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 vgQ 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).
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).
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).
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 nmoP 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).
nemp1 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).
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 nmoadk1 and nmoadk2. Since most of the nmoadk1 and nmoadk2 phenotypes are similar, they will collectively be referred to as nmoadk, 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).
nmoadk alleles displayed several defects in the development of the adult wings that are more severe than those observed in the original nmoP allele. The wings are rounder and shorter than wildtype, contain extra vein material and are held away from the body at a 45° angle. nmoP flies also have smaller wings that are held out 15° to 30° from the body (Choi, 1994). The fully penetrant nmoadk 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 nmoP flies (Verheyen, 2001).
The original nmoP allele has been described as a molecular null allele, since no transcript is detected in homozygous pupae. Genetically, the nmoP allele also behaves as a classic null allele since nmoP/Df(3R)pblNR flies have the same phenotype as nmoP homozygotes. The nature of the nmoadk 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 nmoP is a transcript null allele. Interestingly, the two EMS-induced nmoadk 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 nmoP or nmoadk 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 nmoadk 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 nmoadk 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 nmoadk. Conversely, mutations in the negative regulator Hairless (H/+), which normally exhibit shortening of LV, suppress the ectopic veins seen in nmoadk. In addition to interactions in wing veins, H and nmoadk show a synergistic interaction in the macrochaete bristles of the head and notum (Verheyen, 2001).
nmoadk 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 nmoadk in a H/+ background leads to a dramatic enhancement of the H/+ bristle loss phenotype. Since nmoadk 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 nmoadk 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 nmoadk 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 nmoadk. 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 nmoadk 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;nmoadk and px1;nmoadk double mutants were examined for genetic interactions. Flies doubly mutant for nmoadk 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; nmoadk flies (Verheyen, 2001).
The Dpp pathway is also know to have an essential role in wing vein specification. Flies doubly mutant for nmoadk and dppshv (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).
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).
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 armXP33 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 nmoadk 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 nmoadk 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).
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date revised: 15 October 2007
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