BIOLOGICAL OVERVIEW

Three Activator protein-2 (AP-2) family genes, AP-2alpha, AP-2ß and AP-2gamma, have been found in the mouse. The proteins encoded by these are very similar and can form heterodimers, which may contribute to their ability to regulate a wide variety of target genes. Only one AP-2 gene family homolog exists in Drosophila (Bauer, 1998; Monge, 1998). Drosophila AP-2 displays a great degree of similarity with AP-2 proteins from other organisms, and is slightly more similar to murine AP-2a than to other murine AP-2 family members. The DNA-binding domain is the most conserved part of the protein, and Drosophila AP-2 binds to the same DNA sequence as its mammalian counterparts (Bauer, 1998).

Drosophila AP-2 has essential roles in leg and proboscis outgrowth. AP-2 mutants are defective in leg development (Kerber, 2001 and Monge, 2001) and AP-2 is an important mediator of Notch signaling in joint formation. Loss- and gain-of-function effects of AP-2 on leg development are similar to loss- and gain-of-function effects of Notch signaling components in the leg (Kerber, 2001). The results suggest that AP-2 acts downstream of Notch, perhaps to regulate the genes required for production of the Notch signal. AP-2 is an essential player in the growth organizing properties associated with leg segment boundaries, and may act in regulatory pathways that coordinate limb-growth with development of local and higher order aspects of limb-specific neural circuitry (Kerber, 2001 and Monge, 2001).

A potential role for AP-2 in local neurogenesis in limbs is suggested by the observation that ectopic AP-2 can cell autonomously transform wing vein epithelium into ectopic sensory organs. AP-2 in involved in the development of the adult brain central complex, a higher order center for regulation of locomotor activity. The requirement for AP-2 in brain central complex development suggests an evolutionarily expedient link between growth of limbs and elaboration of their higher order neural circuitry. An ability to couple morphological evolution of body parts to evolution of neural circuits that innervate those parts using shared transcription factors could be an important feature, albeit currently under-appreciated, of gene expression networks. (Monge, 2001).

AP-2 is expressed in the presumptive joints under control of the Notch signaling pathway. AP-2 is required for formation of joints and is sufficient to induce supernumerary joints when ectopically expressed. Unlike Notch mutants, strong AP-2 mutants are viable and produce flies with short legs. The activity of AP-2 in the presumptive joints is required to support survival of cells in the interjoint region. On the basis of clonal analysis it has been inferred that Notch activity in the joints is required for development of the interjoint region and that joints are centers of growth control in the leg. Experiments reported in this study suggest that Notch acts via AP-2 to support survival of cells in the interjoint region of the leg segments. Clonal analysis has shown that AP-2 activity is not required by the interjoint cells themselves, therefore it is suggested that AP-2 might control expression of a secreted factor that is produced by the joint cells and acts non-autonomously to support survival of nearby cells. These observations suggest that AP-2 is an important mediator of Notch signaling in joint formation and leg segment development (Kerber, 2001).

Several observations indicate that AP-2 does not mediate all of the effects on Notch in the leg. (1) AP-2 is not sufficient to induce joints in a Notch mutant leg; (2) other Notch-dependent target genes, including big brain and E(spl) are induced normally in AP-2 mutant leg discs; (3) ectopic activation of the Notch pathway produces supernumerary joints that are often associated with outgrowths of the leg. Like Notch, ectopic AP-2 induces supernumerary joints, but does not cause outgrowths. These observations indicate that AP-2 mediates some, but not all of the activities of Notch in the leg. For example, Fringe is expressed at high levels in the interjoint region. It has been shown that ectopic expression of Fringe can inhibit joint formation. It is possible that the presence of Fringe influences Notch activity to limit joint formation by AP-2. This may provide an explanation for the clustering of ectopic joints when AP-2 is misexpressed (Kerber, 2001).

