InteractiveFly: GeneBrief

nemo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Drosophila nemo was originally identified as a tissue-specific factor required for the establishment of epithelial planar polarity (EPP) in developing ommatidia (Choi, 1994). It was subsequently shown to act downstream of Frizzled signaling in the polarity pathway (Strutt, 1997). Nemo is the founding member of a novel family of serine/threonine protein kinases most similar to MAPKs (Choi, 1994). Homologs have been identified in humans (Dres16), mice (Nemo-like kinase, Nlk) and C. elegans (Lit-1) (Banfi, 1996; Brott, 1998; Meneghini, 1999; Rocheleau, 1999; Verheyen, 2001 and references therein).

Recent studies in C. elegans and vertebrates have uncovered functional roles for Nemo homologs in several Wnt-dependent processes (Rocheleau, 1999; Meneghini, 1999; Ishitani, 1999). Biochemical evidence points to an involvement in canonical Wnt signaling. This Wnt pathway (see Drosophila Wingless) is activated when secreted Wnt ligand binds to a Frizzled-type cell surface receptor, which transduces the signal via the cytoplasmic Dishevelled protein. Wnt signaling in both vertebrates and Drosophila acts to stabilize cytoplasmic ß-catenin/Armadillo (Arm), allowing Arm to translocate to the nucleus where it interacts with a member of the TCF/LEF family of transcription factors (Drosophila homolog: Pangolin), and subsequently controls gene expression (Verheyen, 2001 and references therein).

Homologs of Nemo have roles in regulating Wnt-dependent gene expression through their affects on ß-catenin and TCF homologs. Nlk can phosphorylate TCF and inhibit DNA-binding of the ß-catenin/TCF complex, thereby inhibiting Wnt signaling (Ishitani, 1999). In addition, worm LIT-1 can bind to and phosphorylate WRM-1, a ß-catenin/Armadillo-like protein (Rocheleau, 1999). This interaction leads to phosphorylation of POP-1, and its subsequent export from the nucleus. In C. elegans, interactions between lit-1 and two distinct Wnt signaling pathways, one regulating polarity and one regulating gene expression, have been described (Rocheleau, 1999). In addition, vertebrate Nlk has also been shown to inhibit ß-catenin/Arm function in vivo. Injection of Nlk into Xenopus embryos can suppress the axis duplication phenotype induced by microinjected ß-catenin/Arm (Ishitani, 1999; Verheyen, 2001 and references therein).

Drosophila Nemo plays a role in epithelial plane polarity in the wing and abdomen suggesting it may have a more global role in polarity specification. In addition, nmo is required during wing development for vein patterning, wing shape and morphology. Genetic interactions between nmo and mutations in Wg, Notch and Dpp signaling implicate nmo as an antagonist of Wg. nmo mutations also cause numerous pleiotropic effects during development and the gene appears to be essential during embryogenesis (Verheyen, 2001).

Wings of nmo mutants are rounder and shorter than wildtype, contain extra vein material and are held away from the body at a 45° angle (Choi, 1994). A genetic second-site modifier screen was carried out to identify genes that either participate in Notch signaling or modulate cross-talk between signal transduction pathways. adirondack (adk) mutations, now known to be alleles of nmo, dominantly modify the rough eye phenotype caused by expression of activated Notch under expression of the sevenless promoter. The extra vein phenotype seen in hypomorphic nmo mutant alleles (nmo^adk) suggests that the gene product normally functions in the suppression of the vein fate. The patterning of wing veins has been well characterized and involves many genes at various stages of specification, refinement and maintenance of vein fates. The roles of the EGF receptor and Notch pathways in these processes are well-characterized. One of the earliest markers for presumptive vein cells is the rhomboid gene. rhomboid facilitates signaling via the EGF receptor and its expression is negatively regulated by Notch. To examine the development of the ectopic veins in nmo^adk, pupal wings were dissected and the pattern of Rhomboid mRNA expression was determined. In nmo^adk pupal wings ectopic rhomboid expression is seen in the regions corresponding to where ectopic veins are found in adult wings. These results suggest that nmo normally acts to inhibit rho expression (Verheyen, 2001).

The expression and localization of nmo mRNA was also examined in pupal wings, in order to characterize Nemo's role in vein development. nmo transcript is detected in the intervein regions of the wing blade and enriched in the cells juxtaposed to vein-forming cells. Consistent with the results of the Northern blot, in nmo^adk mutant wings, nmo transcript is still detected although there are regions where the transcript is greatly reduced. These regions correspond exactly to the sites of ectopic vein formation and ectopic rhomboid expression. These findings support the model of a role for nmo in suppression of vein fate, with localized loss of nmo expression leading to formation of ectopic veins (Verheyen, 2001).

Expression of UAS-Nemo in a nmo^adk background suppresses the extra veins seen emanating from the posterior cross-vein (PCV), as well as those near longitudinal veins II and V (LII and LV). Furthermore, in a wildtype background, ectopic expression in intervein cells of UAS-Nemo using 32B-Gal4 and 69B-Gal4 causes loss of posterior cross-vein tissue. These effects depended greatly on the Gal4 driver, suggesting that the precise timing of overexpression is critical to suppression of veins. Interestingly, in the anterior regions of the wing, overexpression of Nemo leads to ectopic vein formation. Ectopic expression of nmo also results in a change in the overall shape of the wing blade. nmo^adk mutant wings are much rounder and shorter than normal. In contrast, ectopic nmo causes the wing blade to elongate and become more narrow and pointed (Verheyen, 2001).

The murine Nemo homolog Nlk has been implicated in regulating Wnt signaling by repressing the Arm/ß-Catenin-TCF complex (Ishitani, 1999), a component of Wingless signaling. Whether such an interaction occurs in flies during wing vein formation was examined. The extra vein phenotypes observed in nmo mutant wings are very similar to those that have been previously described as resulting from overexpression of Armadillo and both vertebrate ß-catenin and plakoglobin in the wing. In addition, ectopic veins are produced as a result of ectopic wg and dsh expression. Constitutively active Armadillo (UAS-Arm^s10) expressed using the 1348-Gal4 driver leads to the formation of moderate ectopic veins emanating from the PCV, in addition to more severe ectopic veins along LII. These are both regions of the wing that are sensitive to nmo mutations and where similar ectopic veins are observed in nmo^adk. The phenotypes seen with overexpression of wg and arm are consistent with the theory that Nemo is a negative regulator of Wingless signaling since loss of nemo mimics extra veins seen with overexpression of arm and wg (Verheyen, 2001).

Overexpression of both wildtype mouse Lef-1 (a TCF homolog) and a constitutive repressor form of dTCF (Pangolin) results in dominant negative phenotypes. The constitutive repressor form of dTCF (UAS-dTCFDeltaN) is unable to bind Arm and represses wg-dependent gene expression. These findings suggest that expression of wildtype dTCF (UAS-dTCFwt) somehow interferes with a wingless-targeted transcription factor in a dominant negative way. Consistent with this, it is found that ectopic expression of UAS-dTCFwt using vestigial-Gal4 results in defects in the posterior wing margin, a phenotype seen with loss of wg signaling. Ectopic expression of the UAS-dTCFDeltaN using the 1348-Gal4 driver is lethal. However, homozygosity for nmo^adk is able to rescue the lethality and the flies that emerged had reduced ectopic wing veins. This finding can be interpreted by taking into account the dual roles TCF plays in the nucleus. In nmo^adk mutants, the negative regulation of endogenous dTCF may be reduced, leading to more wg-dependent signaling and the induction of extra veins similar to those seen with constitutive Arm expression. Expression of UAS-dTCFDeltaN in the nmo^adk background most likely interferes with the de-repressed endogenous dTCF and block the induction of extra veins seen in nmo^adk (Verheyen, 2001).

Since the eye defect in nmo is caused by disrupted polarity of the ommatidial clusters, it was of interest to examine whether other planar polarity processes are disrupted in nmo^adk. The polarity of the wing hairs was examined in nmo^P, nmo^adk1 and nmo^adk2 flies. Normally, each epithelial cell in the wing blade reorganizes its cytoskeleton and produces an actin spike at the distal vertex, which gives rise to the distally oriented wing hairs seen in adults. nmo^P flies do not display any noticeable polarity defects in the wing. A polarity defect is observed in nmo^adk2 wings in those regions where ectopic veins are seen. A milder, variable polarity defect is observed in nmo^adk1 homozygotes. Interestingly, polarity defects are also observed in flies overexpressing UAS-Nemo using the 1348-Gal4 driver. This finding is consistent with studies in which both overexpression and loss-of-function of fz led to polarity defects during eye and wing development. These results have been interpreted to reflect a finely balanced signaling pathway that is easily disturbed by reduced or excessive expression of its components (Verheyen, 2001 and references therein).

Expression of UAS-Nemo using the 69B-gal4 driver results in a number of abnormalities, among them severe patterning defects in the abdomen, including disrupted polarity of microchaete bristles in the tergites. Since other epithelial planar polarity (EPP) mutants also show abdominal phenotypes, it is suggested that Nemo is a part of a conserved signaling mechanism that acts in the establishment of EPP. In addition to polarity disruptions, reduced numbers of bristles and defects in the patterning of the tergites are also seen, most noticeable in the reduction of the pigment band width, with occasional loss of cuticular tissue (Verheyen, 2001).

These results and those of Choi and Benzer (1994) suggest that nmo plays a general role in establishing EPP. Through the analysis of additional alleles and ectopic expression studies, it has been determined that nmo also plays a role in polarity of wing hairs and abdominal bristles. Alterations of signaling by Fz pathway components often leads to unexpected phenotypes, such as the finding that both loss-of-function mutations and overexpression of fz or dsh result in similar phenotypes during eye development. Similarly, in the wing, over-expression of fz leads to a tissue polarity defect that is similar to the loss of function fz phenotype. The finding that both loss of nmo and overexpression of UAS-Nemo cause polarity defects in the wing is consistent with other members of the EPP pathway. The regulation of nmo expression has been shown to be dependent of fz signaling, and it is possible that nmo mutants display aberrant polarity phenotypes as a result of losing some aspect of the downstream events triggered by fz signaling. However, the possibility that Nemo is causing polarity defects through an indirect effect on the regulation of cytoskeletal reorganization cannot be excluded. It is also possible that Nemo's effect on polarity is more indirect and reflects the earlier role for Wg signaling upstream of EPP establishment (Verheyen, 2001).

The nature of the nmo^adk phenotype is somewhat complex, given that it appears stronger than the published null allele, nmo^P (Choi, 1994). It is speculated that nmo^P alleles display the zygotic null phenotype, but that the abundant maternally supplied Nemo permits development through to adult stages. nmo^adk alleles appear more severe by several criteria such as their enhanced affect on wing veins, wing blade angle, fertility and viability. The presence of truncated mRNAs provides a possible mechanism. The mRNAs may encode truncated proteins that have a dominant negative effect on the maternal Nemo and may therefore interfere with development at an earlier stage. The elucidation of the phenotype of a maternal and zygotic null should clarify this issue. It is predicted that embryos both maternally and zygotically null for nmo would be embryonic lethal (Verheyen, 2001).

The role of Nemo in the wing seems quite complex and may reflect interactions between nmo and numerous signaling pathways. The role of nmo in wing vein development is reminiscent of the roles played by the other vein-inhibiting genes such as net and plexus. Both the expression pattern of nmo and the ectopic veins seen in the mutant support a role for nmo in promoting intervein cell fates (Verheyen, 2001).

A relationship is observed between overall wing shape and wing vein patterning. nmo^adk mutants have excess veins and a much rounder, shorter wing. Such a wing shape has been seen in other excess vein genotypes, such as UAS-Argos and extreme px alleles. Overexpressed Nemo results in a longer and more pointed wing shape, in addition to vein loss in the posterior compartment. Such a wing shape is also seen in mutants that reduce vein formation such as vein; in mutants in TGFß signaling such as dpp and gbb-60A, and in flies ectopically expressing dominant negative Raf. This correlation between excess vein and rounder wing shape and loss of veins accompanied by a narrower wing suggests a link between the genes controlling vein/intervein fates and those regulating overall wing morphology. In addition, it has been shown that mutations that have severe effects on vein formation such as double mutant combinations of extra vein mutants result in a larger wing blade. It is therefore interesting that the net¹;nmo^adk2 flies have much smaller wing blades. This phenotype is most likely due to the observed vast expanse of vein tissue, which is known to have a smaller apical surface than intervein tissue (Verheyen, 2001 and references therein).

What is the relation of nemo to the Notch pathway? nemo was originally shown to play a role in planar polarity in the developing eye (Choi, 1994). nmo is required for the rotation of photoreceptor clusters in the final stages of polarity establishment. It has been proposed that, in the eye, nmo functions as a tissue-specific factor downstream of the Fz pathway. The initial step in the establishment of polarity in each ommatidium is the differentiation of the R3/R4 photoreceptors. Recent studies have shown that Notch signaling acts downstream of Fz in this process. Fz signaling leads to expression of the Delta ligand in the R3 cell and through a negative feedback loop Notch signaling is restricted to the R4 cell. In this way, Fz signaling can exert control over Notch activity, by initially biasing the ligand production to one cell type (Verheyen, 2001 and references therein).

