Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg: The joint-suppressive activity of Dve is required to repress dAP-2 expression

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. Evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints (Shirai, 2007).

To achieve specific developmental programs, antagonism between Notch and EGFR signaling has been widely observed. A graded activity of EGFR signaling from the distal tip of a leg disc is crucial for patterning the distal structure, and it should be converted into the segmental activation, which is critical for suppression of inappropriate joint formation. One possible explanation is that P-D patterning genes define the segment boundary, and thereby refine the Notch signaling pathway in the distal region of each segment, where EGFR signaling should be repressed. The expanded expression of argos-lacZ in Nts mutants strongly suggests that the Notch signaling pathway antagonizes EGFR signaling. Interestingly, a similar type of regulation has been reported for Caenorhabditis elegans vulval development. Thus, the antagonistic interaction between EGFR and Notch signaling establishes the complementary activation of these pathways in neighboring cells, and is crucial for both vulval cell fate determination and leg joint formation (Shirai, 2007).

In vertebrates, the early process of body segmentation, i.e., somitogenesis, takes place sequentially from head to tail. Somites are generated from the presomitic mesoderm (PSM), the unsegmented paraxial mesoderm at the tail end of the embryo. A 'clock and wavefront' model has been proposed to explain the mechanism of sequential somite formation. Oscillated gene expression, i.e., the clock, driven by Wnt and Notch signaling in the posterior PSM is translated into the segmental units in the wavefront, which is generated in the anterior PSM in response to the decreased activities of graded Wnt and FGF signaling from the tail end. As an embryo grows caudally, the wavefront moves backwards at a constant rate. Thus, the segment boundary is set at the interface between the Notch-activated and -repressed domains in the anterior PSM (Shirai, 2007).

During vertebrate somitogenesis, it has been shown that the interface between the Notch-activated and Notch-repressed domains is generated on suppression of Notch activity through induction of the lunatic-fringe (Lfng) gene in the segment boundary. This refinement is under the control of the basic helix-loop-helix type transcription factor Mesp2, which is expressed in the rostral half of the anterior PSM, indicating that rostral-caudal polarity within a somite is important for restricted Notch activation. The results indicate that the restricted Notch activation during Drosophila leg segmentation also occurs at the segment boundary rather than the center of each segment, suggesting that a conserved mechanism in both Drosophila legs and vertebrate somites underlies the activation of Notch signaling adjacent to the segment boundary (Shirai, 2007).

A Dve-expressing region straddles the fold of the segment boundary, and the following observations indicate that Dve has joint-suppressive activity: (1) dve mutant legs resulted in extra joint formation and (2) forced expression of dve in the presumptive joint region suppressed joint formation. Thus, the mechanism of joint development can be explained as follows; Notch-mediated Dve repression on the proximal side to the intersegmental fold relieves the above joint-suppressive activity, leading to normal leg joint formation. This is reminiscent of the abdomen-suppressive activity of Hunchback, which is relieved by Nanos to induce the abdominal structure. In contrast, Dve expression on the distal side to the fold should be maintained to suppress inappropriate joint formation, because dve mutation leads to extra joint formation with reverse polarity. It appears that Dve activity is only induced to suppress joint formation and that temporally regulated Dve repression is crucial for normal leg joint formation, because dve mutations did not affect normal joint formation (Shirai, 2007).

Extra joints with reverse polarity (reverse joints) are derived from mutants deficient in the PCP or EGFR signaling pathway. Previous reports have suggested a model in which the Notch signal activation proximal to the Notch ligand-expressing domains is blocked by these signals, only allowing the Notch signal activation in a distally adjacent region, i.e., the distal region of each segment. Based on the expression pattern of the Notch ligand Ser, it is assumed that the center of a segment is highly potent for receiving Notch signaling. This idea can explain the reverse polarity of extra joints, because Ser activates the Notch signaling pathway in two different directions: from proximal to distal for normal joints, and distal to proximal for extra joints. However, it seems unlikely that ectopic activation of Notch signaling is restricted at the center of a segment. A Notch-target gene, dAP-2, is autonomously activated in response to ectopic Notch signaling, and, in pk mutants, ectopic dAP-2 expression has expanded on the distal side to the intersegmental fold, the most proximal but not the central region in a segment. Furthermore, the joint-suppressive activity of Dve is also required to repress dAP-2 expression on the distal side to the intersegmental fold. These results suggest that reverse joints are derived from the distally adjacent region to the intersegmental fold (Shirai, 2007).

