Interactive Fly, Drosophila


Effects of Mutation or Deletion

Table of contents

Notch and neurogenesis: The PNS and bristle formation (part 1/2)

The embryonic peripheral nervous system of Drosophila contains two main types of sensory neurons: type I neurons, which innervate external sense organs and chordotonal organs, and type II multidendritic neurons. Type I neurons are characterized by their single dendrite whose distal part is a modified cilium. In contrast, type II neurons possess several dendrites lacking ciliated structures. Type I neurons are associated with accessory cells (socket and shaft cells, known respectively as tormogen and trichogen cells) that form the non-neuronal part of the sense organ. Type II neurons are not associated with accessory cells. In Notch mutant embryos, the type I neurons are missing while type II neurons are produced in excess, indicating that the type I/type II choice relies on Notch-mediated cell communication. It is proposed that a protoprecursor cell exists (called p0) having both external sense organ and multidendritic cell potentiality. In the absence of Notch, the two daughters of the protoprecursor will adopt the same, multidendritic fate (Vervoort, 1997).

Both type I and type II neurons are absent in numb mutant embryos and also after the ubiquitous expression of tramtrack. This indicates that the activity of numb and the absence of tramtrack are required to produce both external sense organ and multidendritic neural fates. Numb is thought to repress tramtrack, a gene that promotes non-neuronal verses neuronal fate. The analysis of string mutant embryos reveals that when the precursors are unable to divide they differentiate mostly into type II neurons, indicating that the type II is the default neuronal fate. A new mutant phenotype has been described, called X1. It prevents the acquisition of external sense cell fate. In these mutants, ASC-dependent neurons are converted into type II neurons, providing evidence for the existence of one or more genes required for maintaining the alternative (type I) fate (Vervoort, 1997).

Asymmetric divisions allow a precursor to produce the four distinct cells of Drosophila sensory organ lineages (SOLs). The sensory organ precursor (SOP) first divides into two different secondary precursor cells, IIA and IIB, which gives rise to one shaft-producing cell (trichogen) and one socket-producing cell (tormogen), and one neuron and one sheath cell (thecogen), respectively. Although this process requires cell-cell communication via the Notch (N) receptor, mitotic recombination that removes the N ligand Delta (Dl) or Serrate (Ser) in the SOL has mild or no effect. N mutant clones generated on the central region of the adult scutum are devoid of any external bristle structures, such as shafts and sockets, similar to the Nts mutant phenotype at a restrictive temperature. Whereas loss of N function during the process of lateral inhibition produces supernumerary SOPs, this balding phenotype is probably due to the requirement of N in asymmetric divisions. Without N activity the supernumerary SOPs divide symmetrically, giving rise to two IIB cells and, consequently, no external sensory structures. Dl clones typically produce a tuft of densely packed bristles in the interior of the clone. These tufts of bristles are likely due to a failure of lateral inhibition, resulting in overproduction of SOPs. The presence of the external bristle structures in these Dl mutant clones indicates that, unlike N clones, most of the supernumerary SOPs in the Dl mutant clones produce IIA cells that divide to form shaft and socket cells. Clones homozygous for three Ser null alleles produce normal external bristle structures. In contrast, clones with loss of both Dl and Ser function produce epidermal cells but not external bristle structures. This balding phenotype is clearly different from the phenotypes of the Dl or Ser mutant clones but is indistinguishable from that of N mutant clones, suggesting that Ser and Dl have overlapping functions in the N signaling pathway. Dl and Ser also have redundant functions in patterning wing veins. In contrast, Dl and Ser are known to serve distinct functions in specifying dorsal-ventral compartment boundary of the wing (wing margin). Ser in dorsal cells signals to N in ventral cells, and Dl in ventral cells signals to N in dorsal cells. For Dl and Ser to provide distinct signals from one compartment to the other without generating signals among cells within the same compartment, it may be necessary to involve other factors such as those encoded by the dorsally expressed gene fringe (fng), which inhibits a cell's ability to respond to Ser and potentiates a cell's response to Dl. It is concluded that Dl and Ser are redundant in mediating signaling between daughter cells to specify their distinct sensory organ cell fates (Zeng, 1998).