In the wing, ectopic activation of Notch signaling can suppress the extra veins in nmo and mutations in Notch pathway components enhance the extra veins. Notch signaling is also required for vein differentiation, and genetic interactions suggest that nmo acts upstream of Notch and may participate in laying out the initial vein/intervein boundaries. The ectopic veins seen in nmo resemble those seen in other extra vein mutants, and seem to mark the boundaries of para-vein territories. Ancestors of Drosophila possess veins in these para-vein regions, but their formation has been suppressed in Drosophila (Verheyen, 2001).

The specification of wing veins has been shown to involve a number of signal transduction cascades. Roles for Egfr, Notch and Dpp signaling are well established. More recently, a role for Wg signaling in this process has been suggested. The exact molecular nature of Wg signaling in wing vein specification is unknown, but it has been shown that ectopic signaling through Wingless and Armadillo results in the formation of ectopic veins. Dpp signaling also plays a role in specifying vein fates during pupal stages. This is in contrast to the known role for Notch signaling in inhibiting vein fates. A recent study finds that Egfr signaling is regulated developmentally, such that it stimulates veins early in wing development and later inhibits vein formation. Larval expression of dominant negative Ras signaling results in loss of veins but pupal expression of the same construct resulted in ectopic veins. This finding is intriguing in light of the dual effects on vein specification seen with ectopic expression of UAS-Nemo in the wing blade. Whether this effect occurs as a direct consequence of Nemo on the Egfr pathway, through its control of rhomboid expression, or indirectly through the effect of Nemo on Notch or Wg signaling has yet to be determined. The behavior of nmo in the wing is consistent with its playing a positive role upstream of Notch signaling and acting as an antagonist of Wingless signaling, although the possibility cannot be excluded that the effect of nmo on wg may be either positive or negative depending on the developmental stage. It is possible that through repression of Wg signaling that Nemo facilitates Notch signaling. Both genetic and biochemical cross-regulation between Notch and Wg through the Dsh protein have been described (Verheyen, 2001 and references therein).

Wingless signaling has been linked both molecularly and genetically with the Notch, MAPK, Hedgehog, Dpp and Fz planar polarity signal transduction pathways. Such findings have led to the model that Wnt signaling functions as a network, rather than a linear pathway. Such a network could integrate the cross-regulation occurring between numerous developmentally regulated pathways. The network of interactions must be carefully orchestrated, since shifting the levels of any one protein can tip the balance between developmental pathways. Maintaining the equilibrium between differentially activated pools of pathway components has been shown to regulate the function of both Arm and TCF proteins thus enabling them to assume multiple roles during development. Since both proteins are also direct binding partners of the nematode Nemo homolog, this interpretation of the phenotypes induced by loss of nmo must be taken within such a context (Verheyen, 2001 and references therein).

Much has been written about the relationships between signaling pathways during imaginal disc development and patterning. Only recently has insight been gained into the cross-regulation that occurs during the differentiation of vein and intervein fates during pupal stages. It is clear that complex regulatory interactions occur between the Egfr and Dpp vein promoting pathways and that Notch also plays an inhibitory role in Egfr signaling. Wingless signaling has to be added to the equation. It appears that Wg activity promotes veins and that nmo can antagonize this role. Interestingly, during embryogenesis Egfr and Wg signaling act antagonistically in the patterning of the denticle belts of the ventral epidermis, yet in pupal wings, initially both act to promote vein fates (Verheyen, 2001).

Some insight into the upstream regulation of Nemo has come from studies in worms and mice that suggest that the TGF-ß activated kinase (Tak-1) activates Nemo homologs in response to TGF-ß signaling (Meneghini, 1999; Ishitani, 1999; Shin, 1999). The finding that nmo and dpp mutations interact synergistically supports a role for Dpp in regulating Nemo activity in Drosophila, possibly through Tak-1. In addition, the similarity in phenotypes between overexpressed Nemo and mutations in the TGFß-like gene gbb-60A support a model whereby nmo may be differentially regulated by multiple TGFß pathways. Interestingly, another target of Tak-1, p38b, has also been shown to be involved in Dpp signaling in Drosophila and to play a role in wing vein specification (Verheyen, 2001 and references therein).

It is concluded that defects in nmo and genetic interactions have been observed that suggest the gene plays a role in two Wnt dependent processes in Drosophila: those controlling embryonic epidermal patterning and planar cell polarity. This is consistent with studies in C. elegans showing that the nmo homolog lit-1 displays a number of cell polarity defects, and acts in two separate Wnt signaling pathways (Rocheleau, 1999). Nemo homologs have been found to act at an intersection of TGFß and Wnt signaling. These studies suggest that Nemo interacts with other major developmentally pathways such as Notch and Egfr during eye and wing development. Current models of developmental patterning propose that signaling pathways are integrated at multiple levels and that this cross-talk is required for fine tuning of positional information. If these data are interpreted within this context, then Nemo may represent an important developmentally-regulated switch that allows precise spatial and temporal regulation of pattern formation (Verheyen, 2001).

REGULATION

Drosophila Nemo antagonizes BMP signaling by phosphorylation of Mad and inhibition of its nuclear accumulation

Drosophila Nemo is the founding member of the Nemo-like kinase (Nlk) family of serine/threonine protein kinases that are involved in several Wnt signal transduction pathways. Nemo performs a novel function in the inhibition of bone morphogenetic protein (BMP) signaling. Genetic interaction studies demonstrate that nemo can antagonize BMP signaling and can inhibit the expression of BMP target genes during wing development. Nemo can bind to and phosphorylate the BMP effector Mad. In cell culture, phosphorylation by Nemo blocks the nuclear accumulation of Mad by promoting export of Mad from the nucleus in a kinase-dependent manner. This is the first example of the inhibition of Drosophila BMP signaling by a MAPK and represents a novel mechanism of Smad inhibition through the phosphorylation of a conserved serine residue within the MH1 domain of Mad (Zeng, 2007).

This study demonstrates a novel regulatory role for the Drosophila Nlk family member Nemo in a TGF-ß-superfamily signal transduction pathway. Evidence is provided that Nemo is an antagonist of BMP signaling in Drosophila by examining its role in wing development through genetic analysis and monitoring of BMP-dependent gene expression. The genetic interaction studies show that phenotypes caused by activation of the BMP pathway can be suppressed by ectopic nmo and enhanced by loss of nmo. The data suggest that Nemo participates in the BMP pathway by modulating Mad activity. This is seen in the inhibition by Nemo of Mad-dependent gene expression and in the elevated expression of Mad target genes observed in nmo mutant clones. Nemo can bind to and phosphorylate Mad and this phosphorylation has direct consequences on the nuclear localization of Mad in cell culture. The single Nemo target residue maps to serine 25 within the MH1 domain of Mad, a site distinct from those previously implicated in the regulation of Mad activity and nuclear localization (Zeng, 2007).

The vertebrate Mad ortholog Smad1 normally shuttles between the cytoplasm and nucleus in the absence of signal, but upon receptor activation becomes phosphorylated at its C-terminus, binds the Co-Smad and accumulates primarily in the nucleus. Such nucleocytoplasmic shuttling is observed with R-Smads participating in both BMP and TGF-ß signaling. The shuttling provides a tightly regulated mechanism for monitoring the activation status of the receptors. Receptor-phosphorylated Smads are dephosphorylated in the nucleus, most likely causing them to detach from Co-Smads and DNA and allowing them to shuttle back to the cytoplasm. Their nuclear retention is aided by the formation of the R-Smad-Co-Smad complex and DNA binding. Thus, receptor activation leads to elevated nuclear retention. The actual rates of nuclear import are not altered by receptor-mediated phosphorylation (Zeng, 2007).

From these findings it is concluded that under normal conditions, endogenous Nemo acts to modulate the level of active Mad that is retained in the nucleus. Since Nemo is expressed ubiquitously at low levels and is enriched in cells with elevated levels of pMad, it fulfils the requirements for such a molecule involved in fine-tuning the BMP response. The phosphorylation by Nemo might control a delicate balance between promoting cytoplasmic localization of Mad, while allowing certain levels of Mad signaling to proceed in a receptor-dependent manner (Zeng, 2007).

Nemo can inhibit BMP signaling by antagonizing the nuclear localization of Mad in a kinase-dependent manner. Such a mechanism has been attributed previously to crosstalk between Erk MAPK signaling and TGF-ß/BMP signaling. This research presents Nemo as the first MAPK-like protein to attenuate Drosophila BMP pathway activity through phosphorylation of Mad. It has also been found that murine Nlk can bind to Mad, raising the intriguing possibility that this mechanism is conserved across species (Zeng, 2007).

MAPK can repress TGF-ß-superfamily signaling by targeting several Smads. The BMP-specific Smad1 is a target of cross-regulation by EGF signaling through the Erk MAPK pathway. Erk phosphorylates Smad1 in the linker domain and inhibits both the nuclear accumulation and transcriptional activity of Smad1 in cell culture and, in consequence, the in vivo function of Smad1 in neural induction and tissue homeostasis. Ras-stimulated Erk also phosphorylates two R-Smads involved in TGF-ß/Activin signaling and prevents their nuclear accumulation. The phosphorylation sites within these Smads differ, thus providing a mechanism for preferentially selective inhibition of one subtype. Thus, the distinct Nemo phosphorylation site in the MH1 domain represents an additional level of regulation of these proteins (Zeng, 2007).

Interestingly, in these studies, the Drosophila Erk MAPK does not inhibit Mad during wing development. In fact, Erk and Mad appear to synergize in the wing blade, as would be predicted given that both Egfr and BMP signaling are required for vein specification (Zeng, 2007).

The phosphorylation of serine 25 in the MH1 domain of Mad represents a novel site of regulation of Smads. This protein domain is involved in nuclear localization, DNA binding and association with transcriptional regulators. Based on known protein structures of Smads, one can predict that the Mad MH1 domain is composed of several elements. The most N-terminal sequence predicts a flexible region, then a short alpha-helix followed by a linker region and a longer, second alpha-helix. The second alpha-helix contains the predicted nuclear localization sequence (NLS). Serine 25 is located just N-terminal to the first alpha-helix. The added negative charge following phosphorylation by Nemo could modify the interaction between the two alpha-helical regions by potentially neutralizing the positively charged NLS and thereby influencing nuclear localization of Mad. Such a model is also supported by the finding that mutation of serine to alanine renders Mad constitutively nuclear. Interestingly, a similar constitutively nuclear localization has been observed when the Erk phosphorylation sites is mutated in Smad1. This suggests that both Nemo and Erk MAPK are involved in the inhibition of BMP signaling and that their distinct sites of action function to block the nuclear accumulation of Smads. Thus, the cellular factors that induce either Nlk or Erk activity can oppose the functions of BMP signaling (Zeng, 2007).

In addition to the biochemical and cell culture evidence that Nemo targets the MH1 domain of Mad to promote its nuclear export, in vivo evidence is presented that clearly demonstrates that the expression of Nemo or absence of nmo has a measurable effect on the readout of the BMP pathway in terms of Mad target gene expression, wing size, wing vein spacing and vein patterning. Specifically, elevated Nemo can attenuate the expression of vg^Q and salm, whereas nmo somatic clones and mutant discs show elevated or expanded target gene expression. Genetic interaction studies confirm such an antagonistic role, as elevated Nemo can suppress the mutant phenotypes induced by elevated BMP signaling, and reductions in nmo enhanced the penetrance of activated BMP phenotypes. Thus, the phenotypic analyses support and extend the biochemical model of the inhibition of Mad and BMP signaling by Nemo (Zeng, 2007).

Modulation of Nemo does not affect the levels of pMad found at the peaks of the BMP response gradients, suggesting that the effect of Nemo is at the level of the nuclear function of Mad. Studies with leptomycin B (LMB), which acts to inhibit Crm1-dependent nuclear export of Smads, demonstrate that Nemo can affect the nuclear localization of Mad. Thus, it is proposed that Nemo promotes the nuclear export of Mad and that this results in a fine-tuning of the levels of target genes in regions where nmo is expressed (Zeng, 2007).

It is proposed that one role for nmo is in refining the level of BMP signaling regulating proliferation. This early role for BMP signaling also relies on Mad and is therefore a candidate for Nemo-mediated inhibition. The effect on proliferation may affect the spacing, but not levels, of the pMad gradient. It is consistently observed that the genotypes in which wing width is affected do have a mild effect on the spacing of pMad stripes, and it is suggested this might be due to actual changes in cell number in the disc. Additionally, nmo mutations manifest in alterations in wing size, wing shape and cell density (Zeng, 2007).

nmo mutations also affect the later larval and pupal patterning and differentiation functions of BMP, and these can be correlated to changes in target gene expression and with vein patterning abnormalities. Thus, it appears that Nemo can modulate levels of BMP signaling at several developmental stages in wing growth and patterning (Zeng, 2007).

It has been demonstrated that Nemo can antagonize Drosophila Wg signaling during wing development. In this study it was demonstrated that Nemo also acts to attenuate BMP signaling by targeting the activity of Mad. In both of these signaling pathways the net outcome is the inhibition by Nemo of pathway-dependent target gene expression. These results demonstrate that Nemo (and by extension the Nemo-like kinases) play important roles in refining signaling pathways during development (Zeng, 2007).

