and seam cells (Mizumoto, 2007).

Although WRM-1 has armadillo repeats like β-catenin, its sequence is quite diverged from β-catenin, and it does not bind to APR-1/APC or POP-1/TCF. Accordingly, the Wnt/MAPK pathway is sometimes referred to as a 'noncanonical' pathway. However, this study shows that WRM-1 colocalizes with APR-1 and PRY-1/Axin, and that its nuclear localization is negatively regulated by APR-1. Therefore, the pathway may be more relevant to the Wnt/β-catenin pathway in other organisms than previously thought, even if the stability of WRM-1 is not regulated by Wnt (Mizumoto, 2007).

In other organisms, β-catenin and APC localize to the cortex in a variety of cells. In contrast to the observation in C. elegans that cortical β-catenin inhibits Wnt signaling, it has been reported, in Xenopus and Drosophila, that overexpression of a membrane-tethered form of β-catenin activates Wnt signaling. Nevertheless, it has also been reported that overexpression of cadherin, which likely recruits β-catenin to the cortex, inhibits Wnt signaling. Such effects on Wnt signaling are explained by the sequestration of Wnt-signaling components: Axin or APC by membrane-tethered β-catenin, and β-catenin itself. Therefore, the physiological roles of cortical β-catenin in Wnt signaling remain elusive, and it is possible that cortical β-catenin directly inhibits Wnt signaling in other organisms (Mizumoto, 2007).

A function for β-catenin in cell polarity regulation has not been clearly demonstrated in other organisms. However, there are reports on the asymmetric cortical localization of Armadillo/β-catenin and APC in Drosophila neuroblasts that undergo asymmetric divisions (McCartney, 1999; Akong, 2002). Furthermore, APC localizes to the cortex asymmetrically along with β-catenin in germline stem cells and regulates their asymmetric divisions in Drosophila (Yamashita, 2003). Therefore, the function of β-catenin and APC as polarity regulators may be conserved in other organisms (Mizumoto, 2007).

Wnt signaling is involved in the maintenance of stem cells that undergo self-renewing asymmetric cell division. Although there is no evidence that Wnt signals regulate the polarities of stem cell divisions, inappropriate Wnt activation is often correlated with tumor formation, which may be caused by the failure of asymmetric cell divisions. In fact, this study showed that apr-1(RNAi) animals showed overproduction of the seam cells, probably due to defects in asymmetric cell divisions. The data suggest that cortical β-catenin may suppress stem cell tumorigenesis by regulating the polarities of asymmetric stem cell divisions and/or by repressing the nuclear accumulation of β-catenin. Further analyses may shed light on unidentified functions of β-catenin in cell polarity regulation and/or in the Wnt-signaling pathway in other organisms (Mizumoto, 2007).

C. elegans embryos exhibit an invariant lineage comprised primarily of a stepwise binary diversification of anterior-posterior (A-P) blastomere identities. This binary cell fate specification requires input from both the Wnt and MAP kinase signaling pathways. The nuclear level of the TCF protein POP-1 is lowered in all posterior cells. The β-catenin SYS-1 also exhibits reiterated asymmetry throughout multiple A-P divisions, and this asymmetry is reciprocal to that of POP-1. Furthermore, SYS-1 functions as a coactivator for POP-1, and the SYS-1-to-POP-1 ratio appears critical for both the anterior and posterior cell fates. A high ratio drives posterior cell fates, whereas a low ratio drives anterior cell fates. The SYS-1 and POP-1 asymmetries are regulated independently, each by a subset of genes in the Wnt/MAP kinase pathways. It is proposed that two genetic pathways, one increasing SYS-1 and the other decreasing POP-1 levels, robustly elevate the SYS-1-to-POP-1 ratio in the posterior cell, thereby driving A-P differential cell fates (Huang, 2007).

How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. This study establishes a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/β-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. This study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/β-catenin pathway is widely used to control the identity of neuronal lineages (Bertrand, 2009).

Several examples have by now well illustrated that the differentiation of individual neuron types is governed by terminal selector genes that encode transcription factors which directly activate large batteries of terminal differentiation genes. However, how these terminal selector genes are regulated by earlier specification processes, in particular asymmetric divisions, remains poorly understood. This study has uncovered a direct regulatory cascade that links the asymmetric division machinery to the activation of the terminal selector genes ttx-3 and ceh-10 during embryogenesis in C. elegans. These results will first be discussed in the context of the broad concept of progressive regulatory states before analyzing two other general implications of these studies, namely, a common theme of Zic gene function in neural precursors and a potentially broadly conserved role of Wnt signaling in neuronal specification (Bertrand, 2009).

The Zic transcription factor REF-2 is transiently expressed in the SMDD/AIY mother, where it directly activates the expression of the ttx-3 LIM homeobox gene in cooperation with the bHLH transcription factor HLH-2. Following division of the mother cell, TTX-3 is inherited in both SMDD and AIY and activates ceh-10 expression in AIY, but not in SMDD. The difference in ttx-3 activity between AIY and SMDD is due to the Wnt/β-catenin asymmetry pathway. The transcriptional mediators of this pathway, the TCF transcription factor POP-1 and its coactivator the β-catenin SYS-1, are asymmetrically localized after division of the SMDD/AIY mother. In AIY, the POP-1 nuclear concentration is low and SYS-1 concentration is high. This may allow most of the POP-1 proteins to be associated with the coactivator SYS-1 and to activate the transcription of ceh-10 via the predicted POP-1 binding sites present in its promoter. In SMDD, where the POP-1 nuclear concentration is high and SYS-1 concentration is low, most of the POP-1 proteins may not be associated with SYS-1 and therefore repress ceh-10 transcription. Finally, once coexpressed in postmitotic AIY, TTX-3 and CEH-10 directly activate a large battery of terminal differentiation genes responsible for AIY differentiation and specific function. TTX-3 and CEH-10 also maintain their own expression so that the system is locked during larval and adult stages (Bertrand, 2009).

It has been proposed that during development a cell progresses through a succession of 'regulatory states' each characterized by a combination of specific gene regulatory factors. In the case of the AIY terminal division, two regulatory states are observed. The first one (state 1) is characterized by the transient expression of REF-2 and HLH-2 in the SMDD/AIY mother. The second (state 2p) corresponds to the terminal differentiation state defined by the expression of the terminal complex TTX-3/CEH-10 and the battery of terminal differentiation genes. The transition between those two states is driven by a binary decision system based on the Wnt/β-catenin asymmetry pathway (Bertrand, 2009).

These findings provide explicit support for a theoretical model initially proposed by Priess and coworkers (Lin, 1998). In this model a transcription factor 'B' expressed in both daughter cells following the division cooperates with a high POP-1 level in the anterior cell to specify state 2a and cooperates with a low POP-1 level in the posterior cell to specify state 2p. In the case of AIY, this lineage-specific factor 'B' corresponds to the transcription factor TTX-3 (Bertrand, 2009).

