Effects of Mutation or Deletion

Table of contents

Notch can function independent of ligands

The Notch pathway mediates cell fate choice in many species and developmental contexts. In the Drosophila mesoderm, phenotypic differences are observed when different components of the pathway are defective. To determine if these differences reflect variations in the signaling pathway or in the persistence of wild-type maternal products, muscle precursors were examined in embryos that lacked both maternally- and zygotically-derived gene products, called holonull embryos. Most holonull neurogenic embryos have the same number and arrangement of extra muscle precursors, but in Notch holonull embryos many additional cells also become muscle precursors. Holonull E(spl), Delta, big brain and neuralized embryos have approximately the same number of nautilus-expressing cells per myogenic cluster as their zygotic null counterparts. For these neurogenic genes, the mutant phenotypes are not visibly altered by wild-type maternal products. In contrast, Notch holonull embryos have more than twice as many nau-expressing cells per cluster as their zygotic null counterparts. This estimate is probably low, because the depth and roundness of the cluster in the Notch holonull embryos makes discerning the inner cells difficult. In fact, dorsal view give the impression that virtually every mesodermal cell in Notch holonull embryos has adopted a nau-expressing, muscle precursor fate. However, lateral views show that the lateral clusters are discrete. Thus Notch is active in cells where its known ligands and downstream effectors are not active. Delta is the only known ligand for Notch that is expressed in mesoderm. Serrate does not serve as a second ligand for Notch during muscle precursor differentiation. These results indicate that Notch acts in two pathways to determine cell fates in mesoderm: the Delta--> Notch--> Suppressor of Hairless--> Enhancer of split signaling pathway and a second pathway that acts independently (Rusconi, 1998).

The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. Notch acts in two pathways to determine muscle precursor fates. The first is the 'standard' Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts; it was of interest to determine if there is a general requirement for the novel pathway. The novel Notch pathway is required for the development of each of five examined cell types. Holonull Notch mutants (mutants null for maternal and zygotic Notch) have a more extreme phenotype than null mutants for Su(H), Delta, neuralized or mastermind. In Notch holonull embryos, clusters of 10 or 15 eve expressing RP2-like cells are found in place of a normal single RP2. The phenotype for the other neurogenic genes is far less severe. Notch and other neurogenic genes are involved in the determination of the mesectoderm and the visceral mesoderm. The Notch holonull phenotype is more severe in both cases than that of other holonull embryos. These results indicate that the novel pathway is a widespread and fundamental component of Notch function. Both Notch pathways operate in the differentiation of the same cell types. In such cases, the novel pathway acts first and appears to set up or limit the size of equivalence groups. The standard pathway then acts within the equivalence groups to limit individual cell fates (Rusconi, 1999).

Notch can function independenent of Suppressor of Hairless

Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. UbxVMB, a visceral mesoderm-specific enhancer of the Ubx gene, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf (Pangolin) binding sites in the UbxVMB enhancer. Thus, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on UbxVMB. A possible target of this activity is Pangolin. Since Pangolin has been shown to be capable of repressing Wingless targets, these results suggest that this repressive activity may be regulated by Notch. It is suggested that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of Pangolin and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with Pangolin (Lawrence, 2001).

These effects of loss of Notch function are not mediated by the Dpp responsive sites but require the integrity of at least one of two Pangolin binding sites on the enhancer, as do other activities of UbxVMB. This regulatory activity of Notch is likely to be different from that which mediates lateral inhibition since the activity of the enhancer is sensitive neither to Su(H) nor to forms of Notch, such as Nintra, that provide constitutive Notch signaling during lateral inhibition. Altogether, these results suggest the existence of an activity of Notch that antagonizes Wg signaling via Dsh. Thus, the removal of Notch function would lower the requirements for Dsh, as was observed. Similar situations have been described before in the development of muscle and peripheral nervous-system precursors in Drosophila and raise the possibility that, in addition to the Frizzled-mediated events, effective Wg signaling requires the downregulation of a Notch signaling event that might be independent of Su(H). These experiments suggest that a possible target of this event is the activity of Pangolin (Lawrence, 2001).

Members of the Pangolin family interact with Arm/ß-catenin to form complexes that can promote the transcription of Wnt/Wg targets in vitro and in vivo. However, with the exception of LEF-1, Tcf family members on their own do not promote the expression of Wnt target genes, and in some instances they can even repress the expression of these targets (Lawrence, 2001 and references therein).

The UbxVMB enhancer has provided a good model for the analysis of the role of Pangolin in Wg signaling. The optimal activity of this enhancer requires canonical Wg signaling via Dsh, Arm, and Pangolin, and for this reason it was surprising to observe that in the absence of Notch the activity of UbxVMB is independent of the canonical Wg pathway. The ability of the loss of Notch function to reverse the effects of the loss of function of Wingless signaling is most clearly demonstrated in the case of the UbxVMBM2 mutant enhancer. The activity of this enhancer is independent of Dpp but displays an absolute requirement for Wg signaling. However, while UbxVMBM2 is completely inactive in dsh mutants, it directs expression of a reporter in N;dsh double mutants (Lawrence, 2001).

The response of UbxVMB to the loss of Notch function, like that to Wg signaling, requires the integrity of at least one of the Pangolin sites in the context of the full enhancer and thus raises the possibility that these sites, and perhaps the activity of Pangolin itself, are the targets of Notch. It might be that the activity of UbxVMB is repressed by Pangolin in a Notch-dependent manner and that to signal efficiently, Wg must antagonize this repression. In the absence of Notch, this repression would not be implemented, which would lead to enhancer activity that is independent of Wg. This can account for the widespread and premature activity of the enhancer as well as the diminished requirements for Wg signaling that are observed in the absence of Notch (Lawrence, 2001).

Several observations indicate that Pangolin can act as a repressor, and recent results on the regulation of dpp expression in the VM of Drosophila support this possibility. The mutation of Pangolin binding sites in a Wg-dependent enhancer of the dpp gene results in spatially deregulated high levels of activity of the enhancer. This finding suggests that in this case Pangolin acts, primarily, as a repressor and that one function of Wg/Arm signaling might be to promote a nonrepressed state. Pangolin is also likely to act as a repressor at UbxVMB since the mutation of one Pangolin site, although lowering the overall levels of activity, expands the spatial domain of activity of the enhancer. These results provide further evidence for this repressive activity and suggest that Notch might be involved in it. However, in contrast to results with the dpp enhancer, the mutation of both Pangolin sites in UbxVMB abolishes enhancer activity. This finding indicates that at this enhancer Pangolin is also required as an activator, together with Arm (Lawrence, 2001).

In Drosophila, Pangolin can indeed behave as a repressor and an activator through interactions with different molecules. These results raise the possibility that its activity as a repressor, through interactions with transcriptional corepressors such as Groucho or CtBP, is modulated by a signaling event that depends on Notch. Wg signaling can thus lead to gene expression in two ways, by transcriptional activation through a Pangolin/Arm complex or by antagonizing the repressive activities of Pangolin. This dual activity of Wg signaling would explain the observations that in N;dsh double mutant embryos enhancer activity, although higher than in dsh mutants, is lower than that in N mutants. Thus, in N;dsh embryos the activity of the enhancer results from a derepression (inactivation of Pangolin repressor complexes) without the concomitant Arm activation mediated by Dsh (Lawrence, 2001).

The observations on UbxVMB parallel others in which Notch has been shown to antagonize Wg signaling independently of Su(H), and they raise the possibility that effective Wg signaling requires an antagonism of this repression. Interestingly, Wg and Dsh can bind to Notch, and therefore, the antagonism could be mediated by conformational changes in Notch induced at the cell surface through direct interactions between these molecules (Lawrence, 2001).

On the basis of these observations, it is suggest that in Drosophila, Wg signaling operates by regulating two molecular events: (1) repression of the expression of Wg targets implemented by Notch and (2) the Shaggy/GSK3-dependent degradation of Arm promoted by the Axin/APC complex. The regulation of both processes might be linked through the activity of Pangolin: the first event maintains its activity as a repressor, while the second one prevents its becoming an activator. Wg binding to Notch would modulate the repressive activity of Pangolin; then, through the activity of members of the Frizzled family of receptors, it would modulate the activity of Arm. It may be that Arm can only interact with a nonrepressor form of Pangolin and that, therefore, the 'effectiveness' of Wg signaling is determined by the amount of Notch signaling. A combination of antirepression and activation might help explain the observation that dominant-negative Frizzled and dominant-negative Pangolin have no effect on the activity of this enhancer, as they should have if activation was the only way to get expression. In addition, this would explain the observation that lowering Notch signaling increases the effectiveness of Arm signaling. It will be important to understand how Notch modulates Wg signaling (Lawrence, 2001).

One difficulty with this model is the activity of the UbxVMB enhancer in the absence of both Notch and Dsh since, under these conditions, the levels of cytoplasmic Arm are low. It may be that the loss of Notch function alters some parameters of the interaction between Pangolin and Arm that allow very efficient functional association of Pangolin with these low levels of Arm. In this regard there is evidence that the phosphorylation of Tcf can regulate the activity of the Tcf/ ß-catenin complex. Further work should address these issues (Lawrence, 2001).