Delta- and Serrate-mediated signaling can promote the socket cell fate in developing bristle organs. Previous studies have defined roles for Delta in the specification of the sensory organ precursor (SOP), its progeny (pIIa and pIIb), and the daughters of pIIb -- the neuron and thecogen (glia). This paper shows that ectopic expression of Delta or Serrate in neurons within developing bristle organs is capable of non-autonomously inducing the transformation of daughters of pIIa, the pre-trichogen (shaft) cells into tormogen (socket) cells. The frequencies at which Delta can induce these transformations are dependent on the level of ectopic Delta expression and the levels of endogenous Notch signaling pathway components. Delta expression in the cell receiving the Delta signal also has effects on the responsivess of that cell to Delta and Serrate signals. The pre-trichogen cell becomes more responsive to Delta- or Serrate-mediated transformation when the level of endogenous Delta is reduced, and less responsive when the dosage of endogenous Delta is increased, supporting the hypothesis that Delta interferes autonomously with the ability of a cell to receive either Delta or Serrate signal. Thus cell autonomous interactions between Delta and Notch modulate neurogenic signalling in Drosophila. A dominant-negative form of Notch, ECN, is capable of autonomously interfering with the ability of a cell to generate the Delta signal. When the region of Notch that mediates trans-interactions between Delta and the Notch extracellular domain is removed from ECN, the ability of Delta to signal is restored. These findings imply that cell-autonomous interactions between Delta and Notch can affect the ability of a cell to generate and to transduce a Delta-mediated signal (Jacobsen, 1998).

Evidence is presented that the Fringe protein can interfere with Delta- and Serrate-mediated signaling within developing bristle organs, in contrast to previous reports of the converse effects of Fringe on Delta signaling in the developing wing. The fringe gene encodes a pioneer protein, predicted to be secreted, that plays a role in the development of the wing disc by modulating interactions between dorsal and ventral cells that establish the dorsal/ventral boundary and affect specification of the wing margin. One domain of the Fringe product contains motifs similar to the catalytic domain of glycosyltransferases. The primary effect of Fringe on Notch signaling appears to be inhibition of the ability of the Serrate ligand to activate Notch, an effect observed during neuroblast specification within the neuroectoderm and in the developing wing disc. Fringe may act by binding the amino-terminus of Serrate. In the case of Delta and Serrate ectopic expression, coexpression of Fringe with either ligand can interfere with the ability of that ligand to induce trichogen transformation. In this context, Fringe impedes Serrate- and Delta-mediated signaling. The inhibition of Delta and Serrate signaling observed in the developing bristle organ may be context-dependent, i.e., factors present at the wing margin that prevent Fringe from interfering with Delta-mediated signaling may be absent in developing macrochaetae. If Fringe is secreted by the neuron, it could act in a cell non-autonomous fashion to impede the ability of Notch on the pre-trichogen cell to receive ligand-mediated signals. Alternatively, Fringe could function in the neuron in a cell autonomous manner to impede signal generation by interacting with ligand. In either case, Fringe cannot be interfering with Notch-mediated signal reception in a cell autonomous manner in this context. The exact mechanism by which Fringe can operate in the context of bristle development must be the object of future experiments (Jacobsen, 1998).

Specification of cell fate in the adult sensory organs is known to be dependent on intrinsic and extrinsic signals. The homeodomain transcription factor Prospero (Pros) acts as an intrinsic signal for the specification of cell fates within the mechanosensory lineage. The sensory organ precursors divide to give rise to two secondary progenitors: PIIa and PIIb. Pros is expressed in PIIb, which gives rise to the neuron and thecogen cells. Pros expression was first detected among the secondary progenitors in the nucleus of the more anteriorly located cell (PIIb). Interestingly, this was the first cell to divide in all cases examined. Prior to cell division, Pros becomes uniformly distributed on the cortical membrane and throughout the cytosol. Unlike the findings in the central nervous system, Pros is not asymmetrically localized in cells at any stage of the lineage. Asymmetric crescents of Pros immunoreactivity are not observed even after blocking mitosis with colcemid. These data suggest that Pros is expressed first in the nucleus and then generally in the cytosol of PIIb (Reddy, 1999).

Loss of Notch function generated using either a conditional mutant allele or by misexpressing Numb protein results in the ectopic expression of Pros in PIIa. This observation is consistent with a PIIa-to-PIIb conversion by Notch loss of function or nb gain of function. It is not clear whether the apparent negative regulation of Pros by Notch is a direct effect or merely reflects the altered fate of the cells that is caused by other molecular factors. Pros misexpression is sufficient for the transformation of PIIa to PIIb fate (Reddy, 1999).

Pros is not asymmetrically localized in PIIb; following division of this cell, the protein is detected in both the progeny. This is strikingly different from findings in the CNS where Pros, together with Numb and Miranda, is localized asymmetrically in the neuroblasts and inherited by the GMC. Following division of PIIb, Pros is inherited by nuclei of both progeny. Expression in one of the siblings decays rapidly and this cell differentiates as the neuron. Pros immunoreactivity is sustained in the thecogen cell possibly due to de novo synthesis. The requirement for pros function in PIIb precludes analysis of its later role after division. In experiments where Pros was misexpressed in all four cells of the sensory organ, a conversion of external to internal cells is observed, consistent with a PIIa-to-PIIb transformation. Neuronal cells form normally despite the fact that they express the thecogen cell marker. Similarly, Notch loss of function and Numb gain of function results in a conversion of all four cells of the lineage to neurons. Pros is expressed in all these cells under these conditions. These observations together demonstrate that pros activity is not sufficient for identity of the thecogen cell and that neuronal cell differentiation can occur normally despite Pros expression. The elucidation of pros function in the thecogen cell awaits the availability of a hypomorphic mutant allele that can allow loss of function in the thecogen cell without affecting the secondary progenitors. Pros has been shown to be expressed in several CNS- as well as PNS-associated glial cells and it is possible that it plays a role in the later differentiation and/or function of these cells (Reddy, 1999).