Before discussing general principles of Wnt/β-catenin signaling in neuronal specification, ref-2, one specific member of the regulatory network studied here, will be discussed. ref-2 is expressed in several neuronal precursors in the embryo; in contrast, there is no detectable expression of ref-2 in postmitotic neurons at larval and adult stages. Similarly, in Hydra and vertebrates, Zic transcription factors are also expressed in several neural progenitors, while expression in adult postmitotic neurons is only rarely seen. This indicates that Zic transcription factors may have a conserved function in neural precursor development. While in vertebrates Zic transcription factors have been shown to play a role in promoting the proliferation of the progenitors, it is conceivable that they also function as transient initiators of the terminal differentiation program of specific neurons, as observed in the case of AIY. For example, an intriguing parallel can be drawn between the development of the AIY interneurons and the cholinergic projection neurons/interneurons of the vertebrate basal forebrain. These vertebrate cholinergic neurons have an important function in memory formation, as is the case for the cholinergic interneuron AIY. In vertebrates, these postmitotic neurons and their progenitors express the TTX-3-related LIM-homeodomain transcription factor Lhx7/8, which is required for their differentiation. It has been recently reported that the Zic transcription factors Zic1 and Zic3 are also expressed in these progenitors and that inactivation of both genes reduces the number of cholinergic neurons. While these Zic factors seem to regulate primarily the proliferation of the precursors, it would be interesting to test whether, in analogy to ttx-3 initiation by REF-2, they also initiate the expression of Lhx7/8 and endow the progenitors with the ability to generate cholinergic neurons (Bertrand, 2009).

A particular Wnt pathway, the Wnt/β-catenin asymmetry pathway, is involved in many asymmetric blastomere divisions in the early embryo as well as some asymmetric divisions during larval development in C. elegans. Analysis of temperature-sensitive mutants of the upstream kinase gene lit-1(t1512) has shown that this pathway is involved in six successive asymmetric division rounds in the early embryo. However, this pathway has not been shown so far to be implicated in the terminal division of embryonic neuroblasts. This study has observed that the three terminal neuroblast divisions analyzed (giving rise to AIY, AIN, and ASER, respectively) are affected by disrupting this Wnt pathway. Moreover, lit-1(t1512); mom-4(ne1539) embryos shifted at restrictive temperature just before most embryonic neuroblasts undergo their last division give rise to larvae showing strongly uncoordinated movements, suggesting additional defects in motor neuron lineages. These observations predict that the Wnt/β-catenin asymmetry pathway is widely used in terminal neuroblast division in the C. elegans embryo (Bertrand, 2009).

While it was shown that the transcriptional mediators of this pathway, POP-1/TCF and SYS-1/β-catenin, are asymmetrically localized after the terminal division of embryonic neuroblasts, how the asymmetry in this pathway is initially established remains obscure. Both POP-1 and SYS-1 are regulated by this pathway at a posttranslational level (Mizumoto, 2007). In the early embryo POP-1 asymmetry in the AB lineage requires an initial MOM-2/Wnt signal coming from the P1 lineage that may be transmitted among AB blastomeres by a relay mechanism, but POP-1 asymmetry becomes later independent of MOM-2/Wnt. MOM-5/Frizzled is enriched in the posterior pole of early AB derivatives, and in analogy to the planar cell polarity in Drosophila, a Wnt-independent asymmetric Frizzled localization could be responsible for generating asymmetric cell divisions. Additional studies on Wnt requirement and Frizzled localization are required to assess their mode of function in the context of the terminal division of embryonic neuroblasts (Bertrand, 2009).

Neurons are also generated via asymmetric divisions in Drosophila and vertebrates. Recent results suggest a possible role for β-catenin in the asymmetric division of neural progenitors in the mouse brain. For example, it has been proposed that β-catenin may regulate the asymmetric division generating intermediate progenitors from radial glial cells during corticogenesis. A Wnt/β-catenin system, similar to the one shown in this study to operate in terminal neuroblast divisions in C. elegans, may therefore be used in binary cell fate decisions during the development of the nervous system in other organisms (Bertrand, 2009).

Wnt target gene activation in C. elegans requires simultaneous elevation of β-catenin/SYS-1 and reduction of TCF/POP-1 nuclear levels within the same signal-responsive cell. SYS-1 binds to the conserved N-terminal β-catenin-binding domain (CBD) of POP-1 and functions as a transcriptional co-activator. Phosphorylation of POP-1 by LIT-1, the C. elegans Nemo-like kinase homolog, promotes POP-1 nuclear export and is the main mechanism by which POP-1 nuclear levels are lowered. A mechanism is described whereby SYS-1 and POP-1 nuclear levels are regulated in opposite directions, despite the fact that the two proteins physically interact. The C terminus of POP-1 is essential for LIT-1 phosphorylation and is specifically bound by the diverged β-catenin WRM-1. WRM-1 does not bind to the CBD of POP-1, nor does SYS-1 bind to the C-terminal domain. Furthermore, binding of WRM-1 to the POP-1 C terminus is mutually inhibitory with SYS-1 binding at the CBD. Computer modeling provides a structural explanation for the specificity in WRM-1 and SYS-1 binding to POP-1. Finally, WRM-1 exhibits two independent and distinct molecular functions that are novel for β-catenins: WRM-1 serves both as the substrate-binding subunit and an obligate regulatory subunit for the LIT-1 kinase. Mutual inhibitory binding would result in two populations of POP-1: one bound by WRM-1 that is LIT-1 phosphorylated and exported from the nucleus, and another, bound by SYS-1, that remains in the nucleus and transcriptionally activates Wnt target genes. These studies could provide novel insights into cancers arising from aberrant Wnt activation (Yang, 2011).

Function of ß-catenin homologs in other invertebrates

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).

In flies and vertebrates, Armadillo/beta-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signaling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative beta-catenin homologs have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homolog POP-1 mediate Wnt signaling by a mechanism that has challenged current views of the Wnt pathway. BAR-1 is the only beta-catenin homolog that interacts directly with POP-1. BAR-1 mediates Wnt signaling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only beta-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. It is concluded that a canonical Wnt pathway exists in C. elegans. Furthermore, this analysis shows that the functions of C. elegans beta-catenins in adhesion and in signaling are performed by separate proteins (Korswagen, 2000).

It is proposed that the signaling and adhesion functions of Armadillo/beta-catenin have been distributed between separate beta-catenin homologs in C. elegans. Two beta-catenins function in Wnt signaling. WRM-1 is part of a divergent Wnt pathway that, in collaboration with a mitogen-activated protein (MAP) kinase pathway, mediates the asymmetric distribution of POP-1 between daughter cells of many anterior/posterior cells. POP-1 probably acts as a repressor in this pathway and differences in POP-1 expression levels between daughter cells may allow the specification of different fates. BAR-1 is part of a Wnt pathway that is similar to that in flies and vertebrates. BAR-1 can directly associate with POP-1 to activate a Tcf reporter. BAR-1 and POP-1 are required for the expression of the endogenous Wnt target gene mab-5. Of the three C. elegans beta-catenins, only BAR-1 contains a set of four conserved GSK3 phosphorylation sites. These sites are essential for the regulation of beta-catenin by the Wnt pathway and are frequently mutated in cancers. C. elegans contains a single putative APC homolog, APR-11. In vertebrates, APC forms a complex with GSK3, Axin and beta-catenin to downregulate beta-catenin levels in the absence of Wnt signaling. None of the C. elegans beta-catenins bind APR-1 in a yeast two-hybrid assay. Together with the observation that the C. elegans genome does not contain a clear Axin homolog, this indicates that BAR-1 may be regulated differently (Korswagen, 2000).