The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. New alleles of Notch are described that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. The loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. These results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices (Raiman, 2001).

In a screen for flies associated with the loss of microchaetae, a number of mutations in Notch were isolated that result in a dominant loss of thoracic microchaetae, which are called NMcd, where Mcd stands for microchaetae defective. These mutations are lethal, and, for this reason, their behavior was analyzed in mosaics in which clones of mutant cells are juxtaposed with wild-type territories. In these mosaics, mutant cells are recognized by the use of both bristle and epidermal markers. All mutants behave genetically in a similar manner, the strongest alleles, NMcd1 and NMcd5 (collectively NMcd1/5), were chosen for further analysis. In clones for NMcd1 and NMcd5, 99% of the microchaetae are absent, whereas macrochaetae are not affected (Raiman, 2001).

Genetic analysis indicates that the dominant effects of the NMcd alleles are due to antagonism of the wild-type function of Notch. The mutant phenotype of NMcd is enhanced when N+ is lowered and is partially suppressed when N+ is increased. Thus, these gain-of-function alleles of Notch do not induce an aberrant function of the receptor (neomorphism), but rather produce receptors that are more active on the normal function of Notch. NAx alleles exhibit a similar genetic behavior and a similar phenotype to the NMcd alleles. However, several differences distinguish NAx from NMcd. The NAx mutant exhibits a variable loss of both thoracic microchaetae and macrochaetae, leading to irregular patterns. In contrast, NMcd affects only microchaetae. Furthermore, the remaining microchaetae of the NMcd/+ flies are arranged in fewer rows, which are organized in a regular pattern. Finally, NAx/+ flies exhibit broader wings with shortened veins. In contrast, the wings of the NMcd/+ flies appear as those of wild-type flies. In this study of the NMcd alleles, focus was placed on the bristle pattern (Raiman, 2001).

A further demonstration of the specificity of the NMcd mutations for microchaetae is seen by analysis of NMcd1/5clones with impaired function of either hairy or extramacrochaetae (emc), two negative regulators of ac/sc. Flies lacking hairy or its cofactor groucho (gro) exhibit ectopic microchaetae in the scutellum region of the thorax. In clones mutant for NMcd1/5 and lacking gro (NMcd1/5 gro-cells), ectopic microchaetae are absent. In contrast, the NAx mutants again behave differently, since, in Ax59b gro- cells, ectopic microchaetae form. The ectopic macrochaetae, which develop in emc1clones, also arise in NMcd1/5 emc1clones, even when their precursors differentiate simultaneously to those of the microchaetae (Raiman, 2001).

In the absence of any component of lateral inhibition, an excess of neural precursors occurs at the expense of epidermis. In Notch-, Su(H)-, and Dl-clones (mosaic animals), the neurogenic phenotype is extreme; all mutant cells adopt the neural fate, and no cells are left to form epidermis. The lack of epidermal mutant cells leads to a wound partially skinned up by wild-type epidermal-surrounding cells. In gro- and E(spl)-, as well as in the hypomorphic Dl clones, the neurogenic phenotype is less severe, and such clones can differentiate tufts of densely packed sensory bristles accompanied by few epidermal cells. Furthermore, mutant cells for loss-of-function alleles of Notch have an enhanced capacity to produce an inhibitory signal that forces neighboring wild-type cells to adopt the epidermal fate. This signal is mediated by Delta. Thus, along the borders of N mutant clones, no bristles are formed by wild-type cells (Raiman, 2001).

Alleles of Notch encoding constitutively activated receptors show the opposite phenotype, with wild-type bristles forming at the border of mutant territories that adopt epidermal fate. The phenotype of the NMcd mutants resembles that of classic gain-of-function alleles of Notch (among which are the NAx alleles) and therefore might result in an activation of the lateral inhibition function. If this were the case, removal of the function of some or all of the mediators of lateral inhibition will abolish the effects of the NMcdalleles. To test this, double-mutant clones were made using the loss-of-function mutations DlRevF10, Dl9P39, Df(3R)E(spl)b32.2, groE48, and Su(H)IB115. In this case, double-mutant clones for NMcd1,5 and components that mediate lateral inhibition [Delta; E(spl)-C; gro; Su(H)] would be predicted to inactivate lateral signaling; they would be predicted to display the neurogenic phenotypes characterized by the lack of mutant epidermal cells. Surprisingly, in all cases, the double-mutant clones display the NMcd1/5 phenotype with mutant epidermis and no microchaetae differentiated. Therefore, NMcdcells do not require Dl, Su(H), gro, or the E(spl)-C in order to adopt the epidermal fate. In contrast, neurogenic double-mutant clones are observed using Ax59bor AxSX1and at least with Dl, gro, and E(spl)-C. The NMcd Ser and NMcd Dl Ser clones display the NMcdphenotype, suggesting that the NMcdphenotype does not require Serrate, the other ligand of Notch (Raiman, 2001).

The macrochaetae can differentiate normally in clones mutant for NMcd. In the absence of lateral signaling (double-mutant clones for NMcd1,5 and one of the components of lateral inhibition [Dl; E(spl)-C; gro; Su(H)]), mutant clones would be predicted to display tufts of macrochaetae (the neurogenic phenotype). Macrochaetae differentiating as single bristles are observed rather than as a neurogenic tuft. These results confirm that the NMcdmutants affect a function of Notch distinct from lateral inhibition (Raiman, 2001).

Since clones of NMcd cells lack microchaetae, the development of their precursors was examined during pupal stages by means of neural-specific markers. The loss of microchaetae observed in NMcd1/5 is due to the loss of neural cells, as visualized by stainings using the neural-specific antibody 22C10, and to the loss of their precursors, as detected with the reporter neuA101. Since the proneural Ac activity is known to promote the development of the microchaetae precursors, Ac expression was examined in the NMcd mutants. The loss of microchaetae precursors is associated with a severe decrease in Ac expression (Raiman, 2001).

The NMcd phenotype is unlikely to be due to a lack of differentiation of the outer elements of the sensory organs, since 'escaped' microchaetae have a normal morphology. Thus, these results indicate that the NMcdmutations disrupt the early establishment of neural precursors rather than the late lineage that permits the differentiation of the sensory bristle (Raiman, 2001).

Different lines of work have suggested that the existence of Notch-signaling events are independent of the mechanism of lateral inhibition. Some of these experiments suggest that the adaptor protein Deltex (Dx) might be involved in some of these events (Raiman, 2001).

Dx is a cytoplasmic protein that regulates Notch through binding to the ankyrin repeats. Loss-of-function alleles of dx display an excess of microchaetae, whereas overexpression of Dx inhibits neurogenesis. It has been suggested that Dx is involved in a signal transduction event downstream of Notch. Loss-of-function dx alleles behave as dominant suppressors of all the NMcd alleles , and NMcd1/5 dx-clones display a fairly normal microchaetae pattern. The Dx effector, therefore, might represent an essential regulator of the antineural activity revealed by the NMcd receptors (Raiman, 2001).

In contrast, Shaggy, the Drosophila glycogen synthase kinase 3 (GSK3) is a central element in Wingless signal transduction and behaves genetically as a downstream element of the Notch pathway. Mutations in Sgg suppress the effects of NMcd mutants, like mutations in Dx. Altogether, these results indicate that both Dx and Sgg might be involved in the Notch-signaling event that is distinct from lateral inhibition (Raiman, 2001).

Investigations at the molecular level show that all NMcd alleles, except NMcd5, encode receptors with C-terminal truncations. NMcd5 is associated with a single C739Y change that disrupts the median disulphide bridge of the 18th EGF repeat of the extracellular domain. The 114 amino acid common region deleted in all the truncated receptors contains a PEST sequence, which is conserved in the Notch family and is involved in protein degradation. The loss of microchaetae is accentuated with the decreasing length of the NICD. In addition to the PEST sequence, the NICD includes additional elements such as the CcN domain. Deletion of different combinations of these elements might therefore explain differences in the severity of the phenotypes observed (Raiman, 2001).

Since Achaete/Scute expression is required for the establishment of the neural fate, the novel Notch pathway revealed by the NMcd mutants must be repressed during wild-type neural development. One candidate to exert this repression is Dishevelled (Dsh), a component of the Wingless-signaling cascade, which has been shown to bind Notch and block some of its activities. Using a yeast two-hybrid assay, it has been found that Dsh does bind to the C-terminal 114 amino acids of the NICD that are absent in the truncated receptors. Therefore, the Dx-dependent repressive effect of the NMcd receptors appears as the consequence of the loss of the Dsh binding site (Raiman, 2001).