Based on the results, a model is proposed in which joint-forming activity is generated from the intersegmental fold in a bidirectional manner, and that an inappropriate signal having reverse polarity is blocked by Dve activity, and the PCP and EGFR signaling pathways. In this model, Dve activity is required to suppress Notch target genes, such as dAP-2, involved in joint formation. This is very similar to the situation observed in wing discs, where the Notch target gene wg is repressed by Dve in regions adjacent to the Notch-activated D-V boundary. It is an intriguing possibility that the vertebrate somite boundary generates similar bidirectional signals, and that the inhibition of either one is closely linked to the rostral-caudal polarity within a somite. Further characterization of Drosophila leg segmentation is needed to determine whether this model is applicable to vertebrate somitogenesis or other segmentation processes (Shirai, 2007).

dAP-2 and defective proventriculus regulate Serrate and Delta expression in the tarsus of Drosophila melanogaster

The segmentation of the proximal-distal axis of the Drosophila leg depends on the localized activation of the Notch receptor. The expression of the Notch ligand genes Serrate and Delta in concentric, segmental rings results in the localized activation of Notch, which induces joint formation and is required for the growth of leg segments. This study reports that the expression of Serrate and Delta in the leg is regulated by the transcription factor genes dAP-2 and defective proventriculus. Previous studies have shown that Notch activation induces dAP-2 in cells distal and adjacent to the Serrate/Delta domain of expression. Serrate and Delta are ectopically expressed in dAP-2 mutant legs, and Serrate and Delta are repressed by ectopic expression of dAP-2. Furthermore, Serrate is induced cell-autonomously in dAP-2 mutant clones in many regions of the leg. It was also found that the expression of a defective proventriculus reporter overlaps with dAP-2 expression and is complementary to Serrate expression in the tarsal segments. Ectopic expression of defective proventriculus is sufficient to block joint formation and Serrate and Delta expression. Loss of defective proventriculus results in localized, ectopic Serrate expression and the formation of ectopic joints with reversed polarity. Thus, in tarsal segments, dAP-2 and defective proventriculus are necessary for the correct proximal and distal boundaries of Serrate expression and repression of Serrate by defective proventriculus contributes to tarsal segment asymmetry. The repression of the Notch ligand genes Serrate and Delta by the Notch target gene dAP-2 may be a pattern-refining mechanism similar to those acting in embryonic segmentation and compartment boundary formation (Ciechanska, 2007).

The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila.

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).

AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster

Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. This study found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila (Kametaka, 2012).

AP-1 and GGAs are the major clathrin adaptors that function at the post-Golgi compartments in species ranging from yeast to mammals. After a decade of biochemical and cell biological approaches, however, functional specificity of each adaptor at a molecular level still remains to be solved. The present study showed that Drosophila AP-1 and its novel accessory protein Aftiphilin, but not GGA, are required for eye development, suggesting that the Drosophila AP-1-Aftiphilin protein complex is involved in the intracellular trafficking of specific cargo molecule(s) distinct from those regulated by GGA during eye development. It has previously been reported that the GAE domain of Drosophila GGA lacks major conserved amino acid residues potentially required for interaction with the accessory molecules that possess the tetrapeptide PsiG[PDE][PsiLM] motif. Consistent with this, this study showed that Drosophila GGA failed to interact with Aftiphilin, suggesting that the GAE domain of GGA is not structurally conserved. This finding might also reflect the physiological functional diversity between Drosophila AP-1 and GGA. However, the interaction between AP-1 and GGA was detected in the coimmunoprecipitation analysis, thus Drosophila AP-1 might also have a certain functional mode to form a complex with GGA, as implicated in mammalian cells (Kametaka, 2012).

