Delta


Effects of Mutation or Deletion

Table of contents

Delta function in peripheral nervous system differentiation

In the central nervous system (CNS) of Drosophila embryos lacking either cyclin A or Regulator of cyclin A1 (rca1) several ganglion mother cells (GMCs) fail to divide. Rca1 is novel 412 amino acid protein required for both mitotic and meiotic cell cycle progression, Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). GMCs in cycA and rca1 mutants differentiate as neurons: they assume the 'B' fate normally taken by one of their sibling progeny. These GMC fate decisions correspond to Notch pathway mutants ('B/B'), and they oppose the fate changes observed in embryos lacking numb ('A/A'). The loss of zygotic numb or constitutive activation of Notch in a rca1 background allows for a binary fate switch in GMCs: GMCs often differentiate as the 'A' sibling in the context of these mutations. These results indicate that activation of the Notch pathway causes GMCs to adopt the 'A' neuronal fate. Thus, fate choice in non-dividing GMCs appears to occur in much the same way that binary fate decisions occur in sibling neurons. In some models of asymmetric division, a specific factor required to attain one of the sibling fates is produced only upon progression of the cell cycle. The observation that GMCs can attain the fate of either sibling neuron indicates that gene products dependent upon GMC division are not required in this fate decision (Lear, 1999).

Expression of Delta in the mesoderm is sufficient to attain both sibling fates. In Dl mutant embryos, misspecification of neuroectodermal cells results in an excess of neuroblasts and their resulting neuronal progeny. Additionally, binary cell fate alterations are observed at the sibling neuron level. In wild-type embryos, Vnd protein is expressed in pCC but not aCC; in Dl mutants, the numerous GMC 1-1a progeny all lack Vnd expression, indicating that all of these neurons acquire the aCC fate. The twist-GAL4 line was used to drive Dl expression in the embryonic mesoderm. In a wild-type background, expression of ectopic Dl using the twist- GAL4 line appears to have little effect on the embryo; significantly, no effect in CNS cell fate specification is observed. When Dl is expressed in the embryonic mesoderm of a Dl homozygous mutant using twist-GAL4, many sibling neuron pairs attain differential fates ('A/B'). Specifically, at least one aCC and one Vnd-expressing pCC is observed in each thoracic/abdominal hemisegment of these embryos. Thus, cell fate specification of the aCC/pCC sibling pair is rescued by expression of Dl in the mesoderm. Thus, these results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, these results indicate that Delta signal can be received from outside the sibling pair (Lear, 1999).

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).

The magnitude of segregating variation for bristle number in Drosophila exceeds that predicted from models of mutation-selection balance. To evaluate the hypothesis that genotype-environment interaction (GEI) maintains variation for bristle number in nature, the extent of GEI was quantitated for abdominal and sternopleural bristles among 98 recombinant inbred lines, derived from two homozygous laboratory strains, in three temperature environments. There is considerable GEI for both bristle traits. A genome-wide screen was conducted for quantitative trait loci (QTLs) affecting bristle number in each sex and temperature environment, using a dense (3.2-cM) marker map of polymorphic insertion sites of roo transposable elements. Nine sternopleural and 11 abdominal bristle number QTLs were detected. Significant GEI is exhibited by 14 QTLs, but there was heterogeneity among QTLs in their sensitivity to thermal and sexual environments. To further evaluate the hypothesis that GEI maintains variation for bristle number, estimates of allelic effects across environments at genetic loci affecting the traits are required. This level of resolution may be achievable for Drosophila bristle number because candidate loci affecting bristle development often map to the same location as bristle number QTL, including achaete-scute, scabrous, hairy, and Delta (Gurganus, 1998).

The Notch receptor is the central element in a cell signaling mechanism controlling a broad spectrum of cell fate choices. Genetic modifier screens in Drosophila and subsequent molecular studies have identified several Notch pathway components, but the biochemical nature of signaling is still elusive. The results are described of a genetic modifier screen of the bristle phenotype of a gain-of-function Notch allele, Abruptex16. Abruptex mutations interfere with lateral inhibition/specification events that control the segregation of epidermal and sensory organ precursor lineages, thus inhibiting bristle formation. Mutations that reduce Notch signaling suppress this phenotype. This screen of approximately 50,000 flies led to the identification of a small number of dominant suppressors in seven complementation groups. These include Notch, mastermind, Delta, and Hairless , known components in the pathway, as well as two novel mutations: A122 and M285. A122, appears to interact with Notch only during bristle development. M285, displays extensive genetic interactions with the Notch pathway elements and appears, in general, capable of suppressing Notch gain-of-function phenotypes while enhancing Notch loss-of-function phenotypes, suggesting that it plays an important role in Notch signaling. The profile of the genetic interactions documented with M285 is quite similar to that of mutations in other known components of the Notch pathway. Three kismet alleles were isolated as weak suppressors of the Ax16 bristle phenotype. Interestingly, mutations in kismet have been isolated independently as enhancers of the eye phenotype associated with the expression of constitutively activated forms of the Notch receptor. kismet, which may encode a structural component of chromatin, does not display broad genetic interactions with Notch. It has therefore been suspected that the identification of these alleles through the eye screen may reflect its effect on the expression of the transgene by perturbing normal chromatin function rather than significant interactions with Notch signaling. The fact that such alleles were isolated in the bristle screen may be indicative of a link between Notch signaling and kismet function; however, further analysis is necessary before such a relationship can be established (Go, 1998).