Therefore, Notch associates in vitro with Dsh through its C-terminal 114 amino acids. In order to test the functional significance of this C-terminal domain of Notch in vivo, the effect of overexpressed Dsh on the development of microchaetae was examined either in wild-type or in NMcd8 flies lacking the Dsh binding site. Flies carrying four copies of a hsp70-Dsh transgene were analyzed. One 15-min heat pulse (37°C) at the onset of pupariation leads to an increase of 5.8% of the number of microchaetae in a wild-type background. In contrast, the pulse has no effect on NMcd8 flies. These experiments suggest that Dsh binds the 114 amino acid C terminus of Notch in vivo to antagonize the Dx-dependent signaling of the receptor. The effects of overexpressed Dsh were examined in Notch mutant-carrying lesions in the extracellular EGF repeats (nd3; spl;Ax9B2; AxE2). In each case, an increase in the number of microchaetae was observed after heat treatment (Raiman, 2001).

Dsh and Dx display antagonistic activities. Overexpressed Dx inhibits neurogenesis, whereas overexpressed Dsh increases the number of microchaetae in wild-type flies. Furthermore, this latter excess of microchaetae is accentuated when the dosage of Dx is lowered (Raiman, 2001).

NMcd2, NMcd3, NMcd7, and NMcd8 characteristically produce hemizygous escapers showing a strong reduction in the number of microchaetae. However, lateral inhibition is not abolished in these mutants, since the remaining microchaetae are evenly spaced. Consistent with this, Western blot analysis of protein extracts prepared from mutant animals reveals that all NMcd proteins are processed. The resulting NICDs carry intact ankyrin repeats, known to bind Su(H), and therefore could mediate lateral inhibition (Raiman, 2001).

Loss-of-Su(H) alleles behave as dominant enhancers of the NMcd alleles. Dx is a cytoplasmic protein whose activity also relies on binding to the ankyrin repeats. The antagonism between Dx and Su(H) could be explained by a binding competition for the ankyrin repeats of the NICD. Thus, when Su(H) concentration is reduced, Dx signaling is increased and the NMcd phenotype is accentuated. This observation suggests that activity of the Notch receptor depends on the balance between Dx and Su(H) (Raiman, 2001).

Although Deltex has been interpreted as being involved in lateral inhibition, the results of this study make it more likely that it is associated with an alternative signaling event. Dx is a ubiquitous cytoplasmic protein that regulates Notch through binding to the NICD. During lateral inhibition, upon activation by the ligand Dl, the NICD is translocated to the nucleus where it interacts with Su(H) to regulate target genes. However, Su(H) is also present in the cytoplasm, where it displays antagonism with Dx, reflecting a competition to associate to the ankyrin repeats of Notch. Consistently, it has been suggested that Dx may maintain an activated state of Notch indirectly by interfering with the retention of Su(H) in the cytoplasm by virtue of its interaction with the ankyrin repeats of Notch. Moreover, loss-of-functions alleles of Su(H) and loss-of-functions alleles of dx behave, respectively, as dominant enhancers and dominant suppressors of the phenotype of NMcd/+ heterozygous flies. This observation demonstrates that Su(H) and Dx display antagonist activities during N signaling (Raiman, 2001).

The loss of microchaetae in NMcd mutants is accentuated when the number of putative functional domains removed in the NICD is increased. The truncated receptors lack a 114 amino acid fragment required for Dsh to bind to the NICD. This fragment also contains a PEST sequence, which is conserved in the Notch family and which is likely to be involved in protein degradation. Furthermore, a CcN motif is located between the ankyrin repeats and the PEST sequence in the different Notch receptors. It has been shown that the activity of the morphogen Dorsal is negatively regulated by heterodimerization of Dorsal with the ankyrin repeats of the Cactus inhibitor. The proteolysis of Cactus controlled by a PEST domain associated with a CKII site is an essential step for the nuclear translocation of Dorsal and the patterning of the Drosophila embryo. Interestingly, NMcd1 displays the most severe phenotype correlated with the deletion of the CcN motif. The CcN motif contains nuclear-targeting information, and its deletion may explain a reduction of the nuclear import of the NICD, leading to the reinforced cytoplasmic activity of Dx (Raiman, 2001).

When Su(H) concentration is reduced, the cytoplasmic activity of Dx is increased and the NMcd phenotype is accentuated. This observation suggests that the activity of the Notch receptor depends on the balance between Dx and Su(H), and, consequently, any factor that modifies the activity of either pathway would affect bristle pattern. One can speculate that Dsh might play such a role and regulate this balance. Furthermore, the cytoplasmic activity of Su(H) has been reported to stabilize the NICD associated with the membrane, possibly by preventing both Notch ubiquitinylation and the entry of the NICD into the nucleus. Dsh may modulate the phosphorylation status of the NICD, which may favor the binding of Su(H) to the ankyrin repeats and consequently repress the Dx activity (Raiman, 2001).

Potentially, Dsh could exert its repressing effect by modulating the proteasome-dependent proteolysis of Notch or the phosphorylation state versus cytoplasmic/nuclear distribution of the NICD. Interestingly, Dsh contains two proline-rich sequences, PPLP and PPXY, putative binding sites for Su(dx), a cytoplasmic ubiquitin ligase involved in ubiquitinylation/turnover of proteins. When binding to Notch, Dsh could serve as a docking protein for Su(Dx) and could regulate the activity of Dx in targeting the proteasome activity to the C terminus of Notch (Raiman, 2001).

How the Dx-dependent transduction is achieved in the cells is poorly understood. One could speculate that the repressing activity of Dsh may also rely on a direct effect on the Dx-dependent signaling. Thus, Dsh and Dx antagonistically regulate a common target, JNK (JUN N-terminal kinase), and Sgg antagonizes JNK-dependent activation of the JUN transcription factor. dJUN might therefore represent an element mediating the antineural activity of Dx (Raiman, 2001).

The Dx-dependent antineural activity of Notch is regulated by elements of the Wingless-signaling cascade, e.g., the cytoplasmic protein Dsh or the kinase Sgg. Overexpression of Dsh generates extrasensory organs in wild-type flies and fails to elicite ectopic bristles in the NMcdmutants lacking the Dsh binding site. The kinase Sgg is negatively regulated by Dsh in the Wingless-signaling cascade. Dsh and Sgg have opposite effects on the Dx-dependent Notch pathway. Loss-of-function alleles of sgg lead to a constitutive derepression of Wingless signaling and elicit the same number of ectopic bristles in wild-type and NMcd mutant flies (Raiman, 2001).

This analysis of the NMcd mutants supports the idea that Dsh, an effector of the Wingless pathway, directly interacts with Notch in wild-type flies in order to maintain the neural potential. Dsh antagonizes the cytoplasmic activity of Dx and then represses the antineural Dx-dependent function of Notch. In wild-type flies, crosstalks between Wingless and Notch allow stimulation of the ac/sc expression in the equivalent cells of the proneural clusters until a given threshold. It has been reported that Su(H) functions as the core of a molecular switch, acting as a repressor of Notch target genes in the absence of nuclear NICD. Thus, prior to the onset of lateral signaling, the repressive activity of Su(H) is compatible with the activation of ac/sc by the Wingless-dependent pathway. When a given level is reached, ac/sc can activate the Dl gene, and cells can compete with each other for the choice of the neural precursor via lateral signaling. At this stage, the Wg and the Su(H)-dependent Notch signalings have opposite effects on the expression of ac/sc. ac/sc is repressed in the inhibited cells, suggesting that the Su(H)-dependent Notch signaling overrides the Wingless pathway (Raiman, 2001).

Though the NMcd5 allele shares the same loss-of-microchaetae phenotype as other NMcd and affects the same developmental pathway, the NMcd5 mutant receptor carries a single point mutation, leading to the C739Y substitution that disrupts the 18th EGF repeat of the extracellular domain, whereas the other NMcdalleles encode truncated receptors lacking the C terminus of the intracellular domain. Experiments with NMcd5 suggest that the region of the 18th EGF is instrumental for the regulation of alternative Notch signaling. The extracellular EGF domain is known to physically bind Wingless. Further experiments are necessary to determine whether the NMcd5 lesion in the 18th EGF repeat specifically alters the binding of Wingless, Fringe, or other unknown effector(s) (Raiman, 2001).

The present study of NMcd alleles demonstrates that a Deltex-mediated function of Notch represses the proneural activity during establishment of the neural precursors of the thoracic microchaetae. This repressive activity precedes and is distinct from that which mediates lateral inhibition and is constitutively active in NMcd mutants. The NMcd alleles encode truncated receptors that lack the binding domain of the repressor Dishevelled, which is involved in functional interactions between Notch and Wingless signalings. The results suggest a model in which Dishevelled is used to alleviate this initial repressive function of Notch in wild-type development, thereby permitting lateral inhibition to generate the regularly spaced sensory microchaetae. In the absence of ligands or effectors, the repressive function of the Dx-dependent activity of Notch could therefore maintain the cells in an uncommited state. In the presence of effectors like Dsh (Wingless signaling) that repress this antineural activity, cells become competent for further choice between two alternative fates (lateral inhibition). It is proposed that Notch acts during development either as a repressor preventing cell differentiation or as a receptor involved in the choice of cell fate during lateral signaling. This dual function is likely to be regulated in a ligand-dependent manner by crosstalk between the Notch and Wingless pathways. It will be important to find out the different components of this new Dx-dependent repressive cascade of Notch (Raiman, 2001).