In the normal mechanosensory lineage, Notch is involved in the binary choice between thecogen and neuron. In this lineage Notch signaling is experienced by the cell that ultimately becomes the thecogen cell. This is distinct from the scenario at the secondary progenitor stage, where the cell that experiences Notch signaling does not express Pros. This means that, unlike in PIIb, Notch signaling does not downregulate Pros in the thecogen cell. There are several possible explanations for this finding. One possibility is that Pros protein is merely partitioned to the daughters of the PIIb after division. It therefore serves no role in the binary choice of thecogen versus neuron but is synthesized de novo after these cells have acquired their identity. At this later time point, the thecogen cell is no longer experiencing the Notch signal. Another possibility lies in the different effector mechanisms utilized for Notch activity. In three instances (during lateral signaling, PIIa-PIIb choice, as well as in the PIIa lineage) Notch activation results in release of Su(H) from its binding site on the cytoplasmic domain of Notch and its translocation to the nucleus. Su(H) protein can be detected in the nucleus of the socket cell; however, the role of Su(H) cannot be seen in the PIIb lineage. Extra copies of Su(H) do not produce thecogen-to-neuron transformations, suggesting that Notch signaling in the thecogen/neuron choice occurs by a Su(H)-independent mechanism. Thus the regulation of Pros expression could be mediated by Notch through a Su(H)-independent event (Reddy, 1999 and references).

The receptor encoded by the Notch gene plays a central role in preventing cells from making decisions about their fates until appropriate signals are present. This function of Notch requires the product of the Suppressor of Hairless gene. Loss of either Notch or Suppressor of Hairless function results in cells making premature and incorrect cell fate decisions, while increases in Notch signaling prevent cells from making these decisions. The proneural clusters are not established correctly in certain Abruptex mutations of Notch and this failure to establish proneural clusters correctly is not due to increased Notch signaling during lateral inhibition. The overexpression of certain dominant negative Notch molecules can disrupt the initiation of proneural cluster development in a manner similar to the Abruptex mutants (Brennan, 1999b).

Genetic analysis has revealed three regions of the extracellular domain which are functionally important. One is centered around EGF repeats 11 and 12 and is involved in the binding of Delta. A second one, revealed by the Abruptex mutations, is centered around EGF repeats 24 and 25. Mutations within this region have been proposed to either increase Delta signaling via Notch and consequenctly suppressing Achaete expression or, alternatively, to affect a distinct function of Notch that is involved in the initiation of Achaete expression rather than its suppression. The third region is highlighted by mutations in a cysteine-rich region known as the LNG repeats. Mutation or deletion of this region generates gain of function Notch molecules that appear to increase Delta signaling. While work in different systems has indicated a role for Notch in lateral inhibition, analysis of different Notch mutations has raised the possibility that there is a Notch signaling event different from the one that mediates lateral inhibition. This hypothesis stems from the identification, within the Abruptex(Ax) class of Notch alleles, of loss of function mutations, such as the Ax59d and AxM1 alleles. The Abruptex alleles are characterized, in addition to other phenotypes, by the loss of sense organs on the adult thorax. Since many of the Abruptex alleles behave phenotypically as gain of function mutations, the loss of sense organs is typically thought to be due to an increase in signaling during lateral inhibition. However, this hypothesis cannot explain the loss of bristles observed with the loss of function Abruptex alleles. Consequently it has been proposed that the Notch protein has an additional role in the formation of the sense organs and in particular a role in the establishment of the proneural clusters that is disrupted by alleles like Ax59d, which leads to the observed loss of sense organs. This analysis of Notch alleles that reduce bristle number has also highlighted a purely gain of function class of alleles, that includes the l(1)NB mutation. In these mutants the loss of bristles appears to be due to increased signaling during lateral inhibition (Brennan, 1999b and references therein).

It has been shown by (1) examination of the expression of the proneural gene, achaete, in the presence and absence of the Su(H) gene in the third larval instar wing disc of the Ax59d mutant and (2) examination of achaete expression in flies in which Notch function has been disrupted by expressing dominant negative forms of the Notch protein, that the initiation of the proneural clusters is disrupted in Ax59d mutants and flies in which dominant negative forms of Notch are over expressed. In addition, these experiments show that this failure to define proneural clusters correctly is not due to increased Notch signaling during lateral inhibition (Brennan, 1999b).