An intriguing but still incomplete picture is emerging regarding the regulation of both Nlk expression and activity; this regulation represents a potential point of crosstalk between signaling pathways. nmo is transcriptionally regulated by Wg signaling. The kinase activity of Nlk is stimulated by Tak1 after Wnt induction and that Tak1 can be activated by BMP signaling. Activated Nlk can inhibit Tcf/Lef proteins and modulate Wnt-dependent gene expression. In this study, it was found that Drosophila Nlk is playing an important role in modulating BMP signaling and Mad-dependent gene expression, revealing an additional point of cross-regulation and refinement between signaling molecules (Zeng, 2007).

Nemo kinase interacts with Mad to coordinate synaptic growth at the Drosophila neuromuscular junction

Bone morphogenic protein (BMP) signaling is essential for the coordinated assembly of the synapse, but little is known about how BMP signaling is modulated in neurons. This study shows that the Nemo (Nmo) kinase modulates BMP signaling in motor neurons. nmo mutants show synaptic structural defects at the Drosophila melanogaster larval neuromuscular junction, and providing Nmo in motor neurons rescues these defects. Nmo and the BMP transcription factor Mad can be coimmunoprecipitated, and a genetic interaction was found between nmo and Mad mutants. Moreover, this study demonstrated that Nmo is required for normal distribution and accumulation of phosphorylated Mad in motor neurons. Finally, the results indicate that Nmo phosphorylation of Mad at its N terminus, distinct from the BMP phosphorylation site, is required for normal function of Mad. Based on these findings, a model is proposed in which phosphorylation of Mad by Nmo ensures normal accumulation and distribution of Mad and thereby fine tunes BMP signaling in motor neurons (Merino, 2009).

These findings point to a model in which Nmo phosphorylation of Mad promotes its accumulation in the nuclei of motor neurons and thereby ensures effective BMP signaling at the NMJ. nmo mutant larvae show a significant aberration in the accumulation and/or distribution of p-Mad in motor neurons, with elevated levels of p-Mad at the NMJ and decreased levels of p-Mad in the nuclei of motor neurons. In addition, when Mad is mutated at its phosphorylation site for Nmo (MadS25A), it shows an expression pattern that qualitatively resembles that of p-Mad in nmo mutants, with more accumulation at the NMJ and less accumulation in the nucleus compared with wild-type Mad. Consistent with the importance of this phosphorylation, MadS25A fails to rescue synaptic structural defects in Mad mutants effectively. These observations suggest that phosphorylation of Mad by Nmo most likely modulates Mad's function by regulating its distribution and accumulation in motor neurons. Based on these findings, it is tempting to conclude that the reduction in the number of NMJ synaptic boutons in nmo mutants is, to a large extent, caused by the failure of p-Mad to signal to the nucleus effectively. In support of this, a strong trans-heterozygous interaction was observed between nmo and Mad; synaptic defects in nmo mutants can be partially rescued by overexpression of a constitutively active form of Tkv (Tkv-act). Although these findings provide strong support for this model, it cannot be ruled that Nmo is involved in other processes that contribute to the growth of synaptic boutons at the NMJ (Merino, 2009).

In contrast to its critical role in the regulation of synaptic structure, Nmo does not play an important role in the regulation of synaptic function; in the absence of nmo, quantal content remains at normal levels. Consistently, it was found that the MadS25A transgene is capable of rescuing the severe electrophysiological defects of Mad mutants as efficiently as a wild-type Mad transgene. Previously, it has been suggested that structural growth and the homeostasis of neurotransmitter release at the NMJ have different requirements for BMP signaling. Similarly, the current findings highlight the differential requirements for the regulation of synaptic structure and synaptic strength via BMP signaling. Interestingly, although overexpression of Nmo in motor neurons does not influence synaptic structural growth, it does cause a significant reduction in neurotransmitter release. This observation is consistent with those made by Zeng (2007), showing an antagonistic effect of Nmo gain-of-function on Mad in the wing imaginal discs. Nevertheless, although this observation shows a potential for Nmo to act as a negative regulator of Mad, the findings argue against a significant negative regulatory role for Nmo in motor neurons under normal physiological conditions (Merino, 2009).

It is proposed that Nmo exerts its action primarily by modulating Mad's retrograde movement from the NMJ to the nucleus. Nmo has been implicated in the regulation of Mad nuclear export in heterologous cells (Zeng, 2007); however, the current study found no evidence for changes in Mad nuclear export as a consequence of loss of nmo. In contrast, Nmo was found to be required for accumulation of p-Mad in the nuclei of motor neurons. In the absence of nmo, p-Mad levels increase at the NMJ and decrease in the nuclei of motor neurons, suggesting that Nmo is required for normal translocation/trafficking of Mad from the NMJ to the nucleus (Merino, 2009).

Finally, consistent with previous findings (Zeng, 2007), overexpression of Nmo can reduce the proportion of Mad concentration in nuclei versus that in cell bodies of motor neurons. Based on the phenotypic consequences of nmo loss- and gain-of-function, it appears that normal growth of synaptic structures at the NMJ depends on continuous and efficient BMP signaling from the NMJ to the nuclei of motor neurons and is less sensitive to the residence time of Mad in the nucleus. However, it appears that regulation of neurotransmitter release is more sensitive to the residence time of Mad in the nucleus and less dependent on the continuous retrograde signaling from the NMJ. These findings highlight the importance of Nmo phosphorylation of Mad at serine 25 in this process; however, a comprehensive understanding of the regulation of Mad trafficking and movement dynamics in different cellular compartments will require future studies (Merino, 2009).

Finally, an intriguing possibility would be the involvement of the Wg pathway in the regulation of Mad dynamics through Nmo. Nmo has been implicated in the Wg pathway during wing development and has been shown to be a transcriptional target of Wg. As Wg has been shown to participate in the regulation of synaptic growth at the NMJ, it would be tempting to envisage a role for Wg in the regulation of Nmo transcription in motor neurons and thus a link between the Wg and BMP pathways in the regulation of synaptic growth and function at the NMJ (Merino, 2009).

Nemo kinase phosphorylates β-catenin to promote ommatidial rotation and connects core PCP factors to E-cadherin-β-catenin

Frizzled planar cell polarity (PCP) signaling regulates cell motility in several tissues, including ommatidial rotation in Drosophila melanogaster. The Nemo kinase (Nlk in vertebrates) has also been linked to cell-motility regulation and ommatidial rotation but its mechanistic role(s) during rotation remain obscure. This study shows that nemo functions throughout the entire rotation movement, increasing the rotation rate. Genetic and molecular studies indicate that Nemo binds both the core PCP factor complex of Strabismus-Prickle, as well as the E-cadherin-β-catenin (E-cadherin-Armadillo in Drosophila) complex. These two complexes colocalize and, like Nemo, also promote rotation. Strabismus (also called Vang) binds and stabilizes Nemo asymmetrically within the ommatidial precluster; Nemo and β-catenin then act synergistically to promote rotation, which is mediated in vivo by Nemo's phosphorylation of β-catenin. These data suggest that Nemo serves as a conserved molecular link between core PCP factors and E-cadherin-β-catenin complexes, promoting cell motility (Mirkovic, 2011).

The data suggest that Nmo connects the core PCP Stbm-Pk complex to the activity of E-cad-β-cat. Consistent with this, mutations in stbm and pk enhance not only the nmo^P rotation defects but also rotation defects of hypomorphic shg (E-cad) backgrounds. As the presence of the Stbm-Pk complex seems to increase the amount of Nmo at R4 membranes and junctional complexes, it is hypothesized that a rise in Stbm levels would increase the ability of sev>Nmo to cause an over-rotation phenotype. This is indeed the case. These data indicate that Nmo serves as a link from PCP factors to the E-cad-catenin complexes. The data are consistent with a model in which the Stbm-Pk complex helps to recruit and/or stabilize Nmo at membrane regions (where the PCP factors partially overlap with E-cad-β-cat complexes (Mirkovic, 2011).

The effect of Nmo on E-cad-β-cat complexes could be mediated either through the dynamics of lateral clustering (for example, formation or disassembly of higher-order E-cad-β-cat complexes) or through changes in the interaction of β-cat with other associated proteins. An E-cad::β-cat fusion protein (which bypasses a β-cat requirement and provides stable adhesion is not influenced by Nmo, suggesting that once β-cat is part of the E-cad-catenin complex Nmo cannot affect their activity. It is thus possible that phosphorylation of β-cat by Nmo affects the E-cad-β-cat complex activity (as an ArmS10^AAA isoform with the Nmo target sites mutated no longer cooperates with Nmo) and this phosphorylation may also modulate interactions of the complex with other binding partners, such as β-cat. The interactions of adhesion and planar polarity during the early 'convergence-extension' rearrangements in the fly embryo suggest a mechanism in which a polarized pattern of junction remodeling drives cell intercalation. Polarized activity of RhoA and Myosin II (encoded by zipper) regulates adherens junction disassembly along the anterior-posterior axis, primarily by regulating lateral cadherin clustering without affecting surface levels of cadherins. The specific effect of RhoA on rotation, along with the interaction of nmo with zipper, supports the idea that actin-myosin contractility is downstream of Nmo. Loss of maternal contribution or Nmo overexpression in the embryonic epidermis phenocopies shg alleles or ArmS10 cuticle defects, respectively. Thus, Nmo may be generally required in epithelia undergoing morphogenetic movements, where it modulates polarized remodeling of adherens junctions in response to local asymmetries created by, for example, the activity of PCP signaling complexes (Mirkovic, 2011).

In conclusion, this study defines a framework in which Nmo serves as a link between PCP (Stbm) and the regulation of adhesive cell behavior at the level of adherens junction complexes. Although Nmo is recruited and/or maintained apically by the Stbm-Pk complex, other factors must affect Nmo activity or localization as well, because the set of cells requiring nmo (all outer R-cells) is broader than the set of cells requiring stbm (R4). First, the Nmo could regulate rate of rotation independently of the PCP complexes through Notch (N) signaling and/or Egfr signaling, as suggested by genetic data: N^- alleles strongly suppress sev>Nmo, and N functions in all R-cells. Second, an asymmetric input or localization of Nmo by Stbm would provide a direction to rotation. Thus, a Notch-Nmo interaction in all cells and an asymmetric Stbm effect in R4 could combine to regulate both rate and direction of rotation. The observation that zebrafish Nlk enhances the PCP-specific Wnt11 cell migration defects in prechordal plates supports a general Nmo-mediated mechanism in PCP-associated cell movements (Mirkovic, 2011).

NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed

The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of Period (Per) proteins. Using Drosophila, a time-delay circuit based on hierarchical phosphorylation was identified that controls the daily downswing in Per abundance. Phosphorylation by the Nemo/Nlk kinase at the 'per-short' phospho cluster domain on Per stimulates phosphorylation by Doubletime (Dbt/Ck1delta/epsilon) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by Dbt at other more distal sites on Per, including those required for recognition by the F box protein Slimb/β-TrCP and proteasomal degradation. Highly phosphorylated Per has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing Per susceptibility to degradation. These findings identify Nemo as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed (Chiu, 2011).

This study shows that the per-short domain functions as a discrete hierarchical phospho-cluster that delays Dbt-mediated phosphorylation at the Slimb recognition site on Per, providing new insights into how clock protein phosphorylation contributes to circadian timing mechanisms. The cumulative effect of this delay circuit is to slow down the pace of the clock by ~8 hr. It is proposed that Dbt functions in a stepwise manner to phosphorylate clusters on Per that have distinct biochemical functions and effects on the rate of Per degradation, e.g., elements such as the per-short phospho-cluster that delays Per degradation and those such as the Slimb-binding site and global phosphorylation that enhance instability. Nmo plays a major role in the relative timing of Dbt activity at these different elements because it stimulates multisite phosphorylation at the per-short delay cluster by Dbt, which slows down the ability of Dbt to phosphorylate instability elements. Thus, a large portion of the phosphorylation events dictating when in a daily cycle Per is targeted for rapid degradation is not directly linked to binding ofSlimb per se. The current findings demonstrate that presumptive long-range interactions between distinct positively and negatively acting phospho-clusters collaborate to set clock speed and helps to explain why mutations in clock protein phosphorylation sites and/or the kinases that phosphorylate them can yield both fast and slow clocks (Chiu, 2011).

A proposed mechanism for the function of the per-short domain is supported by the congruence between in vitro biochemical studies based on purified recombinant Per protein from cultured S2 cells and in vivo changes in the pace of behavioral rhythms using transgenic models. This suggests that a primary biochemical effect of the per-short domain on clock speed in the fly is via modulating the rate of Dbt-mediated phosphorylation at the Slimb phospho-degron on Per. The physiological role of T583 phosphorylation is not clear, as mutating this site does not have detectable effects on the binding of Per to Slimb in S2 cells. In this regard, it is interesting to note that the original per-short domain was identified as encompassing aa 585-601 of Per (Baylies, 1992). Thus, it is likely that the 8 hr per-short delay circuit is governed by the dynamics underlying the phosphorylated status of three sites (i.e., S596, S589, and S585) (Chiu, 2011).