HMP-2 is the only C. elegans beta-catenin that interacts with the single classical cadherin, HMR-1. This interaction agrees with the hmp-2 mutant phenotype and with the colocalization of HMP-2 with HMR-1 and the alpha-catenin HMP-1 in adherens junctions. It is concluded that HMP-2 functions specifically in adhesion. Vertebrates express a second beta-catenin-like protein, Plakoglobin, which is part of the desmosomal adhesion complex. It is unclear whether Plakoglobin functions specifically in adhesion or also has a role in Wnt signaling. The functions of Armadillo and beta-catenin in Wnt signaling and adhesion are physically separable. It is unclear whether Armadillo/beta-catenin in adherens junctions may directly or indirectly affect the cytoplasmic signaling pool. Mutations in cadherins have been identified in many epithelial tumors. An attractive explanation for the oncogenic potential of cadherin mutations is the release of beta-catenin from adherens junctions, which in turn can interact with Tcf transcription factors to activate the expression of Wnt target genes. These data indicate that, at least in C. elegans, the adhesion and signaling pools of beta-catenin are separate entities (Korswagen, 2000).

The Caenorhabditis elegans embryo provides a model system for studying how cells move and change shape to generate body and tissue morphologies. At hatching, the outermost cellular layer of the body consists of a monolayer of 85 epithelial cells, called hypodermal cells, that are linked together by adherens junctions. During embryogenesis, hypodermal cells are involved in two distinct processes that transform the initially ellipsoidal mass of embryonic cells into a long, thin worm; these processes are called body enclosure and body elongation. The hypodermal cells are born on the dorsal surface of the embryo. As the hypodermal cells develop adherens junction connections, they begin to spread as a sheet across the embryo until the contralateral edges of the sheet meet at the ventral midline. In the anterior of the embryo, ventral hypodermal cells on the periphery of the spreading sheet develop filopodial extensions that may function to draw the contralateral edges of the sheet together. In the posterior of the embryo, the contralateral edges appear to be drawn together by a purse-string-like contraction that completes the enclosure process (Costa, 1998).

In several respects, these processes are similar to epithelial cell movements described in a variety of systems, such as wound healing in vertebrates and dorsal closure in Drosophila. At the completion of body enclosure in C. elegans, the apical surfaces of the hypodermal cells resemble rectangles that are elongated along the circumferential contour of the embryo's body. These apical surfaces begin to change shape, constricting along the circumferential contour of the body and elongating along the anterior-posterior (longitudinal) axis. The coordinate changes in the shapes of the hypodermal cells appear to cause the body to decrease in circumference and to elongate about fourfold along its longitudinal axis. Each hypodermal cell contains an array of actin filament bundles in its apical cortex; these bundles are oriented parallel to the circumferential contour of the body, and are here termed circumferential filament bundles (CFBs). The CFBs appear to connect with the adherens junction that encircles each hypodermal cell. Microtubules in the dorsal and ventral hypodermal cells are aligned parallel to the CFBs. However, few if any microtubules appear to contact the adherens junction; most instead terminate before, or run parallel to, the junction. The parallel filament bundles bridge two opposing sides of each hypodermal cell, apparently connecting to the subapical adherens junction. Contraction of the filament bundles has been proposed as the force that elongates the embryo; the bundles become shorter and thicker during elongation and drugs that disrupt actin filament organization prevent elongation. Apical constriction of cells has been shown in other systems to drive the invagination of epithelial sheets -- because of the closed, cylindrical geometry of the hypodermal sheet in C. elegans, an analogous apical constriction might instead drive body elongation (Costa, 1998 and references).

Mutants defective in morphogenesis have been isolated that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins alpha-catenin, beta-catenin/Armadillo, and classical cadherin, respectively. This putative catenin-cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization (Costa, 1998).

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).

In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Two pathways that control vulval precursor cell fate converge on the Hox gene lin-39: the Ras pathway, functioning downstream of the LET-23 Epidermal growth factor receptor and the Wnt pathway. LIN-39 is an Antennapedia class homeodomain, most similar to those of the Drosophila homeotic genes Deformed and Sex combs reduced. The Wnt signal is transduced through a pathway involving the beta-catenin homolog BAR-1 and controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. These genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target, LIN-39 Hox (Eisenmann, 1998).

ß-Catenins function both in cell adhesion as part of the cadherin/catenin complex and in Wnt signal transduction as transcription factors. Vertebrates express two related proteins, ß-catenin and plakoglobin, while Drosophila has a single family member, Armadillo. C. elegans expresses three ß-catenin-related proteins, BAR-1, HMP-2, and WRM-1, which are quite diverged in sequence from one another and other ß-catenins. While BAR-1 and WRM-1 are known to act in Wnt-mediated processes, and HMP-2 acts in a complex with cadherin/alpha-catenin homologs, it is unclear whether all three proteins retain the other functions of ß-catenin. This study shows that BAR-1, like vertebrate ß-catenin, has redundant transcription activation domains in its amino- and carboxyl-terminal regions but that HMP-2 and WRM-1 also possess the ability to activate transcription. Yeast two-hybrid analysis shows that these three proteins display distinct patterns of protein interactions. Surprisingly, both WRM-1 and HMP-2 can substitute for BAR-1 in C. elegans when expressed from the bar-1 promoter. Therefore, although their mutant phenotypes and protein interaction patterns strongly suggest that the functions of ß-catenin in other species have been segregated among three diverged proteins in C. elegans, these proteins still retain sufficient similarity to display functional redundancy in vivo (Natarajan, 2001).

Re-programming of C. elegans male epidermal precursor fates by Wnt, Hox, and LIN-12/Notch activities

In Caenorhabditis elegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9-11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1°, 2°, or 3°), while eight anterior cells, P(1-8).p, fuse with the hyp7 epidermal syncytium. This study shows that activation of the canonical BAR-1 β-catenin pathway of Wnt signaling alters the competence of P(3-8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1-8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-12 or EGF signaling. While increased Wnt signaling induces predominantly 1° HCG fates, increased LIN-12 or EGF signaling in combination with MAB-5 overexpression promotes 2° HCG fates in anterior Pn.p cells, suggesting distinctive functions of Wnt, LIN-12, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9-11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development (Yu, 2010).

Overall, vulval precursor cell (VPC) and HCG patterning are quite similar: the precise cell fate is generated by progressive restriction through competence, induction, and lateral inhibition mediated by multiple signal integration at different steps, representing a general scenario of complex pattern formation (Yu, 2010).

Specifically, both VPC and HCG competence are established by Wnt signaling and one of the two Hox genes, lin-39 and mab-5, respectively. Expression patterns of both Hox genes are the same in both hermaphrodite and male, with lin-39 expression in P(3-8).p and mab-5 expression in P(7-11).p. However, sex-specific utilization of these two Hox genes, lin-39 and mab-5, determines whether a hermaphrodite vulva or a male hook, respectively, is formed. In hermaphrodites, lin-39 function is favored in the central Pn.p cells, and the ability of mab-5 to prevent P(9-11).p fusion with hyp7 is somehow blocked. As a transcription factor, mab-5 regulates target gene expression. One possibility is that a negative regulator in hermaphrodites sequesters mab-5 from its targets. Alternatively, mab-5 may act with a co-regulator that is missing in hermaphrodites. The Hox genes appear to play a permissive role in VPC and HCG induction because neither multi-vulvae nor multi-hooks are observed when lin-39 or mab-5, respectively, are overexpressed (Yu, 2010).