Characterization of Abruptex mutation of Notch

The Abruptex class of Notch alleles has attracted interest because they exhibit some properties that are best explained in terms of increased activity and others that are best explained in terms of reduced activity in vivo. A comparison was made of the properties of Abruptex[M1] and wild-type Notch as ligand binding receptors. Abruptex[M1] shows less activity than wild-type Notch in its ability to bind Delta and Serrate and is expressed at reduced levels on the cell surface. When differences in expression level are taken into account, Abruptex[M1] is comparable to Notch in its sensitivity to ligand-induced activation of reporter gene expression. Abruptex[M1] is also comparable to Notch in its requirement for modification by Fringe and in being sensitive to cis-dowregulation by co-expressed ligands. By the available criteria Abruptex[M1] exhibits less activity than Notch. To explain the ectopic activity of Abruptex[M1] in vivo, it may be necessary to invoke an altered response to an as yet unidentified ligand or cofactor (Perez, 2005).

When assayed as a soluble protein, the extracellular domain of Ax[M1] binds Delta less well than the comparable domain of Notch. When expressed in S2 cells, full length Ax[M1] shows lower activity than Notch in binding to a soluble form of Delta. Much of this difference can be attributed to reduced cell surface expression of the Ax[M1] protein. When the reduced level of cell surface Ax[M1] expression is taken into account, Ax[M1] shows comparable activity to Notch in terms of ligand-induced activation of a reporter gene (using cell-surface expressed ligand to more normally reflect the in vivo situation). None of these experiments would suggest that Ax[M1] has more activity than Notch in vivo. For example, reduced cell surface expression of Ax[M1] would be more compatible with reduced activity in vivo, rather than higher than normal activity. These findings are consistent with genetic analyses, which reveal that Ax alleles show some characteristics of reduced function (hypomorphic) alleles, even though they also have phenotypes consistent with ectopic activation of Notch (loss of sensory bristles, truncation of wing veins, overgrowth). For example, Ax[M1] results in lethal neurogenic embryonic phenotypes when combined with a deletion of the Notch locus and the bristle loss of Ax[M1] is suppressed by increasing the dosage of wild-type Notch. The loss of function character of Ax[M1] is temperature sensitive, being stronger at 29°C, consistent with the idea that the C999Y mutation results in an alteration in proteins structure that compromises its function. These biochemical assays suggest that the reduced activity of Ax[M1] seen in some genetic tests in vivo is likely to be due to less receptor present on the surface (Perez, 2005).

Despite the hypomorphic component to Ax[M1], this mutant causes many effects indicative of elevated Notch activity, including ectopic expression of Notch targets Cut and Wg in dorsal and ventral cells of the wing disc. Many of the phenotypes are enhanced by an increase in the dosage of Delta, suggesting that Ax could have altered sensitivity to its ligands in vivo. Since the binding assays showed reduced binding of Ax[M1] to the ligands, it is unlikely that the mutant receptors are able to respond to lower concentrations of Delta or Serrate. An alternative explanation is that Ax[M1] has lost the cis-inactivation caused when ligands are present in the same cells as the receptor. In wild type discs ectopic expression of the ligands primarily results in activation outside the domain of expression, an effect explained by cis-inactivation within the ligand-expressing cells. In Ax[M1] the cis-inactivation is less-evident and there is more widespread activation by the ectopic ligands, although they retain their normal dorsal/ventral distinctions with Serrate activating mainly in the ventral compartment and Delta mainly dorsally. Both Ax[M1] and Notch show a similar degree of reduced surface availability when they are co-expressed with Serrate. Ax[M1] is also sensitive to co-expressed Delta, although less so than wild-type Notch. Given that Serrate is thought to be a more potent cis-antagonist than Delta in vivo, the lack of a difference of the effects of Serrate on Notch and Ax[M1] make it difficult to ascribe ectopic activity of Ax[M1] in vivo to a decreased susceptibility to cis-interaction (Perez, 2005).

The glycosyltransferase Fringe modifies O-linked fucose residues that are attached to many of the EGF repeats within the Notch extracellular domain, including those affected by Ax mutations. Since there are striking differences in the effects of ectopically expressing Fringe in wild-type and Ax mutant discs, it has been suggested that the Ax mutations could result in altered glycosylation by Fringe. Fringe is normally present only in dorsal cells where its modification of Notch makes it insensitive to activation by Serrate and more sensitive to activation by Delta. When expressed ectopically Fringe is able to modify Notch in ventral cells, increasing their sensitivity to ventrally expressed Delta and preventing activation by Serrate. In Ax[M1] discs, ectopic expression of Fringe causes very strong ectopic activation of Notch in the dorsal compartment with a more modest effect in the ventral compartment. This observation is striking and suggests the possibility of a difference in the effects of Fringe on Ax[M1] and Notch. However, it has not been possible to measure a difference in the properties of the Fringe-modified receptors in the in vitro ligand-binding assays. Fringe affects Ax[M1] similarly to Notch in terms of altering their binding to Delta. Thus, even though previous studies have reported that the Ax[M1] and Ax[59] mutations can interfere with the interaction between Notch and Fringe, positive effects of Fringe on Ax[M1] function can be detected. In addition, direct assays of Fringe mediated glycosylation of two Ax mutant molecules revealed a loss of glycosylation in only one of the mutants, arguing against this being a primary cause of the hyperactivation of Ax proteins (Perez, 2005).

How can the gain of function characteristics of Ax[M1] be explained? The fact that ectopic Fringe has such different effects in Ax[M1] and that Ax[M1] partially rescues the phenotype of the fng[D4] allele argues that the amino-acid substitution in Ax[M1] activity should change an aspect of Notch function that is influenced by Fringe. One possibility is that Ax[M1] modified by Fringe might be less sensitive to an inhibitor of Notch activation, hence the gain of sensitivity to Serrate and Delta. Alternatively, Ax[M1] might be very sensitive to an activating ligand other than Serrate or Delta. A third possibility is that there may be some component present in the imaginal disc cells that is lacking from S2 cells and that is required for Fringe-modified Ax[M1] to acquire its apparent gain-of-function activity in vivo. A model is favored in which the effects of Ax[M1] depend on some component expressed in the wing disc, because no evidence is found that any aspect of Ax[M1] function as a receptor for the known ligands is increased in a manner that could explain the properties of this allele in vivo (Perez, 2005).

Notch and muscle differentiation

Notch plays a role in many cell fate decisions in the developing Drosophila embryo, often at successive stages during the formation of a single tissue. In the embryonic mesoderm, Notch is involved in the process by which muscle progenitors are selected from a field of equivalent myoblasts. The roles of Notch in somatic myogenesis have been investigated and it has been demonstrated that Notch can affect at least two additional steps in muscle development. Subsequent to the initial specification of progenitors, myoblast identity remains sensitive to mesodermal Notch activity until the time of fusion. Additionally, Notch is capable of suppressing muscle development nonautonomously by regulating a signal that emanates from the ectoderm (Fuerstenberg, 1998).

Shifts of Notchts1 embryos to a restrictive temperature early in embryogenesis cause hyperplasia of progenitor cells from the time they are first detectable in the embryonic mesoderm (assayed by the expression of S59/NK1). In contrast, later shifts to the nonpermissive temperature have no effect on the initial appearance of single S59-positive cells at stages 11 and 12, but nonetheless disrupt the mature muscle pattern. These data suggest that Notch is required to maintain the wild-type pattern of S59 expression subsequent to its requirement in the initial segregation of progenitor cells. Using the pan-mesodermal twist-GAL4 driver to express a constitutively active NotchdeltaE in the mesoderm, S59-expressing and vg- expressing progenitor cells are not specified in 40% and 50% respectively, of embryos examined. This further supports the idea that Notch must affect a later step in somatic myogenesis. NotchdeltaE also suppresses, in many individuals, the expression of progenitor/founder markers before myoblast fusion would normally occur. Expressing NotchdeltaE in the ectoderm also disrupts myogenesis; there are many unfused myoblasts in the mesoderm, and though some identifiable muscles are present, muscle fibers are often inappropriately positioned or formed (thinned). There is some duplication or loss of identifiable muscles in most hemisegments, and some fused syncitia are rounded and unattached to the epidermis. These results demonstrate that the expression of NotchdeltaE in the epidermis has a tissue-nonautonomous effect on patterning in somatic myogenesis (Fuerstenberg, 1998).