One explanation for the suppression of Achaete expression caused by ECNdelta10-12 molecules, that is, expressed proteins in which the extracellular domain of Notch contain a deletion in EGF domains 10-12, might be that they interact with, and 'transactivate', the endogenous full-length Notch protein to generate a strong lateral inhibition signal. To test this possibility the ECNdelta10-12 protein was expressed in a genetic background where lateral inhibition does not occur, such as in larvae homozygous for null mutations of Su(H). In third instar discs that lack Su(H), each proneural cluster contains a large number of cells expressing high levels of Achaete. Expression of the ECNdelta10-12 molecule in Su(H) mutant discs, under the control of ptcGAL4, in many cases eliminates the expression of Achaete in the scutellar cluster. In those cases where Achaete expression is seen the cluster consists of two or three cells that express high levels of Achaete. However, in the same discs, Achaete expression is not affected in clusters not covered by the stripe of ECNdelta10-12 expression and therefore these clusters act as an internal control for the experiment. These results indicate that the suppression of Achaete is not dependent upon Su(H) and therefore it is unlikely that expression of the ECNdelta10-12 protein disrupts Achaete expression by generating a strong lateral inhibition signal. In support of this hypothesis, similar results have been obtained by expressing the ECNdelta10-12 protein in a kuzbanian mutant that also prevents lateral inhibition signaling (Brennan, 1999b).

Previous experiments have shown that the loss of bristles characteristic of Abruptex mutations is suppressed if there is a simultaneous loss of shaggy function; this is likely to be due to the expression of Achaete caused by removing sgg/zw3 function. In light of the similarity observed in the phenotype caused by the Ax59d and AxM1 mutations and the effects of the ECNdelta10-12 molecule on the development of the PNS, the results with shaggy led the authors to test whether or not the phenotype generated by overexpressing the ECNdelta10-12 depends on sgg/zw3. Indeed, the phenotype generated by expressing the ECNdelta10-12 protein can be rescued by reinitiating the expression of Achaete by removing shaggy/zeste white 3 function. This suggests that the phenotype generated by expressing the ECNdelta10-12 is due to the loss of Achaete expression and not due to any secondary effect the ECNdelta10-12 protein may have on the development of the disc (Brennan, 1999b).

The observed loss of Achaete expression in the Abruptex mutants and the overexpression of the ECNdelta10-12 protein could occur in a number of ways. For example, the Notch protein may transduce a signal into the cell that turns on the expression of Achaete in much the same way as it transduces a Delta signal that up-regulates Atonal expression during the development of the eye. In this scenario loss of Notch function should lead to the loss of Achaete expression. This fits well with the expected dominant negative behaviour of the ECN proteins and with the genetic analysis of the Abruptex mutations, which suggests that the Ax59d mutation is a loss of function mutation. However, it is unlikely that this hypothesis is the explanation for the current results, because clones of cells lacking Notch function have a neurogenic phenotype and express Achaete strongly. Alternatively the observed loss of Achaete expression could be due to the ECN molecules and the protein encoded by the Ax59d allele disrupting signaling through a distinct pathway that is required for the initiation of Achaete expression rather than altering signaling through the Notch pathway. In this situation, wild-type Notch function is only required during lateral inhibition and not in the establishment of the proneural clusters. Therefore, the removal of wild-type Notch function would be expected to lead to the failure of lateral inhibition signaling and a neurogenic phenotype, such as is observed in clones of lacking Notch function. One possible pathway with which the ECNdelta10-12 and Ax59d proteins may be interfering is the Wingless signaling pathway, since it is required for the initiation of Achaete expression in both the scutellar and the dorsocentral proneural clusters. The rescue of sensory bristle development by the removal of Shaggy function in Abruptex mutants and in flies in which the ECNdelta10-12 protein has been overexpressed fits with this hypothesis. The removal of Shaggy function from a cell constitutively activates Wingless signaling within that cell regardless of the presence or absence of Wingless. Therefore, the removal of Shaggy should reinitiate a Wingless signal attenuated by the ECNdelta10-12 and Ax59d proteins leading to sense organ development as observed (Brennan, 1999b). Finally, experiments where the ECN and ECNdelta10-12 proteins have been overexpressed throughout the developing wing suggest that these proteins can sequester the Wingless protein (Brennan, 1999a).