A major difference between VPC and HCG development is the major inductive signal used to specify the 1° fate: the EGF pathway induces the 1° VPC fate while Wnt signaling promotes the 1° HCG fate. However, both EGF and Wnt act to induce HCG as well as VPC fates, and it has been observed that excessive Wnt signaling can at least partially substitute for EGF signaling in VPC induction and vice versa in HCG specification. The local abundance of the signal could explain why different inductive signals are utilized in VPC and HCG patterning. The availability of the Wnt and EGF inductive signals differ spatially in hermaphrodites and males, contributing further to the sex-specific bias of Hox gene expression. Although Wnts are present in the central region of the body and the EGF ligand is produced in the tail, the EGF signal emanates from a concentrated source, the gonadal anchor cell, only in the hermaphrodite, while Wnt signaling is more abundant in the tail region as elucidated by extensive tail defects caused by deficient Wnt signaling. As such, only the required Hox gene is promoted in each region in a sex-specific manner -- for example, lin-39 activity in males is not reinforced due to lack of a strong extrinsic signal in the central region. Therefore, different signaling pathways may not be the direct cause of sexually dimorphic organogenesis. The specificity of signaling relies on Hox genes to direct sex-specific pattern formation among competent precursor cells (Yu, 2010).

#946;-Catenin asymmetries after all animal/vegetal- oriented cell divisions in Platynereis dumerilii embryos mediate binary cell-fate specification

In response to Wnt signaling during animal development, β-catenin accumulates in nuclei to mediate the transcriptional activation of target genes. A highly conserved β-catenin in the annelid Platynereis dumerilii exhibits a reiterative, nearly universal embryonic pattern of nuclear accumulation remarkably similar to that observed in the nematode C. elegans. Platynereis exhibits β-catenin sister-cell asymmetries after all cell divisions that occur along the animal/vegetal axis beginning early in embryogenesis, but not after two transverse divisions that establish bilateral symmetry in the trunk. Moreover, ectopic activation of nuclear β-catenin accumulation in Platynereis causes animal-pole sister cells, which normally have low nuclear β-catenin levels, to adopt the fate of their vegetal-pole sisters, which normally have high nuclear β-catenin levels. The presence of reiterative and functionally important β-catenin asymmetries in two distantly related animal phyla suggests an ancient metazoan origin of a β-catenin-mediated binary cell-fate specification module (Schneider, 2007).

While sharing some similarities with other metazoan embryos, the animal/vegetal β-catenin asymmetries in Platynereis are strikingly reminiscent of the anterior-posterior asymmetries of β-catenin activation that control cell-fate specification throughout development in C. elegans. In both species, the animal pole of the early embryo (marked by two polar bodies) produces anterior structures, while the vegetal pole produces posterior structures and endoderm, suggesting a homology of animal/vegetal sister-cell pairs in Platynereis with the anterior-posterior sister-cell pairs in C. elegans. Increased levels of β-catenin specify vegetal-/posterior-pole sister-cell fates, while lower levels specify animal-/anterior-pole sister-cell fates, operating as a binary specification module throughout embryogenesis in both species. Furthermore, an absence of β-catenin asymmetry is observed in both species after bilaterally symmetrical and transverse cell divisions, and after the first two cell divisions. Thus, the overall pattern of β-catenin activation is extraordinarily similar in both species. Finally, while β-catenin-mediated binary specification modules have thus far been described only in Platynereis and C. elegans, the animal/vegetal-oriented cell divisions of some blastomeres in sea urchin embryos produce animal-pole sister cells with low levels of nuclear β-catenin and vegetal-pole sisters with high levels, apparently by using a cell-autonomous mechanism (Schneider, 2007).

There also are differences in the binary cell-fate specification modules that operate in C. elegans and Platynereis. In C. elegans, two highly derived β-catenins (WRM-1 and SYS-1) cooperate to activate target gene transcription in posterior cells. Enrichment of WRM-1 in posterior nuclei promotes the export of most of the TCF homolog POP-1, while increased nuclear levels of SYS-1 in posterior nuclei convert the remaining POP-1 repressor into a transcriptional activator. In contrast, Platynereis binary cell-fate specification uses a highly conserved β-catenin that is negatively regulated by GSK-3β, as in canonical Wnt signaling, while GSK-3 acts positively in the noncanonical C. elegans Wnt pathways. Furthermore, equal segregation of β-catenin to mitotic spindle poles, followed by an increase in nuclear β-catenin levels in vegetal-pole sister cells was found. In C. elegans, SYS-1, but not WRM-1, becomes enriched at both spindle poles, while both WRM-1 and SYS-1 become enriched in posterior nuclei. The similarities between both species suggest a monophyletic protostome origin of binary cell-fate specification, while the distinct molecular mechanisms that operate in each species perhaps favor independent and convergent evolutionary origins (Schneider, 2007).

At fourth cleavage of sea urchin embryos four micromeres at the vegetal pole separate from four macromeres just above them in an unequal cleavage. The micromeres have the capacity to induce a second axis if transplanted to the animal pole and the absence of micromeres at the vegetal pole results in the failure of macromere progeny to specify secondary mesenchyme cells (SMCs). This suggests that micromeres have the capacity to induce SMCs. Micromeres require nuclear beta-catenin to exhibit SMC induction activity. Transplantation studies show that much of the vegetal hemisphere is competent to receive the induction signal. The micromeres induce SMCs, most likely through direct contact with macromere progeny, or at most a cell diameter away. The induction is quantitative in that more SMCs are induced by four micromeres than by one. Temporal studies show that the induction signal is passed from the micromeres to macromere progeny between the eighth and tenth cleavage. If micromeres are removed from hosts at the fourth cleavage, SMC induction in hosts is rescued if they later receive transplanted micromeres between the eighth and tenth cleavage. After the tenth cleavage, addition of induction-competent micromeres to micromereless embryos fails to specify SMCs. For macromere progeny to be competent to receive the micromere induction signal, beta-catenin must enter macromere nuclei. The macromere progeny receive the micromere induction signal through the Notch receptor. Signaling-competent micromeres fail to induce SMCs if macromeres express dominant-negative Notch. Expression of an activated Notch construct in macromeres rescues SMC specification in the absence of induction-competent micromeres. These data are consistent with a model whereby beta-catenin enters the nuclei of micromeres and, as a consequence, the micromeres produce an inductive ligand. Between the eighth and tenth cleavage micromeres induce SMCs through Notch. In order to be receptive to the micromere inductive signal, the macromeres first must transport beta-catenin to their nuclei, and as one consequence the Notch pathway becomes competent to receive the micromere induction signal, and to transduce that signal. Since Notch is maternally expressed in macromeres, additional components must be downstream of nuclear beta-catenin in macromeres for these cells to receive and transduce the micromere induction signal (McClay, 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. 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).

The key steps for specification of endomesoderm can be summarized as follows:

(1) Initial specification of the veg2 domain. The experimental evidence indicates that, under normal conditions, two inputs are required for the specification of veg2 as a field of cells that will execute endomesodermal fates. The first of these is an intercellular signal passed from the micromeres to the adjacent cells, the grandparents and parents of the sixth cleavage veg2 ring. This very early signaling function implies that, at least in some measure, the micromeres are already specified when they are born at fourth cleavage. The second input required for specification of the veg2 lineage is the nuclearization of ß-catenin, a cofactor of the Tcf transcription regulator that is required for it to function as a gene activator. This takes place by a cell-autonomous mechanism for which intercellular contact is not necessary: remarkably, every cell, the progeny of which will express an endodermal or a mesodermal fate, displays elevated nuclear ß-catenin at seventh cleavage, compared to any other cells in the embryo. Furthermore, interference with the ß-catenin nuclearization process by any of several different means completely cancels endomesoderm specification (Davidson, 2002).

(2) The endomesodermal Wnt8 loop. A gene encoding 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).