It has been suggested that CG8538, an ORF in the Drosophila genome, encodes a protein with a limited homology with human Aftiphilin. This study concluded that Drosophila Aftiphilin/CG8538p is a functional counterpart of mammalian Aftiphilin, because of their common characteristics such as the possession of multiple γ-ear binding motifs, specific interaction with the γ-ear of AP-1, and the colocalization with AP-1 at the trans-Golgi compartments. Interestingly, the molecular basis of the interaction between Aftiphilin and the γ-adaptin of the AP-1 complex was also well conserved over species, because ectopically expressed Drosophila Aftiphilin in HeLa cells was also colocalized with γ1-adaptin of AP-1. Thus, the results indicate that Drosophila could serve a good model system to dissect the molecular mechanisms of AP-1 and Aftiphilin functions (Kametaka, 2012).

In the deduced amino acid sequence of Drosophila Aftiphilin/CG8538p, two WxxF-type binding motifs for the α-subunit of AP-2 complex were found. In mammals, Aftiphilin was shown to interact with AP-1 and AP-2, and was also proposed to function with AP-2 at the endocytic pathway in neuronal cells. In S2 cells, Drosophila Aftiphilin is predominantly associated with AP-1-positive Golgi compartments and forms a stable complex with AP-1. Moreover, the molecular interaction between Drosophila Aftiphilin and AP-2 was detected. Although the interaction seems to be minor compared with the interaction with AP-1, it is likely that Aftiphilin has other functions that are not related to AP-1, because the Aftiphilin-depleted fly occasionally showed much smaller eyes with decreased number of ommatidia in addition to the roughened eye phenotype. Precise analysis of the physiological functions of Drosophila Aftiphilin is ongoing (Kametaka, 2012).

Eye-specific depletion of Drosophila Aftiphilin or of any of the sigma1- or mu1-subunits of AP-1 caused misalignment of the photoreceptor neurons due to generation of extra R8 neurons during eye development. A genetic screening for Notch modifier genes suggested that AP47, which encodes the mu1 subunit of Drosophila AP-1, is involved in Notch signaling. Another genome-wide RNAi screening showed that the subunits of Drosophila AP-1 and Aftiphilin/CG8538 are involved in Notch signaling. Recently, it has also been reported that Drosophila AP-1 depletion led to mislocalization of Notch and its regulator Sanpodo (Spdo) to the apical plasma membrane and the adherens junction in the sensory organ precursor (SOP) daughter cells in developing nota in the fly. It was suggested that the altered trafficking of Notch is primarily due to increased recycling of the Notch regulator Spdo from the recycling endosomes to the plasma membrane, and that the mislocalization of Notch to the cell surface caused the gain-of-function phenotype in the AP-1 mutants. By contrast, in the current study a clear loss-of-function phenotype of Notch was observed by depletion of AP-1 or Aftiphilin in the developing eyes (Kametaka, 2012).

This discrepancy is probably due to the different mechanisms by which intracellular trafficking of Notch is regulated in different tissues. This study focused on Scabrous as a candidate for a Notch regulator that is affected by AP-1 or Aftiphilin depletion. Scabrous is a glycosylated secretory protein expressed in the R8 neurons, and sca mutation as well as AP-1-depletion causes duplication of R8 and other photoreceptor neurons. In addition, Scabrous was also shown to bind to the extracellular domain of Notch and to stabilize Notch at the cell surface. Drosophila AP-1 has been shown to function together with clathrin in the biogenesis of mucin-containing secretory granules in the salivary gland (Burgess, 2011). Because Scabrous was shown to accumulate in the intracellular compartments in the AP-1-deficient eye discs, the observations in the current study suggest that a defect in the secretion of Scabrous and/or other regulatory proteins causes the instability of Notch at the cell surface, which leads to degradation of Notch in the endosomal and lysosomal compartments. The decrease in the amount of Notch on the cell surface then causes defects in the lateral inhibition mechanism required for the photoreceptor cell specification during eye development (Kametaka, 2012).