The extracellular domain of Notch is essential for its interaction with extracellular molecules. Previous experiments have indicated that EGF-like repeats 11 and 12 are necessary for the interaction of Notch with its ligands Delta and Serrate. However, at least two aspects of the current results suggest that a region of the extracellular domain centered around EGF repeats 24-26 is required for Notch molecules to disrupt the establishment of the proneural clusters: (1) the Ax59d allele maps to this region within EGF-like repeat 25; (2) the dominant negative Notch molecules will only disrupt the initiation of Achaete expression as long as they contain this domain. It is likely that EGF-like repeats 17-19 and 24-26, present in the ECNdelta10-12 protein are necessary for the ECN molecules to disrupt proneural development. Interestingly, a comparison of EGF-like repeats indicates that the primary sequences of EGF-like repeats 24-26 and 17-19 are similar which suggests that either of these sequences may be sufficient for the ECN molecules to disrupt the establishment of the proneural clusters. Such a possibility can explain the observation that overexpression of ECN molecules deleted for either 17-19 or 24-26 produce antineurogenic phenotypes, similar to those of ECN and ECND10-12. EGF-like repeats 24-26 or repeats 17-19 which are still present in ECND17-19, and ECND24-26 proteins, respectively, would allow the proteins to antagonize the establishment of the proneural clusters (Brennan, 1999b).

Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific 'translational' regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signaling directs the asymmetry between neuronal and non-neuronal lineages, and a zinc-finger transcriptional repressor Tramtrack69 (Ttk69) acts downstream of Notch as a determinant of non-neuronal identity. Repression of Ttk69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3' untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (Msi). Although msi can act downstream of Notch, Notch signaling does not affect Msi expression. Thus, Notch signaling is likely to regulate Msi activity rather than its expression. These results define cell-type-specific translational control of ttk69 by Msi as a downstream event of Notch signaling in asymmetric cell division (Okabe, 2001).

Mechanosensory bristle development in Drosophila is an excellent model system in which to address the molecular mechanisms of asymmetric cell division. Four successive asymmetric cell divisions from a common precursor cell, called the sensory organ precursor (SOP) generate a sensory bristle comprising four different non-neuronal support cells and one neuron. The first asymmetric cell division of SOP into IIa non-neuronal and IIb neuronal precursors is regulated by Notch signaling; specific activation of Notch occurs in IIa owing to inhibition in IIb by Numb, an intracellular negative regulator of Notch. Activation of Notch signaling results in the appearance of Ttk69 protein in the IIa precursor, but not in IIb. The expression pattern of Ttk69, phenotypes of ttk69 loss-of-function mutants, and the overexpression of Ttk69 suggest that Ttk69 is necessary and sufficient to specify the IIa non-neuronal lineage; however, the mechanism through which Notch signaling regulates Ttk69 expression has remained elusive (Okabe, 2001).

Although the translational inhibitory effect of Msi on ttk69 mRNA is specific to the IIb precursors, Msi protein is present in both IIa and IIb precursors. Thus, IIa precursors must somehow be able to escape the action of Msi as a translational repressor of Ttk69, probably through the effect of Notch signaling. Loss of Notch function in the SOP lineage causes the transformation of IIa precursor into IIb, with mutants showing a balding phenotype owing to loss of socket and shaft cells. This phenotype in IIa precursor is dependent on Msi activity; loss of both Notch and msi function results in a dense double-bristle phenotype with no neurons in the subepidermal layer, indicating that the IIb precursor took the non-neuronal fate. msi is thus epistatic to Notch during the asymmetric cell division giving rise to IIa and IIb precursors. Taken together, it is proposed that Notch inhibits Msi activity in the IIa precursor (non-neuronal cell), thus allowing translation of ttk69 mRNA, whereas in the IIb precursor (neuronal cell), where Notch is inactivated by Numb, Msi prevents ttk69 translation (Okabe, 2001).

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from each proneural cluster and then undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. phyllopod (phyl), known to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters (IIb cells are transformed into IIa cells), leading to the fate transformation of neuron and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development (Pi, 2001).

During lateral inhibition, the N pathway is essential to single out the SOP cells. In situ analyses indicate that phyl expression is negatively regulated by N. Also, the supernumerary bristles in N mutants are suppressed by phyl mutation. These results strongly suggest that down-regulation of phyl expression is a major function of N signaling to suppress SOP cell fate (Pi, 2001).

In the SOP lineage, N also plays an important role in the cell fate specification of SOP progeny. Several components of the N pathway, such as Delta, Serrate, Su(H), Hairless, and proteins of the Bearded and E(spl) families, have been shown to be involved in the cell fate specification of all or subsets of progeny. phyl acts epistatically to N in the cell fate specification of SOP daughter cells and is expressed in IIb cells. Also, misexpression of phyl rescues the defects caused by NACT, indicating that N regulates the phyl activity in sensory organ lineage at the transcriptional level (Pi, 2001).

echinoid (ed) encodes an immunoglobulin domain-containing cell adhesion molecule that negatively regulates the Egfr signaling pathway during Drosophila photoreceptor development. A novel function of Ed is shown, i.e., the restriction of the number of notum bristles that arise from a proneural cluster. Thus, loss-of-function conditions for ed give rise to the development of extra macrochaetae near the extant ones and increase the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, loss-of-function of ed reduces the expression of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss-of-function conditions for ed and for different components of the N pathway show clear synergistic interactions manifested as strong neurogenic bristle phenotypes. It is concluded that Ed is not essential for, but it facilitates, N signaling. It is known that the N and Egfr pathways act antagonistically in bristle development. Consistently, it is found that Ed also antagonizes the bristle-promoting activity of the Egfr pathway, either by the enhancement of N signalling or, similar to the eye, by a more direct action on the Egfr pathway (Escudero, 2003).