(3) The micromere Delta signal. During the seventh to ninth cleavage interval, a second signal is transmitted from the micromeres to the adjacent surrounding cells, i.e., now the inner ring of veg2 lineage blastomeres. The result is the specification of these cells as mesodermal precursors. The signaling ligand is Delta, which activates the Notch (N) receptor. In response, the N receptor is activated specifically in the progenitors of the future veg2 mesoderm (i.e., the mesoderm formed from progeny of the 8 sixth-cleavage veg2 cells). This event is specifically required for veg2 mesodermal specification (Davidson, 2002).

(4) Late specification of veg1 endoderm. After midblastula stage, the elevated level of nuclear ß-catenin progressively disappears from the micromere and veg2 progeny, but at late blastula stage (after 24 h), ß-catenin reappears in the nuclei of a ring of cells just outside the veg2 domain. Thereupon, these veg1 progeny begin to express endodermal markers, such as the endo16 gene, the evenskipped gene, and the krox1 gene. These veg1 progeny invaginate and will contribute mainly to the hindgut (Davidson, 2002).

This work suggests that regulatory genes carrying out several different classes of function are likely to be required for endomesoderm specification. These include genes required for micromere functions, genes required for endomesodermal specification that are dependent for activation on the Tcf system, mesodermal genes that are activated downstream of the N system, regulatory genes required for endoderm or for mesoderm cell type specification, and also batteries of downstream genes that encode skeletogenic, mesodermal, and endodermal differentiation products (Davidson, 2002).

Micromeres and their immediate descendants have three known developmental functions in regularly developing sea urchins: immediately after their initial segregation, they are the source of an unidentified signal to the adjacent veg2 cells that is required for normal endomesodermal specification; a few cleavages later, they express Delta, a Notch ligand which triggers the conditional specification of the central mesodermal domain of the vegetal plate; and micromeres exclusively give rise to the skeletogenic mesenchyme of the postgastrular embryo. This paper demonstrates the key components of the zygotic regulatory gene network that accounts for micromere specificity. This network is a subelement of the overall endomesoderm specification network of the Strongylocentrotus purpuratus embryo. A central role is played by a newly discovered gene encoding a paired class homeodomain transcription factor which in micromeres acts as a repressor of a repressor: the gene is named pmar1 (paired-class micromere anti-repressor). pmar1 is expressed only during cleavage and early blastula stages, and exclusively in micromeres. It is initially activated as soon as the micromeres are formed, in response to Otx and ß-Catenin/Tcf inputs. The repressive nature of the interactions mediated by the pmar1 gene product was shown by the identical effect of introducing mRNA encoding the Pmar1 factor, and mRNA encoding an Engrailed-Pmar1 (En-Pmar1) repressor domain fusion. In both cases, the effects are derepression of the delta gene and of skeletogenic genes, including several transcription factors normally expressed only in micromere descendants. Deprepression also occurs in a set of downstream skeletogenic differentiation genes. The spatial phenotype of embryos bearing exogenous mRNA encoding Pmar1 factor or En-Pmar1 is expansion of the domains of expression of the downstream genes over most or all of the embryo. This results in transformation of much of the embryo into skeletogenic mesenchyme cells that express skeletogenic markers. The normal role of pmarl is to prevent, exclusively in the micromeres, the expression of a repressor that is otherwise operative throughout the embryo. This function accounts for the localization of delta transcription in micromeres, and thereby for the conditional specification of the vegetal plate mesoderm. It also explains why skeletogenic differentiation gene batteries normally function only in micromere descendants. More generally, the regulatory network subelement emerging from this work shows how the specificity of micromere function depends on continuing global regulatory interactions, as well as on early localized inputs (Oliveri, 2002).

ß-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, ß-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. In-vivo measurements of the half-life of fluorescently tagged ß-catenin in specific blastomeres has been used to demonstrate a gradient in ß-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3ß-mediated phosphorylation of ß-catenin. Calculations show that the difference in ß-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. Dishevelled (Dsh), a key signaling protein, is required for the stabilization of ß-catenin in vegetal cells; evidence is provided that Dsh undergoes a local activation in the vegetal region of the embryo. GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus (Weitzel, 2004).

Patterning of cell fates along the sea urchin animal-vegetal embryonic axis requires the opposing functions of nuclear ß-catenin/TCF-Lef, which activates the endomesoderm gene regulatory network, and SoxB1, which antagonizes ß-catenin and limits its range of function. A crucial aspect of this interaction is the temporally controlled downregulation of SoxB1, first in micromeres and then in macromere progeny. SoxB1 is regulated at the level of protein turnover in these lineages. This mechanism is dependent on nuclear ß-catenin function. It can be activated by Pmar1, but not by Krl, both of which function downstream of ß-catenin/TCF-Lef. At least partially distinct, lineage-specific mechanisms operate, since turnover in the macromeres depends on entry of SoxB1 into nuclei, and on redundant destruction signals, neither of which is required in micromeres. Neither of these turnover mechanisms operates in mesomere progeny, which give rise to ectoderm. However, in mesomeres, SoxB1 appears to be subject to negative autoregulation that helps to maintain tight regulation of SoxB1 mRNA levels in presumptive ectoderm. Between the seventh and tenth cleavage stages, ß-catenin not only promotes degradation of SoxB1, but also suppresses accumulation of its message in macromere-derived blastomeres. Collectively, these different mechanisms work to regulate precisely the levels of SoxB1 in the progeny of different tiers of blastomeres arrayed along the animal-vegetal axis (Angerer, 2005).

Inderstanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the ascidian mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal β-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ciona friend of GATA gene (Ci-fog) is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. This study has identified a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program (Rothbacher, 2007).

Wnt pathway, ß-catenin and dorsal axis formation in zebrafish

In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. A novel maternal-effect recessive mutation, tokkaebi (tkk), has been identified that influences formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5%-100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos display defects in the nuclear accumulation of ß-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly changed. Injection of RNAs for activated ß-catenin, dominant-negative forms of Axin1 and GSK3ß, and wild-type Dvl3, into the tkk embryos suppresses the ventralized phenotypes and/or dorsalizes the embryos, and restores or induces an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs is disturbed in the tkk embryos. Inhibition of cytoplasmic calcium release elicits an ectopic and expanded expression of chordin in the wild-type, but does not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the ß-catenin-degradation machinery to control the stability of ß-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of ß-catenin is involved in dorsal axis formation in zebrafish (Nojima, 2004).

Using embryos transgenic for the TOP-GFP reporter, this study shows that the two zebrafish β-catenins have different roles in the organizer and germ-ring regions of the embryo. β-Catenin-activated transcription in the prospective organizer region specifically requires β-catenin-2, whereas the ventrolateral domain of activated transcription is abolished only when both β-catenins are inhibited. chordin expression during zebrafish gastrulation has been shown in both axial and paraxial domains, but is excluded from ventrolateral domains. This gene is expressed in paraxial territories adjacent to the domain of ventrolateral β-catenin-activated transcription, with only slight overlap, consistent with the now well-known inhibitory effects of Wnt8 on dorsal gene expression. Eliminating both Wnt8/β-catenin signaling and organizer activity by inhibition of expression of the two β-catenins results in massive ectopic circumferential expression of chordin and later, by formation of a distinctive embryonic phenotype ('ciuffo') that expresses trunk and anterior neural markers with correct relative anteroposterior patterning. chordin expression is required for this neural gene expression. The Nodal gene squint has been shown to be necessary for optimal expression of chordin and is sufficient in some contexts for its expression. However, chordin is not normally expressed in the ventrolateral germ-ring despite robust expression of squint in this domain. The ectopic circumferential expression of chordin and other dorsal genes is completely dependent on Nodal and FGF signaling, and is independent of a functional organizer. It is proposed that whereas the axial domain of chordin expression is formed by cells that are derived from the organizer, the paraxial domain is the result of axial-derived anti-Wnt signals, which relieve the repression that otherwise is set by the Wnt8/β-catenin/vox,vent pathway on latent germ-ring Nodal/FGF-activated expression (Varga, 2007).