At present, it is not clear how phosphorylation in the per-short cluster slows down subsequent phosphorylation by Dbt at Ser47 and other sites. Inactivating the per-short cluster leads to increases in the rate of Dbt-mediated phosphorylation at not only the N terminus, but also the C terminus of Per, suggesting that it is a major control center for regulating the relative efficiency of Dbt phosphorylation at many sites on Per. It is suggested that the per-short phospho- cluster acts as a transient 'temporal trap' for Dbt. Once the sites in the per-short domain are phosphorylated by Dbt, this somehow allows it to continue its normal rate of phosphorylation at other phospho-clusters. Although speculative, progressive increases in phosphorylation at some of these other phospho-clusters might generate time-dependent local/overall conformational changes in Per, possibly via electrostatic repulsion, eventually leading to a more open Per structure that is more accessible to phosphorylation by Dbt at the Slimb-binding site and/or a more efficient substrate for degradation. Thus, the rapid degradation of Per during the early day is likely due to a combination of synchronous increases in the phospho-occupancy of Ser47 and overall phosphorylation of Per. Other factors such as protein phosphatases and the action of Timeless also play major roles in regulating the speed of the Per phosphorylation program (Chiu, 2011).

How might phosphorylation at S596 enhance phosphorylation at S589 and S585 by Dbt? Phosphorylation by the CK1 kinase family is generally enhanced by priming. However, phosphorylation at the per-short domain by Dbt does not follow the consensus priming-dependent recognition motif for the CK1 family of kinases (i.e., S/Tp-X-X-S/T, wherein S/Tp refers to the primed site, X is any amino acid, and the italicized residues the CK1 target site, as the S596 priming site is located C terminal to the Dbt sites. Thus, it is likely that phosphorylation of S596 by Nmo stimulates Dbt phosphorylation at the per-short region in a nonpriming-dependent manner (Chiu, 2011).

Ongoing studies are aimed at understanding the biochemical events underlying the ability of phosphorylation at S596 to enhance phosphorylation by Dbt in the per-short region. The discovery of a delay phospho-circuit also sheds light on why mutations in different phosphorylation sites on Per or Frq proteins, although affecting stability, can speed up or slow down the clock. The current findings also offer a logical explanation for why mutations that lower the kinase activity of CKI, which overall is expected to slow down the rate of PER degradation, can yield fast clocks. For example, although other mechanisms have been offered to explain the short-period phenotypes that are observed for the CKI3tau mutation in hamsters and a CKIdelta mutation associated with familial advanced sleep phase syndrome (FASPS) in humans, it is possible that phosphorylation at a per-short type delay cluster is preferentially compromised by the mutant kinase, which could appear as a substrate-specific gain-of-function mutation (Chiu, 2011).

Negatively acting phospho-clusters are likely to be a general feature of the timing mechanisms regulating the daily abundance cycles of clock proteins such as Pers in animals and Frq in Neurospora. However, other regulatory modules that operate in a phase-specific manner must participate to generate an ~24 hr oscillator. Most conspicuously, clock speed is intimately linked to the Per and Frq abundance cycles necessitating daily phases of de novo synthesis to replenish the pools of previously degraded proteins.Asrecently shown, the transcriptional negative feedback aspect of Per regulating Clk-Cyc-mediated transcription is also a component of the period-setting mechanism in Drosophila. Therefore, the ~24 hr Per abundance cycle is based on a combination of 'time constraints' that are generated using different regulatory modules. It is proposed that the per short- based timer mainly functions once Per has accumulated and begins participating in transcriptional repression, controlling Per abundance once it is disengaged from the dynamics of its cognate mRNA by setting in motion a series of sequential phosphorylation events that are calibrated to stimulate Per degradation in the nucleus at the appropriate time in a daily cycle, enabling the next round of circadian gene expression. In this context, it is interesting to note that a prior study analyzing the per-short domain suggested that it functions with a nearby 'perSD' domain to increase the transcriptional repressor function of Per. It is possible that the same phosphorylation events leading to Per degradation also function to increase its potency within the repressor complex (Chiu, 2011).

These studies also identify Nemo as a clock kinase. Nemo is the founding member of the evolutionarily conserved Nemo-like kinase (Nlk) family of proline-directed serine/threonine kinases closely related to mitogen-activated protein kinases (MAPK). It was originally characterized in Drosophila as required for planar cell polarity during eye development and is now known to function in many pathways. Nmo/Nlk is localized in the nucleus and is another factor in the circadian clock that also functions in the Wnt/Wg-signaling pathway, such as CKI3/Dbt, β-TrCP/Slimb, and GSK-3β/Sgg. It will be of interest to determine whether Nlk functions in the mammalian clock. Intriguingly, the phosphorylation sites on Per are largely clustered, and several of them have the same spatial arrangement as the per-short cluster, with a predicted pro-directed kinase site at the C-terminal end of the phospho-cluster. This suggests that Nmo and/or other pro-directed kinases serve as control points to activate spatially and perhaps functionally distinct phospho-clusters. Indeed, it has recently been shown that phosphorylation at Ser661 of Per by an as yet unidentified pro-directed kinase primes further phosphorylation by Sgg at Ser657 to regulate the timing of Per nuclear entry in key pacemaker neurons (Chiu, 2011).

In summary, a central aspect of circadian clocks is the presence of one or more clock proteins that provide a dual function by behaving as phospho-based timers and linking its timer role to gene expression by operating in a phase-specific manner to recruit repressor complexes that inhibit central clock transcription factors. These studies suggest that a major part of the timing mechanism underlying these phospho-clock proteins is based on spatially and functionally discrete phospho-clusters that interact to impose calibrated and sequentially ordered biochemical time constraints. In the case of Per, the per-short phospho-cluster functions as a central timing module by slowing down the ability of Dbt to phosphorylate instability elements regulating Per degradation and, hence, when Per repressor activity is terminated and the next round of circadian gene expression begins (Chiu, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

It is speculated that Nemo may function at early stages of development since it was found that nmo interacts genetically with genes required for embryonic patterning, such as arm, pangolin and Dl. Choi and Benzer (1994) have shown that the Nemo transcript is expressed abundantly in embryos on Northern blots. The embryonic cellular expression pattern of nmo offers some insight into possible roles in embryonic development. High expression in cellular blastoderm embryos suggests nmo transcripts are maternally loaded. Zygotic expression is seen in most cells, with enrichment during germ band expansion in a segmented pattern. An understained embryo of a later stage shows the segmented pattern and central nervous system staining. nmo transcript is enriched in the cells bordering the tracheal pits. In later stages nmo is enriched in the central nervous system and brain. In an effort to determine what role Nemo may play in wg signaling in the embryo, embryos were double stained to compare nmo mRNA expression with pattern of Engrailed expression, which marks the posterior border of each segment. nmo is found to be expressed in the anterior cells of the segment. These nmo-expressing cells correspond to those that will secrete denticle belt rows 2-6 under the control of wg signaling. The overall pattern and dynamics of expression and ectopic expression phenotypes suggest the possibility that nmo acts as a wg regulator in early embryonic patterning (Verheyen, 2001).

Larval

Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. The mechanisms by which this pattern formation occurs have been investigated and the results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. To determine if nemo expression is regulated by fz, fz mutant flies were generated that carry an enhancer trap in the nemo gene. Expression of beta-galactosidase from the enhancer trap resembles the expression pattern of nemo transcripts. The expression of beta-galactosidase is greatly reduced in fz eye imaginal discs, especially in the morphogenetic furrow. To study further the interaction between fz and nemo, the eyes of nemo;fz double mutants were examined. Interestingly, there was a large number of ommatidia that did not rotate at all compared to either nemo or fz mutants alone, suggesting that fz acts redundantly with nemo to regulate the entire turning of an ommatidium. This suggests that fz and nemo function synergistically in directing rotation (Zheng, 1995).

Nemo function during wing development

The cellular events that govern patterning during animal development must be precisely regulated. This is achieved by extrinsic factors and through the action of both positive and negative feedback loops. Wnt/Wg signals are crucial across species in many developmental patterning events. Drosophila nemo (nmo) acts as an intracellular feedback inhibitor of Wingless (Wg) and it is a novel Wg target gene. Nemo antagonizes the activity of the Wg signal, as evidenced by the finding that reduction of nmo rescues the phenotypic defects induced by misexpression of various Wg pathway components. In addition, the activation of Wg-dependent gene expression is suppressed in wing discs ectopically expressing nmo and enhanced cell autonomously in nmo mutant clones. nmo itself is a target of Wg signaling in the imaginal wing disc. nmo expression is induced upon high levels of Wg signaling and can be inhibited by interfering with Wg signaling. Finally, alterations are observed in Arm stabilization upon modulation of Nemo. These observations suggest that the patterning mechanism governed by Wg involves a negative feedback circuit in which Wg induces expression of its own antagonist Nemo (Zeng, 2004).

To better understand the role of nmo in earlier patterning events, its localization pattern in larval wing imaginal discs was determined in the nmo^P enhancer trap line, nmo-lacZ. The expression of nmo is quite dynamic during larval development. Staining of second instar larval discs reveals very weak expression at the anterior and posterior periphery of the wing disc. Early in the third larval stage, staining at the DV boundary becomes evident and the intensity of the staining increases with age. In late third instar discs, nmo is expressed in two thin stripes flanking the DV boundary. These two stripes of staining are weaker at the point where the anteroposterior (AP) boundary intersects the DV boundary. nmo expression is also seen in a ring encircling the future wing pouch in a tissue corresponding to the future proximal wing hinge, with the expression in the dorsal ring appearing darker than the ventral ring. Staining is also seen in the primordia of longitudinal wing veins 3, 4 and 5, beginning in the late third instar stage. Finally, nmo expression is also detected in spots on the wing imaginal discs that represent sites of sensory organ formation on the future notum. Consistent with such an expression pattern, a role has been shown for nmo in macrochaete bristles, as demonstrated by genetic interactions with Hairless (Zeng, 2004).

That this enhancer trap insertion accurately represents the expression of nmo was confirmed by performing whole-mount RNA in situ hybridization. In addition to the localized staining seen in the enhancer trap, low level ubiquitous staining is detected throughout the disc. This ubiquitous staining is also apparent when anti-ß-galactosidase antibody is used to detect the nmo-lacZ expression pattern (Zeng, 2004).

The nmo-lacZ pattern is reminiscent of the Wg expression pattern in imaginal discs. To examine the relationship between the two expression patterns, double staining was performed for ß-galactosidase and Wg protein. This staining reveals that nmo expression at the DV boundary flanks the Wg protein domain in late third instar wing discs. Wg protein is detected in a narrow stripe along the presumptive wing margin and nmo is seen in the cells directly adjacent to the Wg-expressing cells. In addition, nmo is detected in the ring domain overlapping with the Wg inner ring expression domain that encircles the wing pouch. Such a localization for nmo is also consistent with the observed defect in adult flies in which the wing is held away from the body at an angle and may reflect a hinge defect (Zeng, 2004).

In Drosophila, several examples of Wg feedback inhibition have been identified. (1) It has been shown that Wg downregulates its own transcription in the wing pouch to narrow the RNA expression domain at the DV boundary. (2) Wg signaling can repress the expression of its receptor Dfz2 in the wg-expressing cells of the wing disc. Wg regulation of Dfz2 creates a negative feedback loop in which newly secreted Wg is stabilized only once it moves away from the DV boundary to cells expressing higher levels of Drosophila Fz2. (3) The Wg target gene naked cuticle (nkd) acts through Dsh to limit Wg activity. (4) Wingful (Wf), an extracellular inhibitor of Wg, is itself induced by Wg signaling (Zeng, 2004).

This research adds Nemo to this list of inducible antagonists participating in Wg signaling. Nemo antagonizes the Wg signal in wing development, as evidenced by phenotypic rescue, suppression of Wg-dependent gene expression in discs ectopically expressing nmo, and ectopic expression of a Wg-dependent gene in nmo mutant clones (Zeng, 2004).

Since both wf and nmo expression are positively regulated by Wg signaling in the wing, their expression patterns are relatively similar to that of Wg. Even though nkd also has a similar pattern to Wg in the larval wing disc, unexpectedly, it has no detectable role in wing development. As an intracellular antagonist, Nkd regulates embryonic Wg activity in a cell-autonomous manner by acting directly with Dsh to block accumulation of Arm in response to Wg signaling. Wf apparently has no role during embryogenesis, although both Wf and Nkd can inhibit Wg signaling throughout development when overexpressed. Wf is an extracellular protein that functions non-autonomously to regulate Wg signaling. This mechanism of inhibition parallels that of Argos, a secreted feedback antagonist in the EGFR pathway (Zeng, 2004).

The effect of Nemo on the Wg-dependent reporter gene Dll is confined to regions of endogenous gene expression. In the absence of nmo expression, ectopic Dll expression is only seen at elevated levels within the endogenous expression domain, thus being dependent on Wg activity. This is in contrast to inhibition of the Dpp pathway by Brinker. Brinker acts independently of Dpp in its repression of Dpp target genes, such that in the absence of both brk and Dpp the target genes are expressed ectopically. It is speculated that the role of Nemo in the Wg pathway is analogous to the role of Daughters against Dpp (Dad) in Dpp signaling. Dpp induces the expression of dad, which in turn antagonizes the pathway through an as yet undefined mechanism. These might include either interactions with the intracellular transducer Mothers against Dpp (Mad) or with TGFß receptors (Zeng, 2004).