Twist is required in Drosophila embryogenesis for mesodermal specification and cell-fate choice. The roles of Twist and Notch have been examined during adult indirect flight muscle development. The observations suggest that twist repression is a requirement for the initiation of muscle differentiation in some muscles of the fly. Persistent twist expression aborts the development of these muscles and markers of differentiation such as myosin are greatly reduced. Erect wing, a transcription factor required for indirect flight muscle differentiation begins to be expressed as twist expression declines. Reduction in levels of Twist leads to abnormal myogenesis. It is thought that reduction of Twi levels causes premature differentiation and thus results in fewer myoblasts that are correctly positioned to contribute to muscle development. Notch reduction causes a similar mutant phenotype and reduces Twist levels. Conversely, persistent expression, in myoblasts, of activated Notch causes continued twist expression and failure of differentiation as assayed by myosin expression. The gain-of-function phenotype of Notch is very similar to that seen when twist is persistently expressed. Two models are proposed for Notch function:

Until markers for founder cells in adult myogenesis are identified, it will be difficult to distinguish between these two possiblities. An intriguing result obtained in this study of persistent expression of activated Notch and twist is the significant difference in effects on very closely related muscles: the indirect flight muscles (which are sensitive to Notch and Twist levels), and the direct flight muscles (which are not). These two groups of muscles are clonally related and share progenitors at least until the late third larval instar, and then the progenitors differentiate into very different muscle types. Notch activity might function to delineate myoblast precursors of these two groups of muscles (Anant, 1998).

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col expression is first observed in two promuscular clusters (in segments A1-A7), corresponding to two progenitors and then their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3[A]. This lineage-specific restriction depends on the asymmetric segregation of Numb during the progenitor cell division and involves the repression of col transcription by Notch signaling. In col mutant embryos, the DA3[A] founder cells form but do not maintain col transcription and are unable to fuse with neighbouring myoblasts, leading to a loss-of-muscle DA3[A] phenotype. In wild-type embryos, each of the DA3[A]-recruited myoblasts turns on col transcription, indicating that this conversion, accomplished by the DA3[A] founder cell, induces the ‘naive’ myoblasts to express founder cell distinctive patterns of gene expression, activating col itself. Muscles DA3[A] and DO5[A] (DA4[T] and DO5[T] respectively) derive from a common progenitor cell, the DA3[A]/DO5[A] progenitor. However, ectopic expression of Col is not sufficient to switch the DO5[A] to a DA3[A] fate. Together these results lead to a proposal that specification of the DA3[A] muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles (Crozatier, 1999).

Following establishment of the promuscular clusters, specification of the progenitors is controlled by lateral inhibition, a cell-cell interaction process mediated by the neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos, promuscular Col expression is initiated normally but fails to become restricted to a single cell per cluster, similar to observations previously made for the expression of l’sc. As a consequence, a hyperplasic expression of Col is observed from stage 11. Since it is expressed in promuscular clusters and segregating muscle progenitors, l’sc has been proposed to play a role in muscle progenitor selection similar to the role of achaete and scute in neuroblast specification. However, in embryos lacking l’sc activity, selection of the Col-expressing progenitors occurs normally at stage 11 and muscle DA3[A] forms as in wild type (Crozatier, 1999).

The organization and function of the Notch signaling pathway in Drosophila are best understood with respect to the role of this pathway in the process of selection of neural progenitor cells. However, there is evidence that, in addition to neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these progenitor cells is increased in neurogenic mutants. It has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Additional elements of the Notch signaling pathway participate in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells (Giebel, 1999).

To test whether the Notch signaling pathway is involved in myogenesis, the effects of expression of a constitutively active Notch protein (Notchintra) were examined. A second chromosomal effector line with an UAS-Notchintra construct was used. This construct led to complete blocking of neural development upon activation with daG32. Embryos carrying that construct driven by daG32 or by 24B-Gal4, respectively, do not express any of the muscle founder cell markers S59 and Kruppel in the mesoderm. Therefore, it is assumed that no muscle progenitor cells are specified in these animals. Confirmation of this assumption is provided by the observation that no muscle fibers differentiate in these embryos, as shown by means of the expression of a myosin heavy chain (MHC) reporter gene. In mutants where fusion of myoblasts is blocked, founder cells express corresponding founder cell markers, while the non-founder myoblasts remain as undifferentiated rounded cells, which express certain muscle specific genes like myosin. Since Notchintra expressing mesodermal cells are rounded and many of them express the MHC reporter, it is assumed that the MHC expressing cells are non-founder myoblasts that have failed to undergo fusion due to the lack of muscle founder cells (Giebel, 1999).

In view of the myogenic abilities of the proneural gene lethal of scute, whose gene products function as heterodimers with Daughterless, a test was performed to see whether the effects of constitutive activation of Notch could be compensated by the simultaneous Gal4 mediated expression of daughterless, lethal of scute and Notchintra. No difference was found as compared to embryos that do not express UAS-daughterless and UAS-lethal of scute. These results indicate that the Notchintra-mediated inhibitory signals are dominant over the action of both endogenous and exogenous provided proneural proteins. The Gal4 mediated expression of daughterless and lethal of scute by itself causes just minor defects in the pattern of mature muscles. Duplication of founder cells or muscles after overexpression of both lethal of scute alone or in combination with daughterless is rarely detected. During neurogenesis daG32 mediated expression of UAS- lethal of scute or UAS-daughterless effectors compensates for loss of AS-C or daughterless function, respectively; this suggests that both constructs are functionally expressed in the mesoderm, at least after activation with daG32. Therefore, it has been concluded that the inhibitory signals mediated by exogenously provided Notchintra and those mediated by the endogenous Notch dominate over the function of Lethal of scute and Daughterless. These results are consistent with results obtained on the development of the embryonic nervous-system (Giebel, 1999).

Further evidence for a Notch pathway role in myogenesis was obtained by overexpressing UAS-E(spl) in the mesoderm. Following Gal4 mediated activation of UAS-E(spl), the number of S59 and Kruppel positive cells is strongly reduced. This correlates with a defect in the number of differentiated muscle cells, as shown by MHC reporter gene expression. Again these data fit well with the results obtained on the development of the neuroectoderm, in which Gal4 driven UAS-E(spl) expression leads to strong reduction of CNS and PNS structures (Giebel, 1999).

During neurogenesis Su(H) becomes active if Notchintra is expressed. During imaginal neurogenesis the function of Su(H) is antagonized by proteins encoded by Hairless; this effect is mediated by direct protein-protein interactions. During specification of imaginal sensory organ precursors, overexpression of Hairless counteracts the phenotypic effects of activated Notch. If Su(H) is involved in transducing the inhibitory signals mediated by activated Notch during myogenesis, Gal4 mediated expression of Hairless could theoretically weaken the effect of Notchintra on the course of myogenesis. To test the relationships between active Notch and Hairless during myogenesis, UAS-Hairless and UAS-Notchintra were expressed in the mesoderm of the same embryos. S59 and Kruppel positive cells are present in these embryos, although in much lower numbers than in wildtype embryos. Well differentiated muscles are also present in these embryos. Similar results were obtained in the course of embryonic neurogenesis. Embryos with daG32 driven UAS-Notchintra are completely aneural. After coexpression of UAS-Hairless, structures of the CNS as well as of the PNS differentiate as visualized with 22C10 (see Futsch) and 44C11 antibody stainings. Gal4 mediated expression of UAS-Hairless alone leads to a slight increase in the number of S59 and Kruppel positive cells in the mesoderm. This correlates with an increase in the number of differentiated muscle fibers, shown by the expression of the MHC reporter gene. In correspondence to those data the neuroectodermal Gal4 mediated expression of UAS-Hairless leads to the development of a weakly hyperplasic nervous system, as shown by stainings using the neural antibodies 22C10 and 44C11 (Giebel, 1999).

Muscle founder cells arise from the asymmetric division of muscle progenitor cells, each of which develops from a group of cells in the somatic mesoderm that express lethal of scute. All the cells in a cluster can potentially form muscle progenitors, but owing to lateral inhibition, only one or two develop as such. Muscle progenitors, and the subsequent founder cells, then express transcription factors such as Krüppel, S59 and Even-skipped, all of which confer identity on the muscle. Definition of some muscle progenitors, including three groups that express S59, depends on Wingless signaling. Lateral inhibition requires Delta signaling through Notch and the transcription factor Suppressor of Hairless. Since the Wingless and lateral-inhibition signals are sequential, one might expect that muscle progenitors would fail to develop in the absence of Wingless signaling, regardless of the presence or absence of lateral-inhibition signaling. The development of the S59-expressing muscle progenitor cells has been examined in mutant backgrounds in which both Wingless signaling and lateral inhibition are disrupted. Progenitor cells fail to develop when both these processes are disrupted. This analysis also reveals a repressive function of Notch, required before or concurrent with Wingless signaling that is unrelated to its role in lateral inhibition (Brennan, 2000).

During wild-type development, expression of S59 is first seen during stage 10 in a single muscle progenitor cell either side of the midline in every segment. By stage 11, this pattern has evolved in abdominal segments such that S59 expression is seen both in the nervous system and in two groups of muscle progenitor cells. During stage 12, a third muscle progenitor cell starts to express S59. These muscle progenitor cells give rise to three muscle founder cells that maintain the expression of S59. Fusion of these founder cells with myoblasts results in the S59-expressing muscles seen in late stages of embryogenesis (Brennan, 2000).

Disruption of lateral-inhibition signaling, in either Notch (N) germline-clone, suppressor of Hairless germline-clone or Delta zygotic mutant embryos, increases the number of cells expressing S59 compared with wild type at stage 11. Because of general degeneration of these embryos during germ-band retraction, however, it is difficult to examine the expression of S59 after stage 11, but the mesoderm clusters that can be identified are expanded (Brennan, 2000).