Scribble controls clustering of β-catenin foci in dividing zebrafish neural progenitors

How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. This study shows that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. This study shows that Scribble is required for oriented cell division, and its function in this process is independent of canonical apicobasal and planar polarity pathways. A role is identified for Scribble in controlling clustering of β-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. It is proposed that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, the data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis (Žigman, 2011).

Function of ß-catenin, the Armadillo homolog in Xenopus

Spemann and Mangold first proposed in 1924 that a small region of the amphibian embryo could induce neighboring cells of the host embryo to form a secondary body axis. Spemann called this region located above the dorsal blastopore lip, "the organizer," to indicate that this small group of cells is able to determine completely the initial fate of the embryo. Nieuwkoop demonstrated that the vegetal hemisphere is able to signal the overlying animal hemisphere to convert, by induction, prospective ectodermal cells to various mesodermal lineages. The dorsalizing region, called the Nieuwkoop center, functions to induce the overlying mesodermal cells to become the Spemann Organizer. These two signaling centers can be distinguished according to two criteria: (1) the activity of the Nieuwkoop Center is maximal in the lower vegetal region, while the activity of the Spemann Organizer is restricted to the equatorial region, and (2) the cells of the Spemann Organizer themselves become part of the induced dorsal mesoderm, independently of their origin, while cells expressing Nieuwkoop Center-like activities are not recruited into axial structures but maintain their original fates (endoderm in the case of the vegetal pole). Another intracellular molecule with strong dorsalizing activity is Siamois, a novel Hox-type transcription factor expressed in the region of the Nieuwkoop Center. Its potent activity, its transient expression at the beginning of the midblastula transition before goosecoid (See Drosophila Goosecoid) or Xbrachyury (See Drosophila T-related gene) and Xlim-1 (See Drosophila Apterous) are expressed in the Organizer related marginal zone (presumptive mesoderm and some endoderm), strongly suggest that Siamois is an important component of the Nieuwkoop Center. It is suggested that the role of Siamois in dorsal-vegetal cells is to activate genes encoding diffusible factors that mediate the induction of the Spemann organizer in overlying mesoderm. It is clear that other dorsalizing proteins such as Vg1, Activin and Noggin are not the primary dorsalizing agents of the Nieuwkoop Center, but that the WNT pathway including ß-catenin (Drosophila homolog: Armadillo) serves this function (Fagotto, 1997, Kimelman, 1992, Lemaire, 1995 and references).

In Xenopus embryos, establishment of the dorsal-ventral axis can be traced to the post-fertilization cortical rotation and the subsequent activation of transplantable dorsal-determining information by the time the 8-16 cell stage has been reached. The initial indication that activation of a single signaling pathway is sufficient to trigger formation of a new dorsal axis came from the observation that ectopic expression of Wnt1 promotes duplication of this axis. Supporting the idea that the WNT1 pathway is normally involved in axis specification, transcripts encoding the known components of this pathway are present in Xenopus eggs, including beta-catenin, dishevelled (See Drosophila Dishevelled), GSK3 (See Drosophila Shaggy) and homologs of Drosophila Frizzled. Additionally, their ectopic expression (or inhibition, in the case of GSK3) elicits the expected duplication of the axis (Moon, 1997).

Although Xwnt8b mRNA is present maternally and is the only maternal Xwnt that induces a complete ectopic axis, it might not be required for axis formation, even if it normally participates in this process. This is supported by data showing that expression in fertilized Xenopus eggs of a dominant-negative WNT or a dominant-negative Dishevelled blocks formation of ectopic, but not endogenous, axes. However, ß-catenin is clearly necessary for the formation of the endogenous axis, as depletion of beta-catenin transcripts blocks formation of the endognous axis. ß-catenin promotes axis formation through interaction with the HMG-box transcription factor XTCF3 (Drosophila homolog: Pangolin), resulting in translocation of ß-catenin-XTCF3 complexes into the nucleus. This leads to induction of specific regulatory genes, such as the homeobox gene siamois and others, that are involved in axis formation (Moon, 1997).

Endogenous ß-catenin is enriched in the dorsal cytoplasm by the end of the first cell cycle, with further accumulation in nuclei of dorsal, but not ventral, blastomeres by the 16-cell stage. Remaining questions for investigation include determining whether fertilization of Xenopus activates this dorsal accumulation of ß-catenin in a WNT, or other ligand-dependent or -independent manner, and how the WNT pathway might interact with other maternal signaling pathways, such as Vg1 to initiate gene expression leading to formation of the Spemann gastrula organizer (Moon, 1997 and references).

Plakoglobin interacts with both classical and desmosomal cadherins (See Drosophila Shotgun). It is closely related to Drosophila Armadillo. In Xenopus, WNTs (mammalian homologs of Drosophila Wingless) can produce anterior axis duplications by inducing dorsal mesoderm. To test whether simply increasing the level of plakoglobin mimics the effects of exogenous WNTs in Xenopus, fertilized eggs were injected with RNA encoding plakoglobin; this induces both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein is coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin are suppressed. Mutational analysis indicates that the central armadillo repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm (Karnovsky, 1995).

Plakoglobin is one of two vertebrate proteins closely related to the Drosophila segment polarity gene product Armadillo. Overexpression of plakoglobin induces neural axis duplication in Xenopus and the exogenous plakoglobin is localized to nuclei. A series of experiments has been carried out to test whether the nuclear localization of plakoglobin is required for its inductive effects. Prior to the midblastula transition exogenous plakoglobin is cytoplasmic and concentrated in the cortical regions of blastomeres; after the midblastula transition exogenous plakoglobin accumulates in embryonic nuclei. The addition of a "nuclear localization sequence" does not change the timing of plakoglobin's nuclear localization, suggesting that it is anchored in the cytoplasm prior to the midblastula transition. Next, two "membrane-anchored" forms of plakoglobin were constructed. These are exclusively cytoplasmic; yet both are as effective at producing a "Wnt-like" axis duplication as were "free," unfettered forms of plakoglobin. Moreover, expression of anchored plakoglobins have no apparent effect on the cytoplasmic or nuclear levels of beta-catenin. These data indicate that plakoglobin can act cytoplasmically to generate a WNT-like phenotype. Taken together with the ventralizing effects of a mutant from of the XTcf-3 transcription factor, it is speculated that in the early Xenopus embryo, activation of plakoglobin (or beta-catenin) inhibits rather than activates the activity of XTcf-3 or a XTcf-3-like factor. The experimental result that necessitates this conclusion is the ability of cytoplasmically anchored plakoglobins to induce a WNT-like phenotype. Such cytoplasmically anchored plakoglobin is thought to be unavailable for translocation to the nucleus (Merriam, 1997).