In addition to the tissue-specific regulation of Notch trafficking, Notch signaling could also be regulated in several ways in the intracellular trafficking pathways. In the AP-1-depleted eye antennal discs, Notch was accumulated at the late endosomal-lysosomal compartment upon treatment with the lysosomal inhibitor chloroquine, suggesting that Notch is missorted for its lysosomal degradation. It has recently been showm that defects in endocytic trafficking caused by mutations of vps25, a component of the ESCRT-II complex, caused endosomal accumulation of Notch and enhanced Notch signaling. This suggests that the cellular output of Notch signal could be affected drastically in several ways through alterations in the intracellular transport machineries for Notch protein. Finally, the possibility cannot be excluded that Notch is a cargo molecule for Drosophila AP-1, although no direct interaction between AP-1 and the cytoplasmic tail of Notch has been observed so far (Kametaka, 2012).

In conclusion, Drosophila AP-1 plays a crucial role in Notch stability in vivo. It is inferred that Drosophila AP-1 is involved in the intracellular trafficking of tissue-specific regulators of Notch at the TGN or endosomal compartments, as proposed by Benhra (2011). Notch trafficking can be regulated by several mechanisms, and a particular regulatory mode would predominate according to the context of the development. Further analysis on the precise molecular mechanisms by which Drosophila AP-1 and Aftiphilin are involved in the sorting of these signaling molecules will uncover the physiological functions of these adaptor proteins in vivo (Kametaka, 2012).


Drosophila AP-2 is expressed during early embryogenesis and DAP-2 transcripts are also detected in the adult. Whole-mount in situ hybridizations demonstrate that AP-2 is expressed initially at stage 9 of Drosophila embryonic development and that AP-2 transcripts are detected in regions of the brain, eye-antennal disc, optical lobe, antenno-maxillary complex, and in a subset of cells of the ventral nerve cord (Bauer, 1998).

The developmental expression pattern of AP-2 was analyzed by in situ hybridization of DIG-labeled-AP-2-antisense RNA probe to wild-type Drosophila embryos and dissected larval tissues. Zygotic transcripts are first detected at stage 9 in a restricted area of procephalic neuroectoderm (PNE) (brain anlage). Expression in the PNE expands between stages 10 and 12 to include several discrete subregions. During stages 11-13, AP-2 transcripts are also detected in presumed neuroblasts and daughter cells underlying PNE sites. At stage 14 and thereafter, AP-2-expressing cells are located in the brain, mainly in the anteriormost neuromere, the protocerebrum. When viewed together with procephalic fate map data, the spatiotemporal pattern of AP-2 expression in these dorsal head regions strongly suggests a lineage relationship between AP-2-expressing cells in the PNE and in the brain thereafter. Aspects of this pattern are reminiscent of proneural gene expression, e.g., achaete-scute complex (AS-C) genes expressed in neuroectoderm sites and neuroblasts selected from these sites (Monge, 1998).

AP-2 was expressed in the ventral nerve cord (VNC) beginning at stage 12 in two clusters of cells in every hemineuromere. The relatively late onset of VNC expression indicates that AP-2 is not involved in selection of VNC neuroblasts from ventral neuroectoderm (VNE), but rather functions in a subset of differentiating neurons or glia. Expression is detected in subregions of the maxillary segment beginning at stage 10 and persists as this head segment moves anteriorly to become part of the larval antenno-maxillary complex. The maxillary segment generates larval mouth hooks, maxillary cirri and two chemoreceptive and mechanoreceptive sensory organs of the antennomaxillary complex: the ventral organ (VO) and maxillary sense organ (MxSO or terminal organ). MxSO and VO anlage roughly map to dorsomedial and ventromedial parts of the maxillary lobe at stage 12, and may be flanked by or partly coincident with AP-2-expressing cells. AP-2 expression in the maxillary segment overlaps with expression of proboscipedia (pb) and Deformed (Dfd) (group 2 and 4 Hox genes) which regulate development of adult labial and maxillary palps, and larval mouth hooks, ventral organs and maxillary cirri, respectively (Monge, 1998).