Epistatic and clonal analyses are compatible with Ed facilitating N signaling by acting at a step previous to the release of the NICD. Accordingly, the possibility that Ed might physically interact with N was tested. First, the subcellular localization of both proteins was examined in the wing imaginal disc. Using antibodies that recognize the C terminus of Ed and the zonula adherens marker Armadillo (Arm), Ed was observed to mainly, if not exclusively, accumulate at the zonula adherens where it colocalizes with Arm. This is in sharp contrast to the eye disc, where Ed resides throughout the cell membrane of all cells. Using NICD-specific antibodies, it was further observed that N is mainly colocalized with Ed. Similar colocalization with Ed at zonula adherens can also be detected with NECN-specific antibodies, but Ed is not present in the NECN-containing internalized vesicles (Escudero, 2003).

The colocalization of Ed and N at zonula adherens and the observation that the intracellular domain of Ed is required for the dominant-negative effect prompted a determination of whether the intracellular domain of both proteins might also physically interact with each other. Both GST pull-down and yeast two-hybrid assays were performed. No detectable binding between the intracellular domain of N and either the entire intracellular domain or the last 50 amino acids of Ed was observed. This suggests that the functional interaction between Ed and N is not mediated by a direct interaction between both proteins, although the possibility still remains that a physical interaction might occur via their extracellular domains (Escudero, 2003).

Thus far, the results indicate that Ed cooperates with the N pathway to control the determination of notum macrochaetae. Because Egfr and N pathways act antagonistically in macrochaetae development, the genetic interactions between ed and members of the Egfr signaling pathway were examined. Overexpression of wild-type Egfr (UAS-Egfr) alone by sca-Gal4, has a very weak effect on the number of notum bristles. However, the co-expression of both UAS-edDeltaECD and UAS-Egfr results in a severe tufting phenotype. Similar results were obtained when edDeltaECD and a constitutively activated form of Raf (UAS-rafgof) were co-expressed. As expected, increased number of SOPs were observed in proneural clusters, as detected with anti-Sens antibody. The interaction between Ed and Egfr pathways was verified by observing that a decrease of Egfr activity (overexpression of a dominant-negative form of Egfr, UAS-EgfrDN) partially suppressed the extra bristle phenotype caused by ed1x5/edslH8. Together, these results demonstrate an antagonism between Ed and Egfr signaling pathways in bristle development. However, considering the known antagonism between the Egfr and N pathways in macrochaetae development, these results open the possibility that the Egfr pathway might mediate, at least in part, the interaction between ed and the N pathway. If this were the case, one would expect that modifications of the activity of the Egfr pathway would affect the activity of the N pathway. Apparently, this did not occur. The levels of E(spl)m8 mRNA accumulation in proneural clusters were essentially unmodified by overexpressing either a constitutively activated form of Ras (UAS-ras1V12) or the Egfr-negative ligand Argos (UAS-aos). These conditions mimicked a strong stimulation and an inhibition of the pathway, since they respectively led to formation of many ectopic SOPs or to the removal of most macro and microchaetae. It is concluded that it is unlikely that the interaction of Ed and N is mediated by the Egfr pathway (Escudero, 2003).

Functional dissection of Timekeeper (Tik) implicates opposite roles for CK2 and PP2A during Drosophila neurogenesis

Repression by E(spl)M8 during inhibitory Notch signaling (lateral inhibition) is regulated, in part, by protein kinase CK2, but the involvement of a phosphatase has been unclear. Timekeeper (Tik), a unique dominant-negative (DN) mutation in the catalytic subunit of CK2, was used in a Gal4-UAS based assay for impaired lateral inhibition. Specifically, overexpression of Tik elicits ectopic bristles in N1 flies and suppresses the retinal defects of the gain-of-function allele Nspl. Functional dissection of the two substitutions in Tik (M161K and E165D), suggests that both mutations contribute to its DN effects. While the former replacement compromises CK2 activity by impairing ATP-binding, the latter affects a conserved motif implicated in binding the phosphatase PP2A. Accordingly, overexpression of microtubule star (mts), the PP2A catalytic subunit closely mimics the phenotypic effects of loss of CK2 functions in N1 or Nspl flies, and elicits notched wings, a characteristic of N mutations. These findings suggest antagonistic roles for CK2 and PP2A during inhibitory N signaling (Kunttas-Tatli, 2009).