Unlike the disruption of lateral-inhibition signaling, attenuation of Wingless signaling, by removing either wingless (wg) or dishevelled function, blocks the expression of S59 in the mesoderm. In contrast, increasing Wingless signaling, either by overexpressing the Wingless protein in the mesoderm using the GAL4/UAS system (twist-GAL4>UASwg embryos), or by removing shaggy function (sggm11 germline-clone embryos), leads to enlarged groups of S59-expressing muscle progenitor cells during stage 11. However, during germ-band retraction, the groups are reduced in size. In the twist-GAL4>UASwg embryos the reduction in cluster size leads to a largely normal set of three muscles, whereas in the sggm11 embryos the reduction is more extreme and leads to the loss of S59-expressing muscles (Brennan, 2000).

Since Wingless signaling is required for the initiation of S59 expression in the mesoderm and lateral-inhibition signaling is required for the subsequent restriction of S59 expression to one or two cells within each cluster, it is expected that in the absence of Wingless signaling S59 will not be expressed, even if lateral-inhibition signaling is also blocked. This appears to be the case in wgS107.5;DlFX3 zygotic and wgS107.5,Su(H)SF8 germline-clone embryos. In contrast, mesodermal S59 expression is observed in Df(1)N81k1,dshv26 and Df(1)N81k1;wgCX4 germline-clone embryos, in which Wingless signaling is blocked and Notch function is removed. Finally, as with the single-mutant embryos, the double-mutant embryos degenerate during germ-band retraction, making it difficult to examine S59 expression after stage 11 (Brennan, 2000).

These results first confirm that Wingless signaling is required for the initiation of S59 expression and that a Delta-initiated lateral-inhibition signal is required for the restriction of S59 expression to one or two cells of each initial cluster. They also confirm the prediction that, in the absence of a Wingless signal, S59 is not expressed, regardless of whether lateral-inhibition signaling is occurring. Also, even though hyperactivating Wingless signaling leads to initially enlarged groups of S59-expressing muscle progenitor cells, a reasonably normal muscle pattern is obtained (Brennan, 2000).

The observed S59 expression in Df(1)N81k1, dshv26 and Df(1)N81k1; wgCX4 embryos can be explained if it is assumed that Notch has a repressive function that precedes Wingless signaling. In this situation, removal of Notch function will lead to the derepression of S59 expression before Wingless signaling. Consequently, it does not matter whether or not Wingless signaling occurs. This repressive function cannot be related to Delta signaling, however, because the removal of Delta or Su(H) function in embryos where Wingless signaling is not occurring does not result in S59 expression. The repressive function of Notch uncovered in these experiments must therefore be distinct from its repressive role during lateral inhibition (Brennan, 2000).

The second observation suggests that in response to increased Wingless signaling there is a linked increase in lateral-inhibition signaling. This would mean that increased Wingless signaling will only lead to a significant increase in the number of muscle progenitors if lateral inhibition cannot occur. The observed difference in the final muscle pattern between twist–GAL4>UASwg and sggm11 embryos is probably due to the difference in how Wingless signaling is activated in the different embryos. In the twist–GAL4>UASwg embryos, Wingless signaling is activated only transiently and is restricted to the mesoderm. In contrast, Wingless signaling is activated globally and throughout embryogenesis in sggm11 germlineclone embryos. This difference, along with the proposed linkage between Wingless signaling and lateral inhibition would mean that lateral inhibition is much greater in the sggm11 embryos. This situation would explain the greater reduction in the size of the groups of S59-expressing muscle progenitor cells observed in the sggm11 embryos and the loss of muscles if the restriction is too great (Brennan, 2000).

The link between Wingless signaling and lateral inhibition could occur in a number of ways. For example, Wingless signaling may directly alter a component of the Delta signaling pathway that would then increase the ability of this pathway to transduce the Delta signal. Alternatively, Wingless signaling could affect Delta signaling by altering the transcription of one of the components of the pathway. Either of these mechanisms would allow the organism to generate a lateral-inhibition signal appropriate to the input signal: a strong Wingless signal would lead to a strong lateral-inhibition signal and prevent unnecessary and unwanted development, whereas a weak Wingless signal would lead to a weak lateral-inhibition signal that allows development to proceed even though the input signal is weak. This would allow normal development to occur even if there are fluctuations in the input signal (Brennan, 2000).

It is thought that the muscle progenitor cells develop from a large pool of developmentally equivalent cells that is refined through two steps to produce one muscle progenitor cell. A very large group of cells is initially defined that have the potential to become muscle progenitor cells but are prevented from doing so by the novel function of Notch identified here. Wingless signaling then alleviates this repressive function of Notch within a few cells of the cluster to establish an equivalence group. This triggers the process of lateral inhibition, which subsequently selects a single cell to become a muscle progenitor. In this situation, overexpressing Wingless or constitutively activating Wingless signaling will alleviate the initial repressive function of Notch in all the cells is observed, revealing the larger extent of the initial cluster. The linked increase in lateral-inhibition signaling, however, ensures that the normal number of muscle progenitor cells develop (Brennan, 2000).

This model contrasts with others in which Wingless signaling is instructive and defines the position at which muscle progenitor cells will develop, but can explain why overexpressing Wingless leads to the development of S59-expressing muscles in their normal position. In this model the Wingless signal is permissive and not instructive: it does not define where S59 will be expressed but merely reveals places defined by earlier mechanisms. Finally, these data suggest that the loss of S59 expression in the absence of a Wingless signal is due to the early repression mediated by Notch (Brennan, 2000).

lame duck, termed myoblasts incompetent (minc) in this study, is essential for normal myogenesis and myoblast fusion in Drosophila. myoblasts incompetent is expressed in immature somatic and visceral myoblasts. Expression is predominantly in fusion-competent myoblasts and a loss-of-function mutation in myoblasts incompetent leads to a failure in the normal differentiation of these cells and a complete lack of myoblast fusion. In the mutant embryos, founder myoblasts differentiate normally and form mononucleate muscles, but genes that are specifically expressed in fusion-competent cells are not activated and the normal downregulation of twist expression in these cells fails to occur. In addition, fusion-competent myoblasts fail to express proteins characteristic of the general pathway of myogenesis such as myosin and Dmef2. Thus myoblasts incompetent appears to function specifically in the general pathway of myogenesis to control the differentiation of fusion-competent myoblasts (Ruiz-Gomez, 2002).

Mutations in twist, which eliminate all mesodermal derivatives also completely lack expression of minc, thereby confirming its exclusively mesodermal pattern of expression. Previous work has shown that founder cells segregate from the somatic mesoderm by a process of lateral inhibition that is mediated by the neurogenic genes and the activation of the Notch signaling pathway in neighboring cells, which are thereby prevented from becoming founders themselves. In Notch mutant embryos, there is an overproduction of founder cells at the expense of other adjacent cells. It seems likely that the cells that are inhibited from becoming founders are the cells that will enter the other myoblast class, namely the fusion-competent cells. This view is reinforced by the finding that sns expression, which is characteristic of these cells, is reduced in Notch mutant embryos. If this is so, and if minc is involved in the specification of fusion-competent cells, then it would be expected that its expression too would be reduced in neurogenic mutant embryos. Accordingly, the expression of minc was examined in mutants for Notch and Delta. In both cases there is a striking reduction in the expression of minc. There is some persistent minc expression in these embryos which is likely to be in cells of the visceral mesoderm (Ruiz-Gomez, 2002).

Notch and axon guidance

The central problem in axon guidance is to understand how guidance signals interact to determine where an axon will grow. A specific axon guidance decision in Drosophila embryos has been investigated, the sharp inward turn taken by the ISNb motor nerve to approach its muscle targets. This turn requires Notch and its ligand Delta. Delta is expressed on cells adjacent to the ISNb turning point, and it is known from previous work that Notch is present on axonal growth cones, suggesting that Delta and Notch might provide a guidance signal to ISNb. To induce the turning of ISNb axons, Notch interacts genetically with multiple components of a signal transduction pathway that includes the Abl tyrosine kinase and its affiliated accessory proteins. In contrast, genetic interaction experiments fail to provide evidence for a major role of the 'canonical' Notch/Su(H) signaling pathway in this process. It is suggested that the Notch/Abl interaction promotes the turning of ISNb axons by attenuating the Abl-dependent adhesion of ISNb axons to their substratum, thus releasing the axons to respond to attraction from target muscles (Crowner, 2003).

The receptor Notch is present on axons and growth cones and is required for extension of some early-growing 'pioneer' axons in the fly embryo. More recently, later functions of Notch in axon patterning have been investigated, using a temperature-sensitive Notch allele (Notchts1) to remove Notch activity well after most embryonic neuronal identities have been specified. In temperature-shifted mutant embryos, it has been found that ISNb axons reach their targets via an aberrant bypass trajectory, in which ISNb axons remain associated with the ISN. All Notchts embryos display the bypass phenotype, with 31% of hemisegments affected. Raising the temperature 1 hr earlier in development increases the expressivity of the ISNb bypass phenotype to 73% of hemisegments. Wild-type embryos subjected to the same temperature protocol, or Nts embryos maintained at 25°, displayed few if any defects in ISNb defasciculation. Despite the aberrant pathfinding in Notchts embryos, formation of neuromuscular synapses to ventral longitudinal muscles occur as efficiently in temperature-shifted mutant embryos as in similarly treated wild-types (Crowner, 2003).