In third instar larvae, AP-2 is expressed in discrete parts of the optic lobes, brain, VNC, and antennal and leg imaginal disks. The homeodomain gene Distalless (Dll) is also expressed, like AP-2, in the maxillary segment and antennal and leg disks; and similarities exist between the expression patterns of AP-2 and Dlx (murine Dll-related) family members in the branchial arches, limbs, and forebrain during mouse embryogenesis. The gene is expressed in rings in the leg imaginal disc (Monge, 1998).

In summary, the AP-2 developmental expression pattern strongly parallels the embryonic expression patterns of murine AP-2 family members in the CNS, maxillary (proximal) portion of the mandibular arch, and frontonasal and distal limb regions. Thus, expression in discrete CNS regions and tissues that develop extensive sensory innervation is a conserved feature of Drosophila and mammalian transcription factor AP-2 family genes (Monge, 1998).

Effects of Mutation or Deletion

An allele of AP-2 was isolated as a viable mutation causing a severe reduction in leg length. Because of this phenotype, this mutation was initially called stummelbein (German for 'short leg'). The P-element is integrated 87 base pairs upstream of AP-2. AP-2 has two alternative first exons predicted to produce proteins, which differ in about the first 20 amino acids (Bauer, 1998; Monge, 1998). The stummelbein P-element is inserted close to the first exon (Monge, 1998). The alternative first exon is located about 9kb upstream of the P-element insertion site. To verify that the stummelbein P-element insertion is a mutant in AP-2, stummelbein mutant flies were crossed with flies carrying two different EMS induced mutations in AP-2. The leg defects in the homozygous AP-2 mutants and the AP-2/stummelbein flies were indistinguishable, indicating that stummelbein is defective in AP-2 activity. The defects in homozygous stummelbein or AP-2 mutants were comparable in severity with the defects produced when these alleles were heterozygous with a deletion that removes the AP-2 gene (Dfcroc2). This suggests that the stummelbein and AP-2 mutants behave genetically as strong loss-of-function mutations in the leg. No AP-2 protein expression could be detected in stummelbein mutant leg discs or in AP-22 or AP-215 mutant embryos and discs by antibody labeling (Kerber, 2001).

AP-2 mutants are often pupal lethal and most of the homozygous flies die before eclosion. Comparison with wild-type legs shows that AP-2 mutant legs are severely truncated along the proximal-distal axis and show fusions of leg segments. To better understand the basis for the defects in AP-2 mutant legs, the expression was examined of genes that reflect the primary subdivision of the leg imaginal disc along the proximal-distal axis. Distal-less (Dll), Dachshund (Dac) and Homothorax (Hth) proteins are expressed in broad, partially overlapping domains along the proximal-distal axis of the leg. Dll and Dac are required in the region of the leg affected in AP-2 mutants. Although the AP-2 mutant discs are smaller, it was found that the overlapping pattern of Dll and Dac expression is unaffected in the AP-2 mutant discs. This suggests that AP-2 is unlikely to be involved in the early stages of axial patterning of the leg (Kerber, 2001).

To find out when and where AP-2 might be required in the leg disc, its expression was followed throughout leg disc development. AP-2 protein is not expressed in second instar larval discs. AP-2 is first detectable at the beginning of third instar, slightly later than the onset of Dac expression. AP-2 expression starts as a ring outside the early Dll domain. In mature third instar discs, AP-2 is expressed in a series of rings along the proximal distal axis. These rings coincide with the expression domains of the Notch targets big brain and Enhancer of split [E(spl)] (Kerber, 2001).