Inhibitory N signaling is vital for stereotyped patterning of sense organs such as the eye and the bristles. This signaling pathway is required for proper SOP/R8 selection and involves cell-cell communications. Specifically, the future SOP/R8 cell expresses the highest levels of the N ligand, Delta, which activates N in all cells of the PNC, but the future SOP/R8. This, in turn, elicits expression of the E(spl) repressors, a family of homologous basic-helix-loop-helix (bHLH) proteins. These bHLH proteins, along with the corepressor Groucho, then antagonize ASC/Ato. As a result, cells that receive N signaling are redirected from adopting the default (SOP/ R8) neural fate. This model reflects the findings that loss of inhibitory N signaling leads to excess SOP and R8 specification, which manifest as ectopic bristles and rough eyes, respectively. It is, therefore, important to fully define the mechanisms that regulate this critical step in neural patterning (Kunttas-Tatli, 2009).

Earlier studies suggested that transcription of E(spl) and the ensuing rise in protein levels was, perhaps, sufficient for restriction of the R8/SOP fate. Accumulating evidence, however, suggests that phosphorylation of E(spl) proteins is important for repression. Evidence has so far been obtained for M8 and its structurally related repressor Hairy, and in either case phosphorylation by CK2 augments repression in the eye and/or the bristle. It has, however, remained unclear whether protein phosphatases act to oppose CK2 functions. The characterization of such a regulation would open the possibility that phosphorylation and repression by E(spl) (inhibitory N signaling) is dynamically controlled in vivo. A role for PP2A has been implicated in studies showing ectopic bristle defects upon increased dosage of the regulatory subunits widerborst (wdb) or twins (tws) and in screens for modifiers of N. However, interactions between PP2A and alleles of N, such as Nspl have not yet been described. These studies provide new insights into the genetic behaviors of Tik and its revertant allele TikR, and implicate a tripartite regulatory nexus, involving CK2, PP2A and inhibitory N signaling (Kunttas-Tatli, 2009).

Both Tik and TikR lack CK2 kinase activity (in vitro). The severe clock defect of Tik/1 flies is, however, not observed in TikR/1 animals, and in this sense TikR meets the criteria of a revertant allele. These studies suggest that the TikR protein is not only devoid of kinase activity, but more importantly is deficient for binding CK2b, a prerequisite for CK2-holoenzyme formation and for proper functions in vivo. The most parsimonious interpretation is that misfolding of TikR prevents its incorporation into the holoenzyme. It seems reasonable to, therefore, suggest that the ability of Tik to incorporate into and 'poison' the endogenous holoenzyme (by binding CK2b) underlies its strong DN effects in vivo. However, it has been generally thought that these effects of Tik primarily reflect the M161K, but not the E165D, substitution. These studies on site-specific variants, suggest that these substitutions have additive effects on activity and N signaling, and Tik is likely to therefore be a 'double hit (Kunttas-Tatli, 2009).

The studies in N1 and Nspl backgrounds provide evidence that both substitutions in Tik affect proper CK2 functions. How might one interpret the effects on Nspl? Unlike the bristle, where N signaling occurs only after the specification of the bristle PNCs, the development of patterned founding R8 photoreceptors requires N signaling in a biphasic manner in the MF of the developing third instar eye disc. At the anterior margin of the MF, N elicits ato expression (for R8 specification), whereas in the MF it drives expression of E(spl) enabling refinement of a single R8 cell from the PNCs. Nspl only perturbs the latter. Specifically, Nspl renders R8 precursors hypersensitive to inhibitory N signaling, and consequently impairs R8 differentiation. These impaired R8’s are defective in the presentation of signals such as Hedgehog and Decapentaplegic, whose activities are necessary for ato expression at the anterior margin of the MF. As a result, the reduced ato expression in the MF of Nspl perpetuates throughout retinal histogenesis, and elicits the rough and reduced eye of Nspl. Consistent with the notion that this allele renders R8’s sensitive to inhibitory N signaling, the retinal defect of Nspl are strongly suppressed by conditions that attenuate E(spl) activity, such as halved dosage of Delta or E(spl), or by reduced CK2 activity (Kunttas-Tatli, 2009).

The dominant-negative effects of CK2a-M161K and CK2a-E165D in N1 and in Nspl animals are likely to involve the ability of either variant to robustly interact with CK2b and efficiently incorporate into the endogenous holoenzyme, in a manner akin to wild type CK2alpha. It is suggested that incorporation of the former variant attenuates endogenous CK2 activity. In contrast, the dominant-negative effects of the E165D substitution might not involve impaired CK2 kinase activity, but instead reflect its ability to perturb the interaction of endogenous CK2 with PP2A, an interaction that is increasingly suspect in the regulation of this protein phosphatase. These possibilities are addressed below (Kunttas-Tatli, 2009).