The site of Notch activation was localized by examining the Notch ligand, Delta. Temperature shifts of a temperature-sensitive combination of Delta alleles (Dl6B37/Dlvia1) produced an ISNb bypass phenotype indistinguishable from that induced by Notchts. The Deltats mutant combination is not as 'tight' as Notchts1, so the reduced expressivity of the Delta phenotype relative to that of Notch is not surprising. Antibody staining revealed that at the time when ISNb is pioneered, Delta is expressed on cells very near to the first choice point, most prominently on the ganglionic branch of the trachea. This tracheal branch develops prior to ISNb outgrowth, the ISN grows in close association with the trachea, and ISNb axons separate from the ISN at that point where they first contact the trachea. The highest tracheal acccumulation of Delta protein is on the apical surface of the cells, in the tracheal lumen; however, Delta protein is also found on the basal surface of tracheal cells, available for interaction with ISNb axons. Adding back wild-type Delta to the trachea of temperature-shifted Deltats embryos (with btl-GAL4) rescues the Delta ISNb bypass phenotype. btl-GAL4 is expressed in midline glial cells in addition to tracheal cells; however, midline expression of Delta does not rescue ISNb trajectory in Deltats. Staining with an anti-tracheal antibody demonstrates that the ganglionic tracheal branch develops normally in temperature-shifted Nts embryos (Crowner, 2003).

While tracheal expression of Delta is sufficient to restore ISNb defasciculation, ISNb still defasciculates properly in btl mutant embryos that lack trachea. Delta protein, however, is also detectable on nontracheal cells that abut the first choice point, and it is postulated that the Delta on these other cells might act redundantly with that on the trachea to provide a defasciculation signal for ISNb axons. The positions of these cells are consistent with some of them being peripheral glia, and indeed some of these cells label with Repo, a marker for glial cell nuclei. Embryos lacking glia show a low frequency of ISNb bypasses, and this frequency was substantially enhanced in embryos that simultaneously lack the trachea, consistent with the notion that both glia and trachea contribute to the defasciculation of ISNb at the first choice point (Crowner, 2003).

Two signaling pathways have been described for Notch. Notch controls cell fate and differentiation by a mechanism whereby a fragment of the receptor is released by proteolysis to transit to the nucleus as part of a transcriptional activation complex [the 'Su(H)/mam pathway'] and it controls axon patterning by regulating a signal transduction pathway defined by the Abl tyrosine kinase and its accessory genes, fax, dab, nrt, trio, and ena (the 'Notch/abl pathway') (Giniger, 1998). Genetic interaction experiments were performed; every mutant tested in the abl pathway displayed dominant genetic interactions with Notch in ISNb pathfinding. Removal of just one copy of the abl, neurotactin (nrt), or trio genes from Notchts embryos significantly suppresses the Notch bypass phenotype, and simultaneous reduction of abl and nrt suppresses the Notch phenotype more effectively than either heterozygous mutation by itself. Conversely, increasing Abl activity either by removing one copy of the abl antagonist, enabled, or by overexpression of abl significantly enhances the expressivity of the Notch bypass phenotype. Moreover, a bypass phenotype produced by overexpression of abl in wild-type embryos is suppressed by co-overexpression of Notch. In addition, it was found that reduction of Notch activity suppresses the ISNb zygotic mutant phenotype of abl homozygotes, mirroring the suppression of the Notch phenotype by reduction of abl documented above (Crowner, 2003).

In contrast to the strong genetic interactions of Notch with abl pathway genes in ISNb development, genetic tests failed to provide evidence indicative of a major role for the Su(H)/mam pathway in the Notch-dependent control of ISNb trajectory. Reducing the dosage of either Su(H) or mam did not enhance the Notch mutant phenotype, and expression of a signal-independent Su(H) did not suppress the requirement for Notch in ISNb. While these are negative results, it is noted that mam in particular displays strong, dominant-genetic interactions with Notch in a wide variety of genetic paradigms, while expression of Su(H)-VP16 mimics many of the effects of activated Notch, for example, blocking the development of ~98% of embryonic abdominal sensory neurons when expressed in wild-type embryos under control of the ectodermal GAL4 driver, 69B. Moreover, no evidence was found for aberrations in the identities or differentiation of ISNb neurons in temperature-shifted embryos. Taken together, these data suggest a simple hypothesis for how Notch and Abl cooperate to direct the trajectory of ISNb axons. ISNb guidance is known to reflect a competition between adhesion of these axons to the ISN pathway versus attraction to the target muscle field, and Abl activity is limiting for adhesion to the ISN pathway. It is now seen that defasciculation of ISNb axons requires Notch and Delta. Since the presence of Notch in postmitotic neurons and of Delta in the trachea is sufficient to fulfill their respective roles, it seems that the relevant activation of Notch must occur in the peripheral motor axons, where these cells touch. It is further found that Notch acts by antagonizing Abl activity in ISNb patterning -- mutations in these genes mutually suppress -- and thus it is suggested that the specific turn of ISNb axons arises from ligand-dependent attenuation of Abl pathway activity by Notch. By reducing the Abl-dependent adhesion of ISNb axons to the ISN pathway, Notch evidently makes the axons competent to respond to attraction from their target muscles. Consequently, they can now dive down into the ventrolateral muscle field. It is noted that it has not yet been determined whether the action of Notch, or Abl, is autonomous to the ISNb axons themselves. Cell-cell adhesion, and particularly axon fasciculation, is necessarily a bidirectional interaction. Since all ISN axons, dorsally directed ISN axons as well as ISNb axons, come into contact with the trachea and peripheral glia at the first choice point, regulation of ISNb-ISN defasciculation by Notch and Abl could plausibly reflect the function of these genes in ISNb, in ISN, or both. It is further noted that, in this model, the essential function of Delta and Notch is to prevent ISNb axons from remaining associated with the ISN dorsal to the choice point, not to act in isolation to set uniquely the position where ISNb turns. Delta and Notch function in ISNb patterning mainly to antagonize Abl, but even the strongest abl zygotic mutant does not cause premature defasciculation, presumably reflecting the presence of yet other factors that act in parallel to prevent ISNb axons from entering the muscle field prematurely. Thus, ubiquitous expression of Delta also does not cause premature turning of ISNb (Crowner, 2003).

The mechanism by which Notch antagonizes Abl activity is not yet clear. It is noted, however, that Notch, Abl, Disabled, Fax, Trio, and Enabled are all enriched in axons, and preliminary experiments show that Notch coimmunoprecipitates with both Disabled and Trio from wild-type fly lysates. It may be, therefore, that the Notch/abl genetic interactions observed here reflect a physical complex of Notch with Abl pathway proteins. It also seems remarkable that Notch can interact with the Abl signaling pathway in two apparently opposite ways. Where axons grow along Delta-expressing substrata, as in CNS longitudinal axon tracts, Notch works cooperatively with Abl: partial loss-of-function mutations of Notch and abl interact synergistically to produce synthetic phenotypes and embryonic lethality. In contrast, in ISNb, Notch and Abl act antagonistically: their gain- and loss-of-function phenotypes mutually suppress one another. It is imagined that there must be some additional factor that determines the 'sign' of the interaction between Notch and Abl. This could be a negative signal that prevents ISNb axons from growing along the Delta-expressing trachea, or a positive signal that allows Notch-dependent growth cone motility along other Delta-expressing substrata. The capacity of a single growth cone receptor to be switched between opposite functional modes has become a common observation in recent years, with cyclic nucleotides often playing a crucial role in the process. Perhaps the interaction of Notch with Abl is 'switched' by some analogous mechanism (Crowner, 2003).

In recent years, studies of axon guidance have focused on a relatively small set of receptors that have strong, instructive effects on growth cone trajectory. It has long been clear, however, that many guidance decisions reflect quantitative integration of signals from broadly distributed factors, many of which individually have low specificity. The data reported here provide a paradigm for understanding how subtle modulation of a key signaling pathway allows a combination of relatively low-specificity cellular interactions to produce a precise axonal trajectory (Crowner, 2003).

How Notch establishes longitudinal axon connections between successive segments of the Drosophila CNS

Development of the segmented central nerve cords of vertebrates and invertebrates requires connecting successive neuromeres. This study shows both how a pathway is constructed to guide pioneer axons between segments of the Drosophila CNS, and how motility of the pioneers along that pathway is promoted. First, canonical Notch signaling in specialized glial cells causes nearby differentiating neurons to extrude a mesh of fine projections, and shapes that mesh into a continuous carpet that bridges from segment to segment, hugging the glial surface. This is the direct substratum that pioneer axons follow as they grow. Simultaneously, Notch uses an alternate, non-canonical signaling pathway in the pioneer growth cones themselves, promoting their motility by suppressing Abl signaling to stimulate filopodial growth while presumably reducing substratum adhesion. This propels the axons as they establish the connection between successive segments (Kuzina, 2011).