Notch signaling has been implicated in formation of the joints between segments in the leg. Cells close to the end of each tarsal segment express elevated levels of the Notch ligands Delta and Serrate. Signaling by these ligands through Notch induces the expression of the big brain-lacZ reporter gene in distally adjacent cells. The observation that AP-2, E(spl) and big brain-lacZ expression patterns coincide prompted an examination of whether AP-2 expression also depends on Notch signaling activity. Clones of cells were generated in which Notch signaling was impaired or overactivated. Suppresser of Hairless [Su(H)] is required to activate targets of the Notch pathway. AP-2 is not expressed in the tarsal rings in Su(H) mutant clones induced 48±12 hours after egg laying, indicating that Notch signaling activity is required. Conversely, AP-2 expression is ectopically induced in cells expressing a constitutively active form of Notch in the disc epithelium. These results indicate that AP-2 is expressed in presumptive joint cells under the control of the Notch signaling pathway (Kerber, 2001).

In order to test whether AP-2 is required for joint formation, clones of AP-2 mutant cells were induced. AP-2 mutant clones that are located in the central 'interjoint' region of the segment do not show a phenotype, indicating that AP-2 is not required for normal growth, survival or differentiation of leg cells. However, in all cases when the clones cross between tarsal segments they affect joint formation. Although AP-2 mutant cells appear to be unable to participate in joint formation, wild-type cells adjacent to the clone can form the joint (Note that clones cannot include the entire circumference of the leg because they are restricted to A or P compartments and so cannot include the entire joint). The length of the leg segment is normal, indicating that partial loss of AP-2 expression can be compensated by the wild-type cells that contribute to forming the inter-segmental joint. Very large AP-2 mutant clones can show a reduction in leg length, as is known to occur for clones lacking Notch activity. These observations suggest that AP-2 functions as a mediator of Notch activity in joint formation (Kerber, 2001).

Ectopic activation of the Notch pathway can induce leg repatterning, outgrowths and ectopic joint structures in tarsal segments. Since AP-2 is required downstream of Notch for joint formation it was asked whether ectopic expression of AP-2 would be sufficient to induce ectopic joints. AP-2 was misexpressed in a stripe of cells along the proximal-distal axis of the leg using the patchedGal4 driver (i.e. crossing the endogenous AP-2 rings). Legs from patchedGal4/UAS-AP-2 flies contain many ectopic joints in the tarsal segments. It was note that AP-2 does not produce the other pattern abnormalities associated with expression of activated Notch. The ectopic joints induced by AP-2 in the tarsal region have wild-type morphology. The supernumerary joints tend to be clustered together and not uniformly distributed along the segment. The significance of this observation is unclear. To ask whether AP-2 is sufficient to mediate all of the activities of Notch in joint formation, UAS-AP-2 was expressed using patchedGal4 in a Notchts mutant background. Under these conditions, joints do not form. This indicates that, while AP-2 is required for joint formation it is not able to induce joints in the absence of Notch activity. To further test the requirement for AP-2, Notchintra was expressed with the dppGal4 driver in AP-2 mutant larvae. In the absence of AP-2 activity, activated Notch is not able to rescue joint formation. Together, these observations indicate that whereas AP-2 expression is regulated by Notch signaling, AP-2 is not the only mediator of Notch activity in joint formation and the requirement for AP-2 function cannot be overcome by constitutive activation of Notch. It is suggested that some other Notch-dependent activity may be required to define a region in which joint formation is possible when AP-2 is expressed (Kerber, 2001).