Delta (Dl) and Hairless (H) are two chromosome 3 candidate neurogenic loci that might contribute to naturally occurring quantitative variation for sensory bristle number. To evaluate this hypothesis, quantitative genetic variation in abdominal and sternopleural bristle numbers was assessed among (1) homozygous isogenic third chromosomes sampled from nature and substituted into the Samarkand (Sam) inbred chromosome 1 and 2 background; (2) among homozygous lines in which the wild-derived Dl-H gene region was introgressed into the Sam chromosome 3 background, and (3) among Dl-H region introgression lines as heterozygotes against the Sam wild-type strain and derivatives of Sam into which mutant Dl and H alleles had been introgressed. Variation among the Dl-H region introgression lines accounts for 36% (8.3%) of the total chromosome 3 among line variance in abdominal (sternopleural) bristle number and for 53% of the chromosome 3 sex x line variance in abdominal bristle number. Naturally occurring alleles in the Dl-H region fail to complement a Dl mutant allele for female abdominal bristle number and sternopleural bristle number in both sexes, and an H mutant allele for both bristle traits in males and females. These results are consistent with the hypothesis that naturally occurring alleles at Dl and H contribute to quantitative genetic variation in sensory bristle number (Lyman, 1998).

polychaetoid is required for cell fate specification in the eye. In pyd mutants a slight roughness of the eye is detected. This roughness is due to the presence of mispositioned and duplicated mechanosensory bristles and occasional enlarged, irregularly shaped facets. In addition, the rows of ommatidia are misaligned. In the midpupal stage eyes of pyd mutants, a small fraction of ommatidia are observed to have extra cone cells and/or primary pigment cells. Some ommatidia have too few cone cells. Ommatidia with extra cone cells also have extra photoreceptors. Reduction of Delta or Notch function in pyd mutants strongly enhances both the pyd extra bristle and rough eye phenotypes (Chen, 1996).

A restriction enzyme survey of a 57-kb region including the gene Delta has uncovered 53 polymorphic molecular markers in a sample of 55 naturally occurring chromosomes. A permutation test, which assessed the significance of the molecular markers with the largest effect on bristle variation in four genetic backgrounds relative to permuted data-sets, found two sites that are independently associated with variation in bristle number. A common site in the second intron of Delta affects only sternopleural bristle number, and another common site in the fifth intron affects only abdominal bristle number in females. Under an additive genetic model, the polymorphism in the second intron may account for 12% of the total genetic variation in sternopleural bristle number due to third chromosomes, and the site in the fifth intron may account for 6% of the total variation in female abdominal bristle number due to the third chromosomes. These results suggest the following: (1) models that incorporate balancing selection are more consistent with observations than deleterious mutation-selection equilibrium models; (2) mapped quantitative trait loci of large effect may not represent a single variable site at a genetic locus, and (3) linkage disequilibrium can be used as a tool for understanding the molecular basis of quantitative variation (Long, 1998).

The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).

The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).

The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).

To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999).

Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999).

Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).

The hypothesis was tested that naturally occurring nonsynonymous variants in the Delta ligand of the Notch signaling pathway contribute to standing variation in sternopleural and/or abdominal bristle number in Drosophila melanogaster, for both a large cohort of wild-caught flies and previously described laboratory lines. The transcribed region of Delta was sequenced for 16 naturally occurring chromosomes and 65 SNPs, including 7 nonsynonymous SNPs (nsSNPs). Identified nsSNPs and 6 additional common SNPs, all located in exon 6 and the 3' UTR, were genotyped in 2060 wild-caught flies using an oligonucleotide ligation assay-based methodology and were genotyped in 38 additional natural chromosomes via DNA sequencing. None of the genotyped nsSNPs were significantly associated with natural variation in bristle number as assessed by a permutation test. A 95% upper bound on the additive genetic variance attributable to each genotyped SNP in the large natural cohort was < 2% of the total phenotypic variation. Results suggest that two previously detected genotype/phenotype associations between bristle number and variants in the introns of Delta cannot be explained by linkage disequilibrium between these variants and nearby nonsynonymous variants. Unidentified regulatory variants more parsimoniously explain previous observations (Genissel, 2004).

Table of contents

Delta: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.