The effects of CK2alpha-M161K in N1 or in Nspl are easier to reconcile given its position in the ATP-binding site. This substitution substantially impairs kinase activity, and consequently ectopic CK2alpha-M161K mimics the neural defects of knockdown of this enzyme by RNAi. It would therefore seem to be the case that ectopic CK2alpha-M161K binds CK2beta, efficiently incorporates into the endogenous CK2-holoenzyme and attenuates activity, and this lowered activity impairs phosphorylation of, and repression by, endogenous E(spl). If so, this will reduce the 'strength' of inhibitory N signaling and elicit ectopic bristles in N1, and suppress the eye/R8 defects of Nspl. The effects of CK2alpha-M161K in these three developmental contexts are consistent with this model (Kunttas-Tatli, 2009).

However, the behavior of the E165D substitution was unexpected. The suggestion that this substitution exerts a negative impact on CK2 functions is supported by multiple findings, in addition to the extraordinary conservation of Glu165 in metazoan CK2alpha subunits. First, CK2alpha-E165D elicits ectopic bristles in N1 and suppresses the retinal defects of Nspl, and these effects are observed with multiple independent insertions and with multiple drivers. Second, CK2alpha-E165D restores eye size and the hexagonal phasing of the facets in Nspl, akin to Tik or CK2alpha-M161K. Third, CK2alpha-E165D appears to restore Ato expression anterior to the MF and increases the number of Sens-positive R8 cells at its posterior margin. Therefore, its effects closely correlate, in time and space, to R8 cell specification, which is defective in Nspl. Together, these results suggest that the E165D substitution impairs CK2 functions. These functions, however, might not involve perturbed kinase activity per se, but may instead be related to the interaction of this enzyme with PP2A (Kunttas-Tatli, 2009).

Studies with mts overexpression are of interest, because this is the first demonstration that increased dosage of the PP2A catalytic subunit elicits developmental defects that are hallmarks of loss of N functions. Specifically, mts overexpression elicits ectopic bristles and notched wings in N1 flies, and suppresses the retinal defects of Nspl. Furthermore, its effects on restored ommatidial phasing and eye size (facet numbers) are comparable to those seen with Tik, CK2alpha-M161K or CK2alpha-E165D. These studies lead to the suggestion that interaction of PP2A with CK2 down-regulates phosphatase activity, perhaps by competing with the regulatory subunit such as Wdb, which is essential for target recognition and dephosphorylation. Such a mechanism would reflect the mutually exclusive binding of the catalytic (Mts) subunit of PP2A with Wdb or SV40 t-antigen. If so, ectopic Mts would override the binding capacity of endogenous CK2, and upon recruiting Wdb attenuate repression by E(spl) through dephosphorylation (Kunttas-Tatli, 2009).

This model could account for the dominant-negative effects of CK2alpha-E165D. In this case, ectopic CK2alpha-E165D would bind CK2beta, incorporate into the endogenous CK2-holoenzyme, and impair PP2A binding and downregulation. Its effects should therefore mimic Mts overexpression, a proposal that is consistent with the findings. If so, overexpression of CK2-E165D probably leads to enhanced PP2A activity. In contrast, the effects of ectopic CK2alpha-M161K more likely reflect a negative influence on CK2 activity itself, and suggest that this variant may represent a more precise dominant-negative construct of CK2 (Kunttas-Tatli, 2009).

The possibility arises that a precise regulation of repression by E(spl) proteins involves a balance between the opposing activities of CK2 and PP2A, perhaps involving direct interactions. Indeed, direct interactions between CK2 and PP2A have been identified by proteomic analysis in the mouse model and in cultured cells. While consensus sequences for kinases are easier to identify computationally and biochemically, similar analysis with phosphatases has been less forthcoming. For example, in the case of Period (Per), the central clock protein, coordinated activities of CK2, CK1, and PP2A are required for proper function. While Per is phosphorylated by CK2 and CK1 in vitro and in vivo, evidence for its dephosphorylation by PP2A is lacking especially as it relates to its site preference(s). In the future it will be important to determine whether E(spl) proteins are direct targets of PP2A, and if so how a balance between PP2A and CK2 activities regulates repression. PP2A may play a similar role in the regulation of mammalian Hes6 (the homolog of fly M8), given its phosphorylation by CK2. A reversible switch could be important in neural patterning to confer a rapid and precise temporal control over the onset of repression, or prevent a protracted block to the neural fate once resolution of the PNC has occurred and the SOP’s and R8’s have been selected (Kunttas-Tatli, 2009).

Continued: see Notch and neurogenesis: The PNS and bristle formation part 2/2

Table of contents

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

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