The axons of the longitudinal pioneer interneurons of the Drosophila ventral nerve cord establish the initial connection between successive segments of the animal. The receptor Notch is crucial for making those first connections, performing two parallel, partially redundant but completely separate functions. Canonical Notch signaling in the interface glia constructs an unbroken track for longitudinal pioneer axons to follow by shaping a continuous band of neuronal membrane that bridges from segment to segment. Simultaneously, non-canonical Notch/Abl signaling in the pioneer neurons themselves promotes the motility of their growth cones, suppressing the activity of the Abl tyrosine kinase signaling module to stimulate filopodial development. Either signaling mechanism provides substantial rescue of a Notch mutant, but both are required for full activity in formation of longitudinal connections of the CNS (Kuzina, 2011).

The mechanism that guides the very first axon to establish the path of a nascent nerve is one of the most fundamental problems in neural development. For longitudinal pioneers of the Drosophila CNS, it is now seen that constructing their path requires coordinated contributions from four interacting cell types. First, the axons of commissural interneurons bearing the Notch ligand Delta contact interface glia. The glia respond by activating canonical Notch signaling, enhancing expression of Notch target genes, including prospero. The genetic program stimulated by Notch directly or indirectly allows the glial cells to attract a 'cap' of fine filopodial processes from nearby differentiating neurons, and shapes that cap into a continuous longitudinal band that bridges between segments. The neuronal cap atop the glia bears the Netrin receptor Frazzled (DCC), which in turn recruits soluble Netrin, thus constructing a domain of accumulation of Netrin protein that hugs the surface of the associated glia. Finally, the pioneer growth cones advance along the edge of that domain of immobilized Netrin until they meet and fasciculate with their partners pioneering from the next segment. The consequence of this choreography is a nerve trajectory that follows, indirectly, the shape of the row of interface glia (Kuzina, 2011).

This view suggests plausible explanations for many aspects of longitudinal axon development that have, up to now, been confusing. Previous investigators have documented that the pioneer growth cones extend amidst a thicket of filopodia that cap the interface glia. The provenance and significance of those filopodia were unknown, though it was clear that they did not derive from other axons. It is now seen that these filopodia come from surrounding, differentiating neurons, and that their function is to hold Frazzled, and therefore Netrin, in a pattern dictated by the positions of the overlying glia, creating the Netrin domain along which the pioneers extend. This explains why the positions of the interface glia correlate so closely with the axon trajectory even though the glia are not the direct substratum. The data, along with other recent results, also suggest why previous experiments investigating the guidance function of interface glia might have given such confusing results. Transformation of the glia into neurons in a gcm mutant would be predicted to place a row of DCC-expressing neurons in precisely the position of the wild-type filopodial carpet. Moreover, genetic experiments ablating or displacing the glia have relied on reagents targeting the progeny of the longitudinal glioblast, but it is now known that only nine of the ten interface glia come from this precursor; the tenth, M-ISNG, is from a different lineage. M-ISNG is appropriately positioned to anchor the filopodial carpet in the absence of the other interface glia and preliminary experiments suggest that it is sufficient for this. Moreover, it has been argued that in those rare segments where pioneer axons stall owing solely to manipulations of the interface glia, all ten of them, including M-ISNG, tend to be absent or displaced. Finally, as in previous studies, this stud found that the Netrin zygotic mutant has a mild, and genetically enhanceable phenotype, showing that the null for the gene is not null for the genetic pathway. It might be that there is a maternal contribution to Netrin, as there is for frazzled. Alternatively, because the receptor on longitudinal pioneers presumably recognizes a Netrin-Frazzled complex, it might be that this receptor has some affinity for Frazzled even in the absence of Netrin. Identification of the missing Netrin receptor will be necessary to clarify this point. It also seems likely that other neuronal components cooperate with the Netrin-Frazzled complex on the meshwork to provide substratum function, as expression of Fra in interface glia is not sufficient to rescue the Notch axonal phenotype (Kuzina, 2011).

Once the pathway for an axon has been constructed, there remains the problem of driving the motility of the growth cone along that pathway. Somehow, the information encoded in a pattern of occupancy of cell surface receptors must be transformed into a pattern of cytoskeletal dynamics that drives growth cone motion. At the level of the axon, this is the bedrock problem in axon guidance, and it, too, has resisted analysis. The current data reveal how Notch modulates an elementary property of the actin cytoskeleton to promote motility of longitudinal pioneer growth cones. Through its antagonism of the Abl signaling network, Notch de-represses the Abl antagonist Enabled and suppresses the Rac GEF Trio. Enabled directly promotes filopodial growth; suppressing Rac indirectly promotes filopodia, probably by redirecting various factors away from lamellipodia. Stimulating filopodial development probably promotes longitudinal axon growth in at least two ways. First, converging pioneer growth cones from successive segments need to encounter one another and fasciculate to establish the connection between segments. Extension of filopodia increases the area searched by an advancing growth cone, increasing the probability that it will encounter its partners advancing from the adjacent segment. Second, filopodia promote neurite growth by promoting microtubule invasion of the leading edge (Kuzina, 2011).

In parallel with stimulating filopodia, suppression of Trio, and thus of Rac, is expected to reduce substratum adhesion. When the pioneers are growing towards the segment border, small gaps in the Frazzled-Netrin pattern are not uncommon, so release of the advancing growth cone from the substratum is likely to aid its forward motion. Moreover, initially there is more Frazzled-bound Netrin within the neuromeres than there is at the segment border, requiring advancing pioneers to go down a gradient of Netrin, towards a region with less Netrin. It is possible that both of these properties make it helpful to limit substratum adhesion of the growth cone via reduction of Rac signaling by Notch (Kuzina, 2011).

It might seem paradoxical that Notch promotes axon growth by suppressing Abl signaling when Abl has been the archetype of a motility-promoting signaling pathway. Indeed, genetic studies of Abl in axon guidance have often appeared to be confusing and contradictory. In part, this reflects pleiotropy. Abl appears to act in the glia, and in the cells providing the filopodial carpet, in addition to the pioneers. As the phenotype in a whole-animal mutant reflects the sum of unrelated functions in different cells, seemingly similar experiments can produce contradictory results if different cellular processes become limiting. For longitudinal axons, for example, if pathway establishment is limiting (in Abl- or fra- animals), reduction of Notch interferes with axon growth synergistically; if growth cone function is limiting (in Notch- animals), reduction of the Abl pathway restores axon growth. It was therefore essential in the current work to control gene activity, and analyze phenotypes, in single, identified cells (Kuzina, 2011).

Beyond pleiotropy, however, complexity arises because the effect of signaling molecules in motility is profoundly context dependent. Ena promotes actin polymerization but often restricts cell motility; cofilin severs actin filaments but can promote net actin polymerization and cell migration. As axon growth is achieved by throughput through a cycle of actomyosin dynamics, it requires a balance among the steps of that cycle. Excessive activity or inactivity of any single step in the process inhibits motility by impairing progression through the cycle. The data now reveal that, for Drosophila longitudinal pioneers, an essential aspect of growth cone movement is restraint of Abl activity to allow filopodial development, and perhaps also to limit substratum adhesion (Kuzina, 2011).

The data reported in this study reveal that Notch promotes CNS longitudinal axon growth in two very different ways, constructing a pathway using its canonical signaling mechanism and promoting motility via the Notch/Abl interaction. This dual role bears striking parallels to the dual role of Notch in radial migration of neurons in the mammalian cortex. There, as in the fly, canonical signaling by Notch is essential for the development of glial cells that define a migration pathway, whereas interaction with the Abl pathway protein Disabled controls neuronal motility and adhesion. Further study will be required to assess whether these parallels between Notch function in the fly and vertebrate nervous systems reflect a deeper mechanistic similarity. Similarly, it will be interesting to see whether formation of longitudinal nerve tracts in the spinal cord uses machinery homologous to that which this study has described in the fly (Kuzina, 2011).

Notch and dendrite morphogenesis

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4221) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4221-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).

TFs play critical roles in neurogenesis, and some genes that regulate neurogenesis also affect post-mitotic neuronal differentiation. Because clones of duplicated class I neurons have wild-type dendrite arborization patterns, class I dendritic arbors appear to be insensitive to cell number defects. Indeed, dendrite arborization of class I neurons in embryos carrying the temperature-sensitive neurogenic mutation Notchts (Nts) is unaffected by as much as a fivefold increase in class I neuron number, and is likely insensitive to multiplication of other da neurons as well, since the Nts experiments caused increased numbers of other da neurons. Furthermore, in cases where only one of the class I neurons is multiplied, the dendrites of neighboring class I neuron are unaffected. In contrast laser ablation of ddaD or ddaE or the occasional cell loss caused by RNAi of various genes did not generally cause defects in arborization of neighboring class I neurons. Therefore, analysis of class I neurons should allow study of post-mitotic functions of genes that affect neuron number (Parrish, 2006).

Table of contents

Notch: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | References

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