The Xenopus egg contains maternal dorsal determinants that are specifically located at the vegetal cortex. To study physical and functional properties of the dorsal determinants, advantage was taken of the animal-vegetal reversed embryo, produced by inversion of the fertilized egg. This results in formation of ectoderm and endoderm from the unpigmented and the pigmented halves, respectively. Cytoplasmic transplantation demonstrates that the dorsal activity is specifically localized to the unpigmented cortical cytoplasm of the inverted egg, which is segregated into the future ectodermal lineage. This result suggests that the dorsal determinants are associated with the unpigmented cortex and are not dislodged by the inversion. It was found that two vegetally localized transcripts, Xcat2 and Vg1 mRNAs, are present in the reversed animal pole of the inverted egg, suggesting their association with the unpigmented cortex. In order to compare the dorsal determinant activity with known dorsalizing molecules, the expression pattern of Xnr3 and Siamois were examined in the reversed embryo because these two genes are activated by the Wnt-pathway activators (Xwnt-8, beta-catenin, etc.) but not by other dorsalizing molecules (noggin, BVg1, etc.). Animal cap of the reversed embryo, which receives the unpigmented cortex of the egg, expresses Xnr3 and Siamois. However, Mix.1, a marker expressed in endoderm and mesoderm in the normal embryo in response to mesodermal inducers, is not detected in the animal cap of the reversed embryo. Beta-catenin protein accumulates in the nuclei of unpigmented animal pole cells of the reversed embryo. Thus dorsal determinants in the Xenopus egg are firmly associated with the vegetal cortex and behave like activators of the Wnt pathway (Marikawa, 1997).

The maternal dorsal determinants required for the specification of the dorsal territories of Xenopus early gastrulae are located at the vegetal pole of unfertilized eggs and are moved towards the prospective dorsal region of the fertilized egg during cortical rotation. While the molecular identity of the determinants is unknown, there are dorsal factors in the vegetal cortical cytoplasm (VCC). When injected into animal cells, the VCC factors (which establish the VCC/Wnt/beta-catenin pathway) activate the zygotic genes Siamois and Xenopus nodal-related 3 (Xnr3), suggesting that these genes act along this pathway. Siamois is a homoeodomain protein and Xnr3 is a divergent member of the TGF beta superfamily. Siamois and Xnr3 are activated at the vegetal pole of UV-irradiated embryos, indicating that these two genes are targets of the VCC factors in all embryonic cells. However, the consequences of their activation in cells that occupy different positions along the animal-vegetal axis differ. Dorsal vegetal cells of normal embryos or VCC-treated injected animal cells are able to dorsalize ventral mesoderm in conjugate experiments, but UV-treated vegetal caps do not have this property. This difference is unlikely to reflect different levels of activation of FGF or activin-like signal transduction pathways but may reflect the activation of different targets of Siamois. Chordin, a marker of the head and axial mesoderm, is activated by the VCC/Siamois pathway in animal cells but not in vegetal cells whereas cerberus, a marker of the anterior mesendoderm that lacks dorsalising activity, can only be activated by the VCC/Siamois pathway in vegetal cells. It is proposed that the regionalization of the organizer during gastrulation proceeds from the differential interpretation along the animal-vegetal axis of the activation of the VCC/beta-catenin/Siamois pathway (Darras, 1997).

Gap junctional communication (GJC) is regulated in the early Xenopus embryo and quantitative differences in junctional communication correlate with the specification of the dorsal-ventral axis. To address the mechanism that is responsible for regulating this differential communication, the function of beta-catenin was investigated during the formation of the dorsal-ventral axis in Xenopus embryos by blocking its synthesis with antisense oligodeoxynucleotides. This method reduces the level of beta-catenin in the early embryo, prior to zygotic transcription, and inhibits the formation of the dorsal axis. Antisense inhibition of beta-catenin synthesis also reduces GJC among cells in the dorsal hemisphere of 32-cell embryos to levels similar to those observed among ventral cells. Full-length beta-catenin mRNA can restore elevated levels of dorsal GJC when injected into beta-catenin-deficient oocytes, demonstrating the specificity of the beta-catenin depletion with the antisense oligonucleotides. Thus, endogenous beta-catenin is required for the observed differential GJC. This regulation of GJC is the earliest known action of the dorsal regulator, beta-catenin, in Xenopus development. Two lines of evidence indicate that beta-catenin acts within the cytoplasm to regulate GJC, rather than through an effect on cell adhesion: (1) when EP-cadherin is overexpressed and increased adhesion is observed, embryos display both a ventralized phenotype and reduced dye transfer, and (2) a truncated form of beta-catenin (i.e., the ARM region), that lacks the cadherin-binding domain, restores dorsal GJC to beta-catenin-depleted embryos. Thus, beta-catenin appears to regulate GJC, independent of its role in cell-cell adhesion, by acting within the cytoplasm through a signaling mechanism (Krufka, 1998).

The Armadillo family is formed by proteins that possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat. This domain was initially described for the Drosophila segment polarity gene product Armadillo. For the family members Armadillo, beta-catenin and plakoglobin, the Arm domain serves in protein-protein interactions to mediate required cell-cell adhesion and Wnt/Wingless signaling. Similarily, the Arm domain containing src substrate, p120cas, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function for p120cas in either adhesion or signaling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos it was shown by expression of murine p120cas1B (mp120cas1B) in ventral blastomeres that this catenin cannot replace beta-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120cas1B into dorsal blastomeres leads instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures, indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin is altered in the injected embryos it is assumed that mp120cas1B influences motility or orientation of migrating mesodermal cells (Geis, 1998).

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120ctn (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120ctn contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. p120ctn is likely to serve unique functions within the cadherin-catenin complex. For example, it does not bind alpha-catenin and thus does not link cadherins to the actin cytoskeleton in a manner similar to beta-catenin and plackoglobin. Little is known about the function of p120ctn within the cadherin complex. The role of p120ctn1A was examined in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120ctn1A, in contrast to beta-catenin, does not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor does p120ctn affect mesoderm induction. Rather, dorsal misexpression of p120ctn specifically perturbs gastrulation. Lineage tracing of cells expressing exogenous p120ctn indicates that cell movements are disrupted, while in vitro studies suggest that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120ctn are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120ctn has a role in cadherin function and that heightened expression of p120ctn interferes with appropriate cell-cell interactions necessary for morphogenesis (Paulson, 1999).

Xenopus embryos develop dorsal/ventral and anterior/posterior axes as a result of the activity of a maternal Xwnt pathway, in which beta-catenin is an essential component, acting as a transactivator of transcription of zygotic genes. However, the questions of where and when beta-catenin is required in early embryogenesis have not been addressed directly, because no loss-of-function method has been available. A novel antisense approach has been used that allows targeting of depletion of protein to individual blastomeres. When a 'morpholino' oligo (this acts, not by degrading RNA, but by preventing translation of protein) complementary to beta-catenin mRNA is injected into early embryos, it depletes beta-catenin protein effectively through the neurula stage. By targeting the oligo to different cleavage blastomeres, beta-catenin activity was blocked in different areas and at different times. Dorsal vegetal injection at the 2- and 4-cell stages blocks dorsal axis formation and at the 8-cell stage blocks head formation, while A-tier injection at the 32-cell stage causes abnormal cement gland formation. This approach shows the complex involvement of Xwnt pathways in embryonic patterning and offers a rapid method for the functional analysis of both maternal and early zygotic gene products in Xenopus (Heasman, 2000).