It is intriguing that Nemo does not play a role in regulating wg expression; however, this is most probably because of the point of action of Nemo within the Wg pathway. The self-refinement of wg expression in the wing is dependent on Dsh but independent of Arm. Recent work has raised some questions about the factors involved in Wg self-refinement, specifically postulating a role for dTCF in this process. dTCF (pan) somatic clones have elevated Wg protein, suggesting that TCF plays an active role in repressing Wg gene expression. However no distinction was made between increased wg gene expression and stabilized Wg protein. Another study examined regulation of Wg signaling by Twins (tws), a protein phosphatase subunit, and found that it is required for Arm stabilization (Bajpai, 2004). Modulation of tws results in aberrant Wg signaling, as monitored by Dll expression, that are not accompanied by alterations in wg gene expression. The current data are consistent with the findings of Bajpai (2004), and suggest that the mechanism of wg refinement most probably does not involve Arm or dTCF. Genetic analyses support the placement of Nemo at or below the level of Arm within the pathway. The apparent absence of a role for Nemo in regulating wg expression contrasts with the other inducible feedback inhibitors. Modulation of either the extracellular inhibitor Wf or the Dsh-antagonist Nkd can influence wg gene expression in wing discs and embryos, respectively. Neither loss of nor ectopic expression of nmo during imaginal disc development has an effect on the pattern of Wg expression (Zeng, 2004).

The developing wing is bisected by a narrow stripe of Wg-expressing cells. Wg protein has a short half-life near the DV boundary, which causes a rapid decrease in Wg concentration and forms a steep symmetric gradient of the Wg protein. Radiating out from the source of Wg, there are three concentric domains of Wg-dependent gene expression: (1) a very narrow domain of cells adjacent to the highest concentration of Wg expresses achaete (ac); (2) Dll is expressed in a median range domain of Wg, and (3) a long-range domain expresses vg. The current results suggest that nmo is a short-range target, like ac, the activation of which is limited by the high threshold of Wg signal. This may be the explanation for the very narrow pattern of enriched nmo expression at the DV boundary and the ring domain and the cell-autonomous induction of nmo in the ectopic DeltaArm clones (Zeng, 2004).

If higher levels of Wg protein induce nmo expression, it raises the question of why nmo is not expressed in DV boundary cells. One possibility is that there are genes that are expressed between the two stripes of nmo that prevent its expression. vg-Gal4, which is mainly expressed at the DV boundary, drives UAS-fluDeltaarm to induce ectopic nmo expression. In this case, the ectopic expression of nmo fills the gap between the two endogenous bands. This observation supports a model in which there is a suppressor(s) located along the DV boundary to silence nmo expression. The balance between the Wg signal and the suppressor(s) would refine nmo expression into two thin stripes flanking the DV boundary. In the case of ectopic UAS-fluDeltaarm, the Wg signal may overpower the suppressor, thereby allowing nmo to be expressed at the boundary. In a similar mechanism, it has been shown that Wg can direct the expression of ac at the margin but that this expression is prevented, at least partially, by the activity of Cut (Zeng, 2004).

Although the wing margin, ring expression and low level ubiquitous staining of nmo in imaginal wing discs reflects regulation by Wg signaling, the other developmental expression patterns, such as staining in primordia of wing veins, may reflect regulation by other signaling pathways. For example, the staining in the wing vein primordia that emerges in late third instar and the gene expression pattern observed in pupal wings reflects the later role of nmo in wing vein patterning, which may involve interactions with EGFR and TGFß signaling (Zeng, 2004).

In further support that Wg signaling regulates the transcription of nmo, several dTCF consensus binding sites have been found in the 5' region of the nmo gene that may represent enhancer elements. Indeed, two sites match 9 out of 11 bp (GCCTTTGAT) of the T1 site (GCCTTTGATCT) in the dpp BE enhancer that has been shown both in vitro and in vivo to bind and respond to dTCF. The presence of these sites suggests that the observed transcriptional regulation of nmo by Wg may involve direct binding to the nmo DNA sequence by dTCF (Zeng, 2004).

As a result of comparing the endogenous expression pattern of nmo with stabilized Arm, it was noticed that the highest levels of Nemo exclude Arm stabilization, while high levels of Arm are present in cells in which nmo levels are lower. Since Arm protein stabilization is a direct consequence of Wg pathway activation, attempts were made to examine whether Nemo may function to inhibit Wg by promoting Arm destabilization and subsequent breakdown. Indeed, ectopic expression of Nemo can lead to cell-autonomous reduction in Arm protein levels. This preliminary result suggests a mechanism in which Nemo may contribute to the destabilization of Arm that involves the Axin/APC/GSK3 complex. One explanation to account for such a finding would concern the interaction with TCF in the nucleus and the role of dTCF as an anchor for Arm. Given what is known about NLKs, it is likely that Nemo may act on the ability of the dTCF/Arm complex to bind DNA and activate transcription. It has been proposed that dTCF acts as an anchor for Arm in the nucleus. It remains to be determined how efficient this anchor is and whether there are conditions in which the interaction may become compromised, such as is seen with elevated Nemo. NLKs have been shown to affect the DNA-binding ability of TCF/ß-catenin. Perhaps in the absence of DNA binding, this complex is less stable and Arm could be free to shuttle to the cytoplasm where it could associate with Axin or APC and become degraded. It is proposed that the ectopic nmo leads to destabilization of the dTCF/Arm/DNA complex, thus causing Arm to exit the nucleus and be degraded through interaction with Axin, APC and GSK3. The observation that ectopic expression of full-length Arm cannot induce any activated Wg phenotypes has been explained by the hypothesis that even these high levels of protein are not sufficient to overcome the degradation machinery. Thus, the finding that there is no elevated Arm in nmo clones is consistent with an inability to overcome the endogenous degradation machinery; even though less Nemo could lead to more stabilized DNA interactions, this would not lead to higher levels of stabilized Arm than are normally found (Zeng, 2004).

Studies of homologs of Nemo in other species have provided clues to its function, although it is still not clear if the same mechanism in used in Drosophila. The studies in this paper establish that Drosophila Nemo does in fact play a negative regulatory role in canonical Wg signaling. Although nmo was originally identified as playing a role in the non-canonical Fz pathway that regulates tissue planar polarity, its precise role in that pathway has not been further defined (Zeng, 2004).

In addition to the findings that NLKs can bind to and phosphorylate TCF and LEF-1 proteins and thereby decrease the DNA-binding affinity of the TCF/ß-catenin complex, a model is emerging that NLKs regulate multiple HMG-box containing proteins. Recently, it was shown that Xenopus NLK (xNLK) binds to a novel HMG-domain containing protein HMG2L1, which can inhibit Wnt signaling in several assays. In addition, xNLK binds to xSox11, another HMG-box containing transcription factor, and they cooperatively induce neural development in Xenopus (Zeng, 2004).

Although the results do not directly address the molecular mechanism, it is speculated that activated Nemo can inhibit the interaction of the Arm-dTCF complex with DNA. The genetic data presented in this paper support the molecular mechanism that Nemo acts downstream of or at the same level as Arm. Indeed, the finding that increased levels of nmo can block accumulation of Arm is intriguing as it suggests that Nemo may regulate Wg at the level of Arm stabilization and dTCF function. At this point, further biochemical experiments are in progress to address these issues. They should shed light on the exact mechanism of function that allows Nemo to be an inducible antagonist of canonical Wg signaling in Drosophila (Zeng, 2004).

EFFECTS OF MUTATION

nem^p1 was isolated among P[lacZ,w⁺] insertion lines that were generated (B. Mozer, K.-W. Choy and S. Benzer, cited in Choi, 1994). Eyes of nmo flies are slightly narrowed anterior-posterior, and the facets are square rather than hexagonal. Tangential sections show that the pigment cell lattice is abnormal, especially at corners, which have disarranged bristle and secondary pigment cells. Occasional ommatidia are fused. The phenotype is recessive and fully penetrant. In the mutant, there is an initial turning of approximately 45 degrees, but further rotation is blocked. Genetic mosaic analysis indicates that the nmo gene acts upon each cluster as a whole; normal nmo function in one or more photoreceptor cells appears to be sufficient to induce full rotation. The nmo gene sequence encodes a serine/threonine protein kinase homolog, suggesting that the kinase is required to initiate the second step of rotation. In another mutant, roulette, excessive rotation through varying angles occurs in many ommatidia. This defect is suppressed by nmo, indicating that nmo acts upstream in a rotation-regulating pathway (Choi, 1994).

The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development

A genetic second-site modifier screen was carried out to identify genes that either participate in Notch signaling or modulate cross-talk between signal transduction pathways. Mutations were recovered that could dominantly modify the rough eye phenotype caused by expression of activated Notch under expression of the sevenless promoter. The adirondack (adk) complementation group was found to modify the rough eye; homozygous mutant adk flies display numerous phenotypes suggesting that cell fate determination is disrupted. The molecular and phenotypic characterization of adk was initiated, in an effort to understand its possible role in Notch signaling. The adk mutation maps to the location of the nmo gene. nmo is required for the correct rotation of photoreceptor clusters during eye development, and has been placed downstream of the frizzled pathway in the process of planar cell polarity. adk alleles fail to complement the nmo eye phenotype, and are referred to as nmo^adk1 and nmo^adk2. Since most of the nmo^adk1 and nmo^adk2 phenotypes are similar, they will collectively be referred to as nmo^adk, except where noted. The eye phenotype seen in all nmo alleles is characterized by reduced overall eye size, a narrower eye and defects in the structural organization of the ommatidia (Choi, 1994). The normally hexagonal ommatidia are square and generally are flanked by four interommatidial bristles, instead of the usual three bristles at alternating corners (Verheyen, 2001).

nmo^adk alleles displayed several defects in the development of the adult wings that are more severe than those observed in the original nmo^P allele. The wings are rounder and shorter than wildtype, contain extra vein material and are held away from the body at a 45° angle. nmo^P flies also have smaller wings that are held out 15° to 30° from the body (Choi, 1994). The fully penetrant nmo^adk extra vein phenotype is characterized by excess vein tissue originating from the posterior cross-vein (PCV) and extending distally between longitudinal (L) veins LIV and LV. Additional vein material can also form parallel to LII and posterior to LV. Slight extra vein material near the posterior cross-vein (PCV) is also found in a small percentage of nmo^P flies (Verheyen, 2001).

The original nmo^P allele has been described as a molecular null allele, since no transcript is detected in homozygous pupae. Genetically, the nmo^P allele also behaves as a classic null allele since nmo^P/Df(3R)pbl^NR flies have the same phenotype as nmo^P homozygotes. The nature of the nmo^adk alleles were investigated, since they appear to be more severe alleles of the nmo locus. They display more severe eye and wing phenotypes, in addition to reduced viability and infertility. Northern blot hybridization used to detect nmo transcripts confirm that nmo^P is a transcript null allele. Interestingly, the two EMS-induced nmo^adk alleles display alterations in their transcript sizes, reflecting the production of truncated mRNAs. The nmo gene produces numerous transcripts at different stages during development, and whether any of the embryonically-expressed transcripts are affected in either nmo^P or nmo^adk homozygous alleles could not be determined. It is speculated that the truncated transcripts may lead to the production of dominant negative Nemo molecules that interfere with the maternally loaded nmo and thus cause a more severe phenotype then that zygotic null allele (Verheyen, 2001).

The Notch pathway is known to act during initiation and differentiation of wing veins to refine the adult vein pattern. Since nmo^adk was identified as a modifier of Notch in the eye, the link between nmo and Notch signaling in the wing was investigated. Genetic interactions between nmo^adk and mutations in several components of the Notch pathway were characterized. Mutations in the ligand Delta (Dl/+) cause a mild vein thickening phenotype. This phenotype is synergistically enhanced by homozygosity for nmo^adk. Conversely, mutations in the negative regulator Hairless (H/+), which normally exhibit shortening of LV, suppress the ectopic veins seen in nmo^adk. In addition to interactions in wing veins, H and nmo^adk show a synergistic interaction in the macrochaete bristles of the head and notum (Verheyen, 2001).

nmo^adk flies have a mild bristle loss phenotype, and occasionally display bent bristles or duplicated bristles. H/+ flies display a characteristic dominant loss of macrochaetes. Homozgosity for nmo^adk in a H/+ background leads to a dramatic enhancement of the H/+ bristle loss phenotype. Since nmo^adk mutations are enhanced by Dl, and are suppressed in the wing by H, whether nmo acts upstream of Notch was examined. It was asked if the nmo^adk extra vein defect could be rescued through ectopic activation of Notch signaling. Delta and E(spl)mß were ectopically expressed with the 32B-Gal4 driver, which is expressed in the wing blade. E(spl)mß is normally expressed in the cells flanking the presumptive veins and acts to suppress rhomboid expression to the narrow band of vein progenitors. Ectopic expression of UAS-E(spl)mß leads to mild vein thinning and a shortening of LV. Both UAS-Delta and UAS-E(spl)mß specifically suppress the extra veins associated with nmo^adk mutations. Thus, both ectopic activation of the Notch pathway and loss of a negative regulator as seen with H¹/+ can lead to suppression of ectopic veins caused by nmo^adk. These results suggest that Nemo is upstream of Notch and acts in a common vein regulatory pathway (Verheyen, 2001).