AP-2 is expressed in joint cells. In addition to being required for joint formation, AP-2 activity appears to be required to support normal development of the intervening 'interjoint' tissue. It can be inferred that this requirement is indirect because clonal analysis shows that AP-2 mutant cells contribute to normal development of interjoint tissue. Development of the interjoint region is compromised in cases where AP-2 mutant clones are large enough to remove joints. The small size of the segments in the AP-2 mutant legs could be due to reduced growth or increased cell death. No difference was observed in the amount of cell division in AP-2 mutant and wild-type leg imaginal discs labeled with antibody to the phosphorylated form of histone H3 (which labels mitotic cells). In contrast, AP-2 mutant leg imaginal discs show a considerable increase in the amount of cell death, as visualized by acridine orange and TUNEL labeling. Double labeling for TUNEL and beta-galactosidase of AP-2 mutant discs that carry the big brain-lacZ reporter, reveals that much of the cell death occurs in the interjoint region. It is noted that AP-2 activity is not required for big brain-lacZ expression in the presumptive joints. These observations indicate that although AP-2 is required for joint formation, it is not required for expression of the other known Notch targets in the presumptive joints. These genes are expressed in well-resolved rings in third instar. This indicates that the loss of tissue due to cell death does not compromise the ability of Notch ligands to activate Notch signaling and target gene expression in the mutant disc. By pupal stages, the interjoint regions appear to have been lost or reduced so that the tarsal rings of E(spl) expression fuse into a continuous band of expression. Taken together, these observations indicate that AP-2 activity is required in joint cells both for joint formation and to support cell survival in the interjoint region (Kerber, 2001). Proboscis shortening is evident for all hemizygous dAP-2 mutant alleles, and roughly correlates with the severity of leg shortening. Null mutants show a substantial reduction in length and width of the labellum (distiproboscis or labial palp) and number of pseudotracheal rows (chemosensory sensilla). The prestomal cavity is still present and newly eclosed null mutants usually can move the proboscis and can drink if provided with water. The clypeus, maxillary palps and antennae (derivatives of the clypeolabral and antennal imaginal discs) lack overt external defects; however, increased spacing between antennae in null mutants suggests undergrowth of proximal antennal segments at the expense of head cuticle. The proboscis of dAP-29 mutants is nearly normal, while that of dAP-210 mutants is shortened but normal in width. Proboscis and leg defects in AP-2 mutants correlate with AP-2 expression in imaginal disc primordia of these structures. In third instar wild-type larvae, AP-2 is expressed in several radial stripes in labial discs (paired primordia of the distiproboscis) and in concentric rings in leg imaginal discs. Upon leg disc eversion, the rings of AP-2-expressing cells correspond to presumptive joint regions (Monge, 1998; Kerber, 2001). AP-2 null and partial-loss phenotypes indicate that AP-2 is required for joint development and also for elongation of leg segments. Wings and halteres of AP-2 mutants develop normally consistent with lack of AP-2 expression in imaginal disc primordia of these (Monge, 2001).

Although AP-2 is expressed in the embryonic maxillary segment and in the embryonic and larval central nervous system in wild-type (Monge, 1998), AP-2 mutants survive embryogenesis and larval development, indicating that zygotic dAP-2 is not essential during these stages. Also, AP-2 mutants lack gross defects in the embryonic brain. However, loss of Drosophila AP-2 activity in the central nervous system could potentially have critical consequences for adult viability and other adult functions. To begin to address this question, brains of AP-2 mutant adults were examined in frontal paraffin sections to identify morphological changes. This analysis reveals a major defect that is reproducibly present in both null and hypomorphic AP-2 mutants. The defect entails a disruption of the central complex, a prominent central neuropil region in the protocerebrum. Abnormalities in nerve tracts of the optic lobes (antenno-glomerular tract, antennal nerve and median bundle) and an unusual number of large cell somata around the neuropil were also noted. The central complex is comprised of four substructures, namely the protocerebral bridge, the fan shaped body, the paired noduli and the ellipsoid body. In AP-2 mutants, the fan shaped body is bisected instead of being continuous across the midline as in wild type. The ellipsoid body and associated substructures are also disrupted relative to wild type. Lesions and mutations that disrupt the central complex are associated with loss of locomotor activity. In preliminary locomotion studies, AP-2 heteroallelic flies display reduced walking activity compared with wild-type flies, raising the possibility that AP-2 is required for some aspect of central complex development crucial for locomotion (Monge, 2001).


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AP-2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 February 2017

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