These results confirm that the dorsalizing Xwnt pathway is active on the dorsal side of the embryo and indicate that there is a sensitive period from the one- to the four-cell stage when oligo injection can completely interrupt signaling. From the eight-cell stage onward, anterior patterning is affected by morpholino injection. There are several possible interpretations of these data. There may be two separable pathways in the early embryo, one determining dorsoventral and posterior pattern (defined here by siamois and Xnr3) and the second regulating anterior structures (defined by Xhex and cerberus), both dependent on beta-catenin activity, but acting at different times or at different concentrations of beta-catenin. This view is supported by the recent finding that Xhex, which is expressed in animal cells of the blastula stage embryo and dorsal vegetal cells at the gastrula stage, endows vegetal cells with anterior signaling properties, independent of neural and mesoderm differentiation. Alternatively, there may be one pathway activated by beta-catenin, regulating different genes according to different nuclear concentrations or affinities of the beta-catenin/XTcf3 complex. When morpholino is injected at the eight-cell stage there may be sufficient protein translated before it acts to ensure activation of dorsoventral and tail regulating genes, but insufficient protein to activate Xhex (Heasman, 2000).

Thus blocking beta-catenin activity at the 2 and 4 cell stages prevents the formation of neural tissue and prevents the expression of the head marker Xhex. In contrast, blocking its activity in animal cap cells at the 32 cell stage (A-tier progeny) enhances neural tissue formation and also Xhex expression. The simplest explanation of this is that the effect of the 32-cell stage injection is on a different pathway also dependent on beta-catenin, responsible for downregulating otx, XAG1, Xnrp1, and Xnr3 and upregulating Xhex. It will be of interest to determine whether XTcf3 or zygotic LEF1 interacts with beta-catenin in this repressive pathway. These results are in agreement with another study in which overexpression of the negative regulator of Xwnt pathways, GSK3beta, causes ectopic cement gland formation. Since Xnr3 is known to be regulated by XTcf3, the downregulation of Xnr3 by the loss of beta-catenin in animal cells is consistent with a role for XTcf3 in this pathway. The results are not in agreement with overexpression studies in animal cells, in which Xnr3 has been shown to cause neural induction. The results shown here suggest the opposite; that this Xdsh/GSK/beta-catenin pathway actively suppresses neural tissue formation (Heasman, 2000). \

A growing body of work indicates that neural induction may be initiated prior to the establishment of the gastrula mesodermal organizer. Here, neural induction has been examined in Xenopus embryos in which mesoderm induction has been blocked by Cerberus-short, a reagent that specifically inhibits Nodal-related (Xnr) signals. Extensive neural structures with cyclopic eyes and brain tissue are formed despite the absence of mesoderm. This neural induction correlates with the expression of chordin and other BMP inhibitors (such as noggin, follistatin, and Xnr3) at the blastula stage, and requires beta-Catenin signaling. Activation of the beta-Catenin pathway by mRNA microinjections or by treatment with LiCl leads to differentiation of neurons, as well as neural crest, in ectodermal explants. Xnr signals are required for the maintenance, but not for the initiation, of BMP antagonist expression. Recent work has demonstrated a role for beta-Catenin signaling in neural induction mediated by the transcriptional down-regulation of BMP-4 expression. The present results suggest an additional function for beta-Catenin, the early activation of expression of secreted BMP antagonists, such as Chordin, in a preorganizer region in the dorsal side of the Xenopus blastula (Wessely, 2001).

In Xenopus, axis development is initiated by dorsally elevated levels of cytoplasmic ß-catenin, an intracellular factor regulated by GSK3 kinase activity. Upon fertilization, factors that increase ß-catenin stability are translocated to the prospective dorsal side of the embryo in a microtubule-dependent process. However, neither the identity of these factors nor the mechanism of their movement is understood. The GSK3 inhibitory protein GBP/Frat is shown to bind kinesin light chain (KLC), a component of the microtubule motor kinesin. Upon egg activation, GBP-GFP and KLC-GFP form particles and exhibit directed translocation. KLC, through a previously uncharacterized conserved domain, binds a region of GBP that is required for GBP translocation and for GSK3 binding, and competes with GSK3 for GBP. A model is proposed in which conventional kinesin transports a GBP-containing complex to the future dorsal side, where GBP dissociates and contributes to the local stabilization of ß-catenin by binding and inhibiting GSK3 (Weaver, 2003).

The formation of the dorsoanterior axis in Xenopus is dependent upon a series of events that occur during the first cell cycle after fertilization. Sperm entry initiates a rotation of the peripheral layer of the egg, called the cortex, relative to the inner core cytoplasm. This event, called cortical rotation, results in a 30° displacement of the vegetal cortex toward the future dorsoanterior region. Cortical rotation coincides with the translocation of a 'dorsalizing activity' that also moves from the vegetal pole up toward the prospective dorsal side of the embryo. Translocation of the dorsalizing activity is both necessary and sufficient for the formation of the Spemann organizer, which regulates the formation of the embryonic axes during the gastrula stages (Weaver, 2003 and references therein).

Although the molecular identity of the dorsal determinants is not clear, it is known that their translocation leads to the dorsal accumulation of ß-catenin, which then activates the expression of dorsal organizer genes at the onset of zygotic transcription. Cytoplasmic transplant experiments using ß-catenin-depleted embryos have shown that ß-catenin is not the endogenous dorsalizing activity, but that instead this activity probably consists of proteins involved in ß-catenin stabilization. ß-catenin is normally phosphorylated by the serine-threonine kinase glycogen synthase kinase 3 (GSK3) within a protein complex that also includes Axin and the adenomatous polyposis coli gene product (APC), and this phosphorylation targets ß-catenin for degradation by the ubiquitin-proteosome pathway. Work from many laboratories has led to a model in which the localized inhibition of GSK3 in the dorsal region causes the dorsal accumulation of ß-catenin. How GSK3 becomes locally inhibited by the dorsal determinants, however, is still an open question (Weaver, 2003).

A strong candidate component of the translocating dorsalizing activity is GBP, a vertebrate-specific GSK3-binding protein. Depletion of endogenous GBP from the embryo with antisense oligonucleotides causes a loss of dorsal axial structures, showing that GBP is required for dorsal axis formation. GBP inhibits GSK3 activity by preventing its binding to Axin, thus preventing GSK3 from phosphorylating ß-catenin. When microinjected ventrally, GBP mimics the endogenous dorsal signal and induces the formation of a secondary dorsal axis, and overexpression of GBP also leads to GSK3 degradation in the cortical shear zone. In addition to binding GSK3, GBP also binds directly to Dsh, a positive effector of the canonical Wnt signaling pathway. Together, these two proteins potently synergize to stabilize ß-catenin (Weaver, 2003).

Thus, both GBP and its binding partner Dsh have characteristics that strongly suggest that they are part of the endogenous dorsalizing activity. Furthermore, Dsh-GFP has been shown to form particles in the shear zone that exhibit directed movement on microtubules, and endogenous Dsh accumulates dorsally by the end of cortical rotation. However, no direct molecular link has yet been established between either GBP or Dsh and the microtubule array. In this study, it has been demonstrated that GBP binds kinesin light chain (KLC), a component of the plus end-directed microtubule motor kinesin. Like Dsh, GBP-GFP and KLC-GFP form particles that exhibit fast, directional translocation in the shear zone during the period of cortical rotation. These results suggest a model in which GBP acts initially as a link between the transport apparatus and the dorsalizing activity, and subsequently as an inhibitor of GSK3 in the ß-catenin degradation complex (Weaver, 2003).

Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-ß factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localized throughout the vegetal pole, and ß-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, a minimal promoter has been defined that mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional ß-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that ß-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a ß-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. A model is proposed in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of ß-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of ß-catenin/XTcf-3 (Hilton, 2003).

Table of contents

armadillo continued: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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