Vein phenotypes like those seen in nmo^adk alleles are also seen in members of the 'plexus group' of extra vein mutants, including net and plexus (px). px¹ is a loss of function allele and does not represent a null allele, while net¹ is a null allele. net¹;nmo^adk and px¹;nmo^adk double mutants were examined for genetic interactions. Flies doubly mutant for nmo^adk and net¹ show a very strong synergistic interaction and massive excess of vein tissue at the expense of intervein. Such a phenotype is more severe than that seen with net;px double mutants, and more closely resembles the interactions seen between Dl and px or net. A milder, but similar synergistic interaction is seen in px¹; nmo^adk flies (Verheyen, 2001).

The Dpp pathway is also know to have an essential role in wing vein specification. Flies doubly mutant for nmo^adk and dpp^shv (a mild vein loss allele) were examined, and a synergistic enhancement of ectopic vein specification was observed, especially in the anterior of the wing blade near LII (Verheyen, 2001).

The existing nmo alleles do not show embryonic phenotypes and this is attributed to the large maternal contribution that may obscure the true zygotic null phenotype. To investigate the potential role of nmo in the embryo, a dsRNA interference (RNAi) assay was carried out to deplete the embryo of nemo expression. RNAi has been successfully used to mimic loss-of-function phenotypes of Wnt pathway components, including C. elegans lit-1 and Drosophila fz. Injection of nmo dsRNA into wildtype embryos reduces viability of the injected embryos to 5.9%, as compared to 40% viability in controls. As a positive control for RNAi, dsRNA injections were performed to inhibit Egfr function. These resulted in viability of 6.5%, and gave typical Egfr loss of function mutant phenotypes (Verheyen, 2001).

Variable phenotypes were observed in nmo RNAi-induced dead embryos. Defects in the proper patterning of the denticle belts of the ventral epidermis were observed in 40% of dead embryos, and these included variable transformation of denticle rows to naked cuticle. This phenotype is opposite that seen with loss of wg, but similar to that seen with overexpression of Fz or Fz2, or constitutive Armadillo. In addition, defects in head involution were seen in 14% of dead embryos and dorsal closure defects were observed in 6% of dead embryos. Overexpression of two copies of UAS-Nemo with 69B-Gal4 also results in embryonic lethality with variable early phenotypes, including denticle belt defects, failure to produce cuticle and defects in dorsal closure (Verheyen, 2001).

The role of the Drosophila TAK homologue dTAK during development

An examination was carried out to see whether directed overexpression of TGF-ß activated kinase 1 in the eye imaginal disc of third instar larvae (at the time of planar polarity Fz/JNK signaling) can interfere with the correct establishment of planar polarity. To this end UAS-Tak1 was expressed in photoreceptor precursors R3/R4 in the eye imaginal disc (under the sev-enhancer GAL4 driver: sev>Tak1). This type of overexpression creates specific eye planar polarity phenotypes with Fz, Dsh and other components of planar polarity signaling. Weak Tak1 expression (by rearing the flies at 18°C) causes a specific phenotype reminiscent of that caused by the components of planar polarity signaling, with polarity defects affecting both rotation and chirality, and also some loss of photoreceptors. This phenotype is already evident with the appropriate markers (e.g. svp-lacZ) at the time of planar polarity establishment in the third instar eye disc, indicating that it is a primary defect in polarity establishment, and not due to late differentiation defects (Mihaly, 2001).

The GOF sev>Tak1 phenotype provides a tool to test for genetic interactions with mutations in components of the Fz/planar polarity pathway and other signaling cascades. In such genetic interaction assays, it was found that reducing the dosage of the JNK signaling components (hep, bsk and D-jun) causes a strong suppression of the sev>Tak1 phenotype. These results are consistent with Tak1 acting upstream of the JNK module in polarity signaling, and support the notion that Tak1 can act generally upstream of JNK signaling (Mihaly, 2001).

Several other signaling pathways and kinases were tested for genetic interaction with sev>Tak1. Whereas no interaction with components of the Dpp signaling pathway was found, dominant suppression, comparable to that of the JNK components, was found with deficiencies removing the p38 kinases (p38a and p38b) and mutant alleles of nemo. These interactions suggest that the sev>Tak1 eye phenotype depends in part on the activities of these other MAPKs as well and is consistent with the previously reported tissue culture experiments. The observation that the sev>Tak1 phenotype is less sensitive to the dosage of msn/STE20 might indicate that msn is acting upstream of Tak1 (Mihaly, 2001).

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGF-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. Ectopic expression of Nemo using the Gal4-UAS system results in embryonic lethality associated with defects in patterning and head development. An analyses of nemo phenotypes of germline clone-derived embryos is described. Lethality is observed associated with head defects and reduction of programmed cell death and it is concluded that nemo is an essential gene. Data is presented showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Genetic data from the adult wing are presented that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFß and JNK signaling at multiple stages of development (Mirkovic, 2002).

Nemo is conserved across species and its role in various Wnt pathways has been studied. It is thought that Nemo inhibits Wnt-dependent gene expression and plays an inhibitory role in morphogenesis. Since Wg signaling has a pro-survival role in many tissues, it is not surprising that Nemo, as a negative regulator of Wg, would induce apoptosis (Mirkovic, 2002).

Mutation of the C. elegans nmo-like gene lit-1 leads to a maternal-effect embryonic lethal phenotype, suggesting that lit-1 is required for sustained organismal survival. In addition, lit-1 RNAi which eliminates both maternal and zygotic lit-1 results in embryonic lethality. Targeted disruption of Nlk in mice has pleiotropic effects that are strongly influenced by the genetic background in which the mutants are studied. In one case, Nlk^-/- mice died in utero due to uncharacterized causes. In a background in which the Nlk^-/- mice survived, they were found to be growth retarded, have defects in mesenchymal stem cell differentiation and died in the second month of life. While these data do not establish Nlk to be essential, the fact that in certain genetic backgrounds the animals are inviable strongly suggests that Nemo plays important roles in early development. The studies of the embryonic lethal phenotype of Drosophila nmo germline clones all strongly implicate Nlks as being very important regulators of cell growth, patterning and death. It can also be argued that elimination of maternal and zygotic Nemo-like proteins results in organismal demise in all three organisms, albeit not under all conditions (Mirkovic, 2002).

The finding that Drosophila nmo germline clones are lethal is also significant because it establishes an embryonic loss of function phenotype. The observation that in the embryo both loss of Nemo function and ectopic expression can modulate programmed cell death strongly supports the model in which Nemo normally is involved in promoting apoptosis. This role for Nemo is consistent with the proposed model in which Tak1, the only known activator of vertebrate Nlk, is also able to induce apoptosis. The studies establishing a role for dTak1 in apoptosis have relied on expression of transgenes, which sometimes generate effects that are not physiologically relevant. dTak1 are viable and only manifest defects in host innate immunity. This finding is surprising in light of both the effects seen upon ectopic expression of wild-type and mutant forms of dTak1, and the biochemical evidence for roles in numerous vertebrate signaling pathways. It is possible that loss of dTak1 can be compensated for by another kinase molecule. The strong evidence from analysis of C. elegans Tak1(mom-4) and lit-1 indicating that these genes function together in modulating Wnt signaling and the findings that Drosophila Nemo, like ectopically expressed dTak1, regulates cell death suggest that it is still possible that these genes function in a developmentally important signaling cassette (Mirkovic, 2002).

During analysis of the phenotype of germline clones of nmo and ectopic expression of Nemo it was determined that Nemo regulates cell death. Ectopic Nemo expression causes apoptosis in the embryonic epidermis as detected by elevated levels of acridine orange (AO) staining, especially in the head region. Conversely, germline nmo clones display reduced levels of AO staining in the head region. This finding is significant because mutations in other genes involved in controlling apoptosis, such as hid, result in defects in the head (Mirkovic, 2002).

It has also been determined that Nemo plays a role in apoptosis during retinal development, since nmo loss of function alleles contain additional secondary and tertiary pigment cells, which are normally removed through programmed cell death during retinal maturation. The ectopic expression of the pro-apoptotic gene reaper in the developing eye disc results in elevated levels of cell death as evidenced by a severely reduced and abnormal adult eye. Heterozygosity for several alleles of nmo can suppress the phenotype resulting in a larger adult eye. The ability of nmo to suppress the cell death caused by GMR-rpr expression supports the idea that both rpr and nmo are involved in promoting cell death and may act in parallel pathways that converge on regulation of the caspases. The data strongly implicate Nemo in the modulation of cell death within the retina and are consistent with observations in the embryo (Mirkovic, 2002).

Nemo proteins have been found to play a role in regulating Wnt signaling. Thus, it is interesting that characterization of segment polarity mutants revealed that both wg and arm mutant embryos have elevated levels of cell death. These findings imply that those gene products normally act to inhibit cell death at least during embryogenesis. Further evidence that Wnt signaling in Drosophila acts to promote cell viability comes from a genetic screen in which dominant modifiers of arm were identified. In this screen, genes were identified that could rescue the severe arm^XP33 embryonic cuticle phenotype. Heterozygosity for hid shows a dominant suppression of aspects of the phenotype, suggesting that at least some of the arm phenotypes are due to excess apoptosis. Furthermore, expression of the baculoviral caspase inhibitor p35 in the arm mutant background also rescued the phenotype, establishing that the rescue observed in both cases was due to lowering the amount of apoptosis. These data which imply that Wnt signaling opposes apoptosis, combined with the finding that Nemo promotes apoptosis, suggest a possible mechanism whereby Nemo can promote cell death by inhibiting Wnt signaling (Mirkovic, 2002).

It is possible that Nemo exerts a pro-apoptotic effect through repression of EGFR activity in either eye or wing development. Nemo mutant phenotypes mimic EGFR gain of function phenotypes and nmo^adk pupal wings display ectopic rhomboid expression. Rhomboid acts to facilitate EGFR signaling and Nemo can inhibit rhomboid expression, thus by extension Nemo may inhibit EGFR activity in general. Thus, ectopic expression of Nemo could cause cell death through inhibition of the pro-survival role of EGFR (Mirkovic, 2002).

During wing patterning, relative levels of Wg and Dpp morphogen gradients are crucial for establishment of correct positional information and cell fate specification within the wing blade. A connection has been observed between the relative levels of Wg and Dpp during wing formation and the activation of JNK-mediated apoptosis, such that modulation of Dpp signaling can both inhibit and stimulate programmed cell death. An important aspect of these findings is that JNK signaling does not normally act in the wing, but that perturbation in levels of Dpp and Wg triggers its activity. It is possible that Nemo signaling normally also does not modulate JNK activation. However, reduction in JNK activity would present a sensitized background for nmo^adk alleles and it is proposed that at least a subset of Nemo roles may require JNK signaling. It is important to note that JNK-mediated apoptosis in the wing is specific to Wg and Dpp signaling, since mutations in other pathways such as Ras and Raf induce apoptosis through JNK-independent mechanisms. The finding of phenotypic rescue by JNK pathway members of the nemo phenotype supports the model in which Nemo is positioned at the intersection of these pathways (Mirkovic, 2002).

Nemo is required in a subset of photoreceptors to regulate the speed of ommatidial rotation

Both dramatic and subtle morphogenetic movements are of paramount importance in molding cells and tissues into functional form. Cells move either independently or as populations and the distance traversed by cells varies greatly, but in all cases, the output is common: to organize cells into or within organs and epithelia. In the developing Drosophila eye, a highly specialized, 90° rotational movement of subsets of cells imposes order by polarizing the retinal epithelium across its dorsoventral axis. This process was proposed to take place in two 45° steps, with the second under control of the gene nemo (nmo), a serine/threonine kinase. While this analysis confirms that these subsets of cells, the ommatidial precursors, do stall at 45°, it was demonstrated that nmo is also required through most of the first 45° of rotation to regulate the speed at which the ommatidial precursors move. In addition, although the precursors reach only the halfway point by the end of larval life, this work demonstrates that patterning events that occur during pupal life move the ommatidial units an additional 15°. A re-analysis of nmo mosaic clones indicates that nmo is required in photoreceptors R1, R6 and R7 for normal orientation. This work also demonstrates that two major isoforms of nmo rescue the nmo^P1 phenotype. Finally, a dominant modifier screen of a nmo misexpression background identified genomic regions that potentially regulate rotation. The results presented here suggest a model in which a motor for rotation is established in a nemo-dependent fashion in a subset of cells (Fiehler, 2008).

nmo/NLK/lit-1 regulates transcription in both vertebrates and invertebrates. In vertebrates, NLK modifies the ability of the transcription factor Tcf/Lef to bind DNA. In the Drosophila wing, nmo modifies the capacity of wg to regulate the transcription of its target genes. As reported in this study, GFP-tagged Nmo becomes prominent in the nuclei, where it likely regulates transcription of key ommatidial rotation genes (Fiehler, 2008).

The mechanism by which nmo expression is regulated in the eye likely differs from its mode of regulation in the wing, where nmo is a direct target of wg. In the eye, nmo is unlikely to be a direct target of wg for two reasons. (1) nmo expression in the eye does not flank wg expression as it does in the wing. Instead, nmo is expressed in a stripe posterior to the morphogenetic furrow, whereas wg is expressed at the dorsal and ventral poles ahead of the furrow. (2) Overexpression of wg in the eye fails to produce ectopic nmo. Non-canonical Wnt signaling, however, has been implicated in driving nmo expression in the eye (Fiehler, 2008).

The target of nmo activity also remains unclear. Possible targets, based on both published data as well as data provided in this study, include Tcf/Lef and JNK. The data point towards JNK as the best candidate as a target for nmo in the eye. Alternatively, nmo may target a distinct transcription factor, or it may play a novel role in directing rotation, independent of transcriptional regulation (Fiehler, 2008).

The net movement of nmo mutant ommatidia in a positive direction during pupal eye development is curious, and, at this point, it is not known what mechanism underlies this directed movement. However, the fact that ommatidia do exhibit a net movement indicates that the overall direction of movement is not due to chance. Rather, perhaps there is an internal compass with which the ommatidia attempt to align. This phenotype is reminiscent of the sev > nmo phenotype, in which ommatidia also exhibit a tendency to align along the north-south axis (Fiehler, 2008).

At the commencement of rotation, the precluster consists of photoreceptors, R8, R2, R5, R3 and R4 (Fiehler, 2007). Since nmo is not required in these five cells, and rotation does initiate in nmo mutants, this suggests the mechanism that starts rotation is nmo-independent. This nmo-independent mechanism appears to be capable of driving rotation for 45°, as ommatidial precursors rotate 45° in nmo^P1 mutants. However, the rate of rotation is significantly slower in nmo mutants relative to wild type. This indicates the nmo-independent mechanism is inefficient in its capacity to turn ommatidia at a normal pace in the absence of nmo. It further suggests that nmo supplements the nmo-independent mechanism, apparently by accelerating the rate. Rotation stops altogether at row 11 (45°) in the nmo mutant, suggesting that the nmo-independent mechanism does not operate beyond this point (perhaps because the mechanism gets blocked), and that, in wild type, the nmo-dependent mechanism is a non-redundant driving force from rows 11 to 15. If a similar, supplementary mechanism operates from row 11 on, it apparently has no compensatory effect in the absence of nmo function, as movement stalls at this stage (Fiehler, 2008).

The specific role for nmo in ommatidial rotation has not yet been uncovered. A model is proposed for nmo function in which nmo helps to establish the 'rotation machinery', or 'motor', at the interface between moving and stationary cells, as follows. This model is based on two hypotheses (Fiehler, 2007): (1) it is hypothesized that the motor resides at the interface between those cells that rotate (the photoreceptors and cone cells) and those that remain stationary (the undifferentiated cells between ommatidial precursors); (2) since this interface is dynamic, changing as cells are recruited into the assembling ommatidium, it is hypothesized that the driving force for rotation should shift to the outermost rotating cells as new cells are incorporated into the cluster. In the model described below, it is important to note that details regarding both the rows at which photoreceptors are added and the number of degrees ommatidial precursors have rotated at the time subsets of cells are recruited are 'best estimates'. The inherent nature of eye development - in particular the equator-lateral and anterior-posterior gradients - precludes the possibility of providing precise values, since these gradients introduce variability in terms of the timing of events. Given this variability, the relative timing of events is emphasized in discussing the model that follows rather than tying events to specific points in time (Fiehler, 2008).

Placing the results of the mosaic analysis and the rates of rotation in nmo mutants in the context of (1) what is currently known about eye development and (2) the hypotheses outlined above leads to the following model. It is proposed that the nmo-independent component of the motor is housed in photoreceptors R8, R2, R5, R3 and R4, and that this motor resides at the interface between all or a subset of these five photoreceptors and the interommatidial cells. In this model, as additional cells get recruited, they block the nmo-independent mechanism and consequently affect the output of this nmo-independent motor. For example, the recruitment of R1, R6 and R7 at approximately row 5 (one row after initiation of rotation) completely blocks R8, and partially to completely blocks R2 and R5, from contact with the stationary cells. This creates a new interface between rotating and stationary cells. Given that the photoreceptor cluster continues to move an additional ~38° once R8, R2 and R5 are blocked, it seems likely that R3 and R4 play a more significant role in the first 45° of rotation than do R8, R2 and R5 (Fiehler, 2008).

The mosaic analysis presented in this study indicates a requirement for nmo in photoreceptors R1, R6 and, to a less significant extent, R7. In addition, rotation proceeds more quickly for the first 45° in wild type than in nmo mutants, and in nmo mutants, rotation stalls at 45°. The data and arguments presented thus far suggest that R1, R7 and R6 actively take place in rotation via a regulatory mechanism distinct from R3 and R4, and that this secondary, R1/R6/R7, nmo-dependent mechanism not only speeds up rotation during the first 45° but is also essential to move ommatidial precursors the second 45°. It is therefore proposed that nmo is involved in either setting up or transferring the motor from R8/R2/R5 to R1, R7, and R6, and later from the photoreceptors to the cone cells, as described below (Fiehler, 2008).

Genetically wild-type R1, R6 and R7 cells do not completely rescue rotation, raising the interesting possibility that other cells also play a critical role in ommatidial rotation. The anterior and posterior cone cells are recruited following R1, R6, R7, at approximately row 6/7 in wild type (~55°). In nmo, the anterior and posterior cone cells are added well before 55° since nmo mutant ommatidia rotate more slowly. Addition of the anterior and posterior cone cells occludes contact between photoreceptors R1, R6 (and, in some cases, minor portions of R2/R5 and R3/R4) and the interommatidial cells, so at this point in development, R3, R4, R7 and the anterior and posterior cone cells constitute the rotation interface. The polar cone cell is recruited at approximately 80% rotation in wild type, but, given the slower rate of rotation in nmo, this translates to only 45° of rotation in nmo mutant discs. Given that the polar cone cell blocks R3 and R4 from their contact with the interommatidial cells, what is proposed to be the only functional motor in nmo, the nmo-independent motor in R3 and R4, can no longer provide the driving force for rotation in nmo mutants once the polar cone cell is recruited. It is therefore argued that the mechanism underlying the derailment of rotation is that nmo either fails to relocate or otherwise establish the motor in the new interface cells, initially R1, R6 and R7, and ultimately the anterior, posterior and polar cone cells (Fiehler, 2008).

It is also interesting to note that rotation in wild type consists of an early, fast phase and a later, slow phase, and that the slow phase correlates with addition of the anterior and posterior cone cells (Fiehler, 2007). This observation is consistent with the model in that addition of the cone cells in wild-type blocks cells that are proposed to house the more efficient or faster rotation machinery (Fiehler, 2008).

Drosophila nemo promotes eye specification directed by the retinal determination gene network

Drosophila nemo (nmo) is the founding member of the Nemo-like kinase (Nlk) family of serine-threonine kinases. Previous work has characterized nmo's role in planar cell polarity during ommatidial patterning. This study examined an earlier role for nmo in eye formation through interactions with the retinal determination gene network (RDGN). nmo is dynamically expressed in second and third instar eye imaginal discs, suggesting additional roles in patterning of the eyes, ocelli, and antennae. Genetic approaches were used to investigate Nmo's role in determining eye fate. nmo genetically interacts with the retinal determination factors Eyeless (Ey), Eyes Absent (Eya), and Dachshund (Dac). Loss of nmo rescues ey and eya mutant phenotypes, and heterozygosity for eya modifies the nmo eye phenotype. Reducing nmo also rescues small-eye defects induced by misexpression of ey and eya in early eye development. nmo can potentiate RDGN-mediated eye formation in ectopic eye induction assays. Moreover, elevated Nmo alone can respecify presumptive head cells to an eye fate by inducing ectopic expression of dac and eya. Together, these genetic analyses reveal that nmo promotes normal and ectopic eye development directed by the RDGN (Braid, 2008).

This study describes novel roles for nmo in early eye patterning that are distinct from its known role in planar polarity during late larval development. The RDGN is composed of a highly complex cascade of positive feedback loops. The fundamental refinement of this delicate system is apparent from the dramatic defects resulting from reducing or ectopically expressing even a single component. Through loss-of-function and misexpression analyses, genetic evidence is provided that nmo contributes to patterning events orchestrated by the RDGN during eye development (Braid, 2008).

Co-expression of the RD genes is spatially and temporally regulated and confers cellular identity through the consequential formation of selector complexes. For example, So and Eya complex to activate dac expression. Subsequently, Dac can complex with So or Eya to direct expression of complex-specific gene targets. In addition, Ey and So complex to activate ato in cells entering the MF. Repression of ey in, and posterior to, the MF limits this interaction to the pro-neural cells. Spatio-temporal regulation of the RD genes is imperative for normal eye and head development, given the deleterious effects of their misexpression on normal eye development. It has been proposed that the availability and relative concentrations of these cofactors affect which protein-protein complexes form. As such, misexpression of the RD genes alters the pool of available cofactors, resulting in mis-specification of cell fate (Braid, 2008).

Interestingly, reducing any of the eye-specification factors also results in patterning defects, culminating in cell death and loss of tissue. Thus, reducing an RD factor may be analogous to its misexpression since the relative levels of RD factors are similarly perturbed, leading to abnormal development and hyperactivation of apoptosis. The data support such a model, since loss of nmo restores eye- and head-patterning defects associated with loss of ey and eya, as it does with early misexpression of these genes. The ey and eya alleles used in this study are not nulls and therefore may retain some level of activity. These interactions imply that reducing nmo can modulate the transcriptional output of RD complexes, restoring developmental integrity. Moreover, inhibiting apoptosis with co-expression of the caspase-inhibitor p35 did not phenocopy this rescue, further supporting the hypothesis that Nmo may contribute to eye development by affecting the activity of RD selector complexes rather than by generally promoting cell death (Braid, 2008).

Although driving nmo throughout the eye disc in all stages of development with ey-Gal4 has minimal effects on its own, and misexpression of ey or eya causes only small eyes, the combined presence of Nmo and Ey or Nmo and Eya is not compatible with eye and head development. This dramatic synergy, together with the rescue mediated by reducing nmo, is consistent with a model in which Nmo affects the function of one or more of the RD cofactors, thereby affecting the balance of selector factors. This study established that Nmo does not regulate Ey, so, Eya, or Dac levels in somatic clones, supporting the hypothesis that the observed genetic interactions occur at the protein level. Whether nmo is itself regulated by the RDGN is yet to be determined (Braid, 2008).

The context-specific nature of Nmo's role in mediating RD activity was revealed in the ectopic eye induction assay. Misexpression of ey using dpp-Gal4 not only induced ectopic eyes in the antennal, wing, and leg discs, but also interfered with endogenous eye development. Ectopic nmo rescued the dorso-ventral reduction in dpp>ey compound eyes, suggesting that Nmo promotes eye development. It further implies that Nmo may differentially affect Ey activity through cell-specific factors, since early co-expression of nmo with ey>ey had the converse effect, resulting in ablation of the eye and head. Spatial restriction of cofactors to achieve different outcomes is a common developmental strategy. nmo's dynamic pattern of co-expression with Ey, and their complementary expression in the third instar eye and head fields, respectively, supports the hypothesis that Nmo may promote early Ey activity to specify the eye field, while later contributing to patterning of the eye field by antagonizing Ey (Braid, 2008).

Using ectopic eye induction assays, Nmo's contribution to eye development was investigated in cells expressing exogenous Ey, Eya, and Dac. Endogenous nmo potentiates the induction of ectopic eyes in the antennal disc, as well as in the leg and wing. Interestingly, it was found that loss of nmo restricts the ability of Ey, more than Eya or Dac, to induce ectopic eyes. Ey is most potent inducer of ectopic eyes as it can effectively activate transcription of the downstream RD targets. Eya, Dac, and So are much less effective in ectopic eye assays because their transactivating potential is limited by the number of available RD cofactors. Thus, it is expected that misexpressed ey would have the least requirement for nmo in the dpp>ey assay. This finding suggests that Nmo may contribute to deployment of the RDGN by Ey, since cells with exogenous Eya or Dac more readily compensate for loss of endogenous nmo than Ey in the induction of ectopic eyes (Braid, 2008).

The most convincing evidence for Nmo's role in early eye specification is Nmo's ability to respecify a specific set of head cells as retinal cells when misexpressed alone. Importantly, these are the same subsets of cells able to be transformed by ectopic expression of RD genes and Tsh, which induces ey expression. Ectopic eyes induced by other factors such as Optix or Eyegone (Eyg), which promote eye specification through Ey-independent mechanisms, occur in different subsets of cells. This study determined that dac and eya are inappropriately activated in cells transformed by misexpressed nmo. It is tempting to speculate that ectopic Nmo perturbs the basal protein-protein interactions that normally repress them, resulting in deployment of the RDGN in the head primordia. Consistent with this model, loss of Hth was observed in cells ectopically expressing dac. (Braid, 2008).

The ectopic eye induction assay has been utilized to determine epistasis among the RD factors. Although loss of Hth was observed in dpp>3xnmo wing discs, this repression does not culminate in activation of any of the retinal genes. This is consistent with clonal analyses that demonstrate that nmo is not required for expression of the RD genes in the eye disc. Moreover, Nmo antagonizes Dpp and Wg signaling in the wing disc, both of which contribute to regulation of hth expression in the wing hinge. Thus, the observed loss of Hth in dpp>3xnmo eye and wing discs may be the result of different mechanisms. For example, elevated Nmo may promote Eya function to repress hth in the antennal disc. Repression of Hth is not sufficient to deploy the RDGN; therefore Nmo requires the presence of an unidentified factor in the antennal disc to activate eye development (Braid, 2008).

This study showed that nmo is not required for expression of Ey, so, Eya, or Dac or the secreted morphogen dpp. In the eye disc, Wg actively represses eya, so, and dac to antagonize progression of the eye field and promote head development. It has been previously showed that nmo is an inducible feedback inhibitor of Wg signaling in the wing imaginal disc. Although nmo expression is not coincident with wg in the ME during eye development, it was important to verify that the observed genetic interactions between Nmo and the RDGN are not due to repression of Wg signaling. Using mutant clonal analysis, it was confirmed that, as in the wing, Wg levels are unchanged in both somatic and flp-out nmo clones. Furthermore, no change was observed in Wg activity as assayed by stabilization of cytoplasmic Arm. These observations are consistent with a previous study indicating that nmo does not modulate Arm stability in the eye imaginal disc. It will be interesting to determine what unidentified factors are affected by loss of nmo, and how they contribute to patterning of the eye field (Braid, 2008).

Novel targets and modes of regulating RDGN activity are rapidly emerging. Recent studies have expanded the repertoire of transcriptional targets regulated by specific RD complexes beyond the scope of the RDGN itself. Moreover, additional proteins have been identified that modify activity of the canonical retinal factors by various mechanisms. For example, Ey acts as a transcriptional activator when bound to So. However, Ey represses the very same target genes when complexed to Tsh and Hth. Alternatively, the So-Eya interaction is physically inhibited when So is in complex with the transcriptional corepressor Groucho (Gro). In addition, Distal antenna (Dan) and Distal antenna related (Danr) were recently identified as retinal factors that complex with Ey and Dac to promote retinal specification through activation of ato. Whether Nmo directly modulates RDGN output through protein-protein interactions that alter the stoichiometry of available RD cofactors (through post-translational modification of their activity by phosphorylation or indirectly by interactions with noncanonical RDGN regulators) is being investigated. Further characterization of the molecular interactions between Nmo and the RD factors will aid in understanding how cells integrate multiple signals to achieve a specific outcome (Braid, 2008).

NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator

The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by Clock (Clc) and Cycle (Cyc) and repressed by Period (Per) and Timeless (Tim). The timing of Clk-Cyc activation and Per-Tim repression is regulated posttranslationally, in part through rhythmic phosphorylation of Clk, Per, and Tim. Although kinases that control Per and Tim levels and subcellular localization have been identified, additional kinases are predicted to target Per, Tim, and/or Clk to promote time-specific transcriptional repression. A screen was carried out for kinases that alter circadian behavior via clock cell-directed RNA interference (RNAi) and the proline-directed kinase nemo (nmo) was identified as a novel component of the circadian oscillator. Both nmo RNAi knockdown and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. Clk levels increase when nmo expression is knocked down in clock cells, whereas Clk levels decrease and Per and Tim accumulation are delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering Clk, Per, and Tim expression, thereby contributing to the generation of an ~24 hr circadian period (Yu, 2011).

This study has identified nmo as a new component of the Drosophila circadian oscillator. The short-period behavioral rhythms of nmo^P1/Df and nmo RNAi flies indicates that Nmo acts to slow the pace of the circadian oscillator, consistent with the lengthening of circadian period when nmo is overexpressed in clock cells. A nmo P[lacZ] enhancer trap line reveal nmo expression in the sLN_v and other brain clock cells, consistent with the short-period behavioral rhythms that result from expressing nmo RNAi in LN_vs. Nmo is present in complexes with Per, Tim, and Clk when Clk-Cyc transcription is repressed and alters Per, Tim, and Clk levels and/or phosphorylation state. These results suggest that Nmo acts within Per-Tim-Clk regulatory complexes to lengthen circadian period. These results are likely relevant to the mammalian circadian oscillator because mPer and Clock are also highly phosphorylated when transcription is repressed and a single nmo ortholog, Nemo-like kinase (NLK), is present in mice and humans (Yu, 2011).

EVOLUTIONARY HOMOLOGS

In C. elegans, the early embryo contains five somatic founder cells (known as AB, MS, E, C and D) which give rise to very different lineages. Two simply produce twenty intestinal (E) or muscle (D) cells each, whereas the remainder produce a total of 518 cells, which collectively contribute in a complex pattern to a variety of tissues. A central problem in embryonic development is to understand how the developmental potential of blastomeres is restricted to permit the terminal expression of such complex differentiation patterns. lit-1 appears to play a central role in controlling the asymmetry of cell division during embryogenesis in C. elegans. Mutants in lit-1 suggest that its product controls up to six consecutive binary switches which cause one of the two equivalent cells produced at each cleavage to assume a posterior fate. Most blastomere identities in C. elegans may therefore stem from a process of stepwise binary diversification (Kaletta, 1997).

During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. The WRM-1 protein binds to LIT-1 in vivo and WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. These findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway (Rocheleau, 1999).

The signaling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signalling downregulates POP-1 activity in one posterior daughter cell called E. The genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1: Drosophila homolog TGF-ß activated kinase 1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. It is concluded that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, since TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1 (Meneghini, 1999).

In C. elegans, a Wnt/WG-like signaling pathway down-regulates the TCF/LEF- related protein, POP-1, to specify posterior cell fates. Effectors of this signaling pathway include a beta-catenin homolog, WRM-1, and a conserved protein kinase, LIT-1. WRM-1 and LIT-1 form a kinase complex that can directly phosphorylate POP-1, but how signaling activates WRM-1/LIT-1 kinase is not yet known. mom-4, a genetically defined effector of polarity signaling, encodes a MAP kinase kinase kinase-related protein that stimulates the WRM-1/LIT-1-dependent phosphorylation of POP-1. LIT-1 kinase activity requires a conserved residue analogous to an activating phosphorylation site in other kinases, including MAP kinases. These findings suggest that anterior/posterior polarity signaling in C. elegans may involve a MAP kinase-like signaling mechanism (Shin, 1999).

The Wnt signaling pathway regulates multiple aspects of the development of stem cell-like epithelial seam cells in C. elegans, including cell fate specification and symmetric/asymmetric division. This study demonstrates that lit-1, encoding the Nemo-like kinase in the Wnt/β-catenin asymmetry pathway, plays a role in specifying temporal identities of seam cells. Loss of function of lit-1 suppresses defects in retarded heterochronic mutants and enhances defects in precocious heterochronic mutants. Overexpressing lit-1 causes heterochronic defects opposite to those in lit-1(lf) mutants. LIT-1 exhibits a periodic expression pattern in seam cells within each larval stage. The kinase activity of LIT-1 is essential for its role in the heterochronic pathway. lit-1 specifies the temporal fate of seam cells likely by modulating miRNA-mediated silencing of target heterochronic genes. It was further shown that loss of function of other components of Wnt signaling, including mom-4, wrm-1, apr-1, and pop-1, also causes heterochronic defects in sensitized genetic backgrounds. This study reveals a novel function of Wnt signaling in controlling the timing of seam cell development in C. elegans (Ren, 2010).

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

Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. Nlk, a murine homolog of the Drosophila nemo (nmo) gene, has been cloned. The Nlk amino acid sequence is 54.5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4.0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60%-70% of Nlk is localized to the nucleus, whereas 30%-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks (Brott, 1998).

The stromal compartment of the bone marrow is composed of various cell types that provide trophic and instructive signals for hematopoiesis. The mesenchymal stem cell is believed to give rise to all major cellular components of the bone marrow microenvironment. Nemo-like kinase, Nlk, is a serine-threonine kinase that connects MAP kinase and Wnt signaling pathways; its in vivo function in mouse is unknown. Mice have been generated with a targeted disruption of Nlk; the complex phenotype significantly varies with the genetic background. Whereas C57BL/6 mice lacking Nlk die during the third trimester of pregnancy, the 129/Sv background supports survival into adolescence; such mice are growth retarded and suffer from various neurological abnormalities. The Nlk deficiency syndrome includes aberrant differentiation of bone marrow stromal cells. Varying degrees of morphological abnormality, such as increased numbers of adipocytes, large blood sinuses and absence of bone-lining cells are observed in the bone marrow of mutant mice. Nlk deficient mice thus provide a novel model to study the genetic requirements for bone marrow stromal differentiation (Kortenjann, 2001).

Non-canonical Wnt signalling involving TAK1 (TGF-beta-activated kinase) and NLK (Nemo-like kinase) and MAPK

The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and C. elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. This study shows that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway (Ishitani, 1999).

Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor [beta(2)AR-Rfz-2] containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor [beta(2)AR] and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling (Ishitani, 2003a; full text of article).

The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. A specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. This study investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. It was found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity (Ishitani, 2003b; full text of article).

The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. This study reports that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps (Kanei-Ishii, 2004; full text of article).

Genetic studies on endoderm-mesoderm specification in C. elegans have demonstrated a role for several Wnt cascade components as well as for a MAPK-like pathway in this process. The latter pathway includes the MAPK kinase kinase-like MOM-4/Tak1, its adaptor TAP-1/Tab1, and the MAPK-like LIT-1/Nemo-like kinase. A model has been proposed in which the Tak1 kinase cascade counteracts the Wnt cascade at the level of beta-catenin/TCF phosphorylation. In this model, the signal that activates the Tak1 kinase cascade is unknown. As an alternative explanation of these genetic data, whether Tak1 is directly activated by Wnt was explored. It was found that Wnt1 stimulation results in autophosphorylation and activation of MOM-4/Tak1 in a TAP-1/Tab1-dependent fashion. Wnt1-induced Tak1 stimulation activates Nemo-like kinase, resulting in the phosphorylation of TCF. These results combined with the genetic data from C. elegans imply a mechanism whereby Wnt directly activates the MOM-4/Tak1 kinase signaling pathway. Thus, Wnt signal transduction through the canonical pathway activates beta-catenin/TCF, whereas Wnt signal transduction through the Tak1 pathway phosphorylates and inhibits TCF, which might function as a feedback mechanism (Smit, 2004; full text of article).

Wnt/ß-catenin signaling regulates many aspects of early vertebrate development, including patterning of the mesoderm and neurectoderm during gastrulation. In zebrafish, Wnt signaling overcomes basal repression in the prospective caudal neurectoderm by Tcf homologs that act as inhibitors of Wnt target genes. The vertebrate homolog of Drosophila nemo, nemo-like kinase (Nlk), can phosphorylate Tcf/Lef proteins and inhibit the DNA-binding ability of ß-catenin/Tcf complexes, thereby blocking activation of Wnt targets. By contrast, mutations in a C. elegans homolog show that Nlk is required to activate Wnt targets that are constitutively repressed by Tcf. Overexpressed zebrafish nlk, in concert with wnt8, can downregulate two tcf3 homologs, tcf3a and tcf3b, that repress Wnt targets during neurectodermal patterning. Inhibition of nlk using morpholino oligos reveals essential roles in regulating ventrolateral mesoderm formation in conjunction with wnt8, and in patterning of the midbrain, possibly functioning with wnt8b. In both instances, nlk appears to function as a positive regulator of Wnt signaling. Additionally, nlk strongly enhances convergent/extension phenotypes associated with wnt11/silberblick, suggesting a role in modulating cell movements as well as cell fate (Thorpe, 2004).

These results support a role for nlk in the activation of Wnt targets during zebrafish embryogenesis. Overexpressed nlk downregulates two tcf3 homologs, tcf3a and tcf3b, that repress activation of Wnt target genes during neural patterning. This functional interaction with Tcf3 homologs requires wnt8 signaling, and thus probably ß-catenin, consistent with previous data indicating that Nlk specifically interferes with the DNA-binding ability of ß-catenin/Tcf complexes, not that of Tcf alone. Interference with endogenous nlk function reveals important roles in two processes that are regulated by canonical Wnts, mesoderm patterning by wnt8, and patterning of midbrain and forebrain by wnt8b. Since loss of nlk enhances or phenocopies loss of function of these two Wnts, it is concluded that nlk functions as an activator of some canonical Wnt targets in zebrafish. nlk also interacts, directly or indirectly, with non-canonical Wnt pathways, since inhibition of nlk strongly enhances convergent extension phenotypes associated with loss of wnt11 function. A role was uncovered for an unusual wnt8 homolog, wnt8 ORF2, in regulating cell movements during gastrulation (Thorpe, 2004).

Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization

Hippo signaling controls organ size by regulating cell proliferation and apoptosis. Yes-associated protein (YAP; see Drosophila Yorkie) is a key downstream effector of Hippo signaling, and LATS-mediated phosphorylation of YAP at Ser127 inhibits its nuclear localization and transcriptional activity. This study reports that Nemo-like kinase (NLK; see Drosophila Nemo) phosphorylates YAP at Ser128 both in vitro and in vivo, which blocks interaction with 14-3-3 (see Drosophila 14-3-3) and enhances its nuclear localization. Depletion of NLK increases YAP phosphorylation at Ser127 and reduces YAP-mediated reporter activity. These results suggest that YAP phosphorylation at Ser128 and at Ser127 may be mutually exclusive. It was also found that with the increase in cell density, nuclear localization and the level of NLK are reduced, resulting in reduction in YAP phosphorylation at Ser128. Furthermore, knockdown of Nemo (the Drosophila NLK) in fruit fly wing imaginal discs results in reduced expression of the Yorkie (the Drosophila YAP) target genes expanded and DIAP1, while Nemo overexpression reciprocally increased the expression. Overall, these data suggest that NLK/Nemo acts as an endogenous regulator of Hippo signaling by controlling nuclear localization and activity of YAP/Yorkie (Moon, 2016).

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Search PubMed for articles about Drosophila nemo

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