Boundary regions have been implicated as centers of growth control in a variety of developmental processes. Compartment boundaries serve as sources of secreted signaling proteins required to support growth of the wings and legs. At later stages of development, additional subdivisions occur, including wing veins and leg segments. These too are implicated in growth control. These observations provide some insight into the mechanism by which inter-segmental joints influence the growth of leg segments. AP-2 mutant flies show a severe reduction in the length of the leg, whereas clones of mutant cells in the interjoint region have no effect. It is noted that the extra joints induced by ectopic expression of AP-2 do not cause overgrowth of the leg. Thus AP-2 does not appear to produce a growth factor per se. One possibility is that AP-2 expression is required in joint cells to produce a survival factor to support development of the leg segments. Alternatively, the cell death observed in AP-2 mutant leg discs might be a secondary consequence of pattern abnormalities, as has been observed in embryos mutant for segmentation genes (Kerber, 2001).

Based on analyses of mouse, frog and chick AP-2 family members, vertebrate AP-2 transcription factors appear to play conserved roles in similar developmental contexts. The expression domains of AP-2 that seem most evidently conserved between fly and vertebrates are those in the nervous system, head and limbs. AP-2alpha mutant mice show a highly penetrant loss of the radius and transformation or loss of the first digit in the forelimb. AP-2alpha and AP2gamma are both expressed in the limb bud mesenchyme, with AP-2gamma showing an earlier onset than AP-2a. As limb bud outgrowth occurs, AP-2a is expressed in the distal limb bud (progress zone). Given the potential redundancy between AP2alpha and AP-2gamma in limb development, it is perhaps not surprising that the limb phenotype in AP-2 knockout mice is relatively mild. Interestingly, duplications of limb structures have been observed in AP-2alpha chimaeric mice. Since these are not seen in the null mutant mice, it appears they arise as a result of interactions between mutant and wild-type cells in the mosaic limbs. Limb duplications in Drosophila AP-2 loss-of-function mutants have not been observed. However, small outgrowths are sometimes seen in homozygous AP-2 mutant legs and the sex combs are sometimes expanded. This could be due to aberrant healing in areas where extensive cell death has occurred. Interestingly, ectopic joints are observed when AP-2 is expressed ectopically, suggesting that in the fly the interaction between AP-2 expressing and non-AP-2 expressing cells might also be important. This could indicate a functional similarity between vertebrate and Drosophila AP-2 in limb development (Kerber, 2001).

In AP-2 mutant mice, the radius (bone) is sometimes missing and the axial skeleton is abnormal -- it is therefore possible that AP-2 plays a role in bone development. This is supported by the observation that ossification occurs more slowly in AP-2 mutant mice than in wild-type mice. The Notch signaling pathway plays a role in endochondral bone development and thus indirectly in joint formation in the chicken limbs. In this process, Notch signaling is required to regulate the differentiation of chondrocytes and to downregulate their proliferative activity. Since signaling pathways are often conserved between species, it is possible that vertebrate AP-2 factors are also regulated by the Notch signaling pathway (Kerber, 2001 and references therein).

Segment-specific regulation of the Drosophila AP-2 gene during leg and antennal development

Segmentation involves subdivision of a developing body part into multiple repetitive units during embryogenesis. In Drosophila and other insects, embryonic segmentation is regulated by genes expressed in the same domain of every segment. Less is known about the molecular basis for segmentation of individual body parts occurring at later developmental stages. The Drosophila transcription factor AP-2 gene, dAP-2, is required for outgrowth of leg and antennal segments and is expressed in every segment boundary within the larval imaginal discs. To investigate the molecular mechanisms generating the segmentally repetitive pattern of dAP-2 expression, transgenic reporter analyses was performed and multiple cis-regulatory elements were isolated that can individually or cooperatively recapitulate endogenous dAP-2 expression in different segments of the appendages. An enhancer specific for the proximal femur region, which corresponds to the distal-most expression domain of homothorax (hth), was analyzed in the leg imaginal discs. Hth is known to be responsible for the nuclear localization and, hence, function of the Hox cofactor, Extradenticle (Exd). Both Hth and Exd were shown to be required for dAP-2 expression in the femur, and a conserved Exd/Hox binding site was found to be essential for enhancer activity. These loss- and gain-of-function studies further support direct regulation of dAP-2 by Hox proteins and suggest that Hox proteins function redundantly in dAP-2 regulation. This study reveals that discrete segment-specific enhancers underlie the seemingly simple repetitive expression of dAP-2 and provides evidence for direct regulation of leg segmentation by regional combinations of the proximodistal patterning genes (Ahn, 2011).

The segmentally repeated expression of dAP-2 in the developing leg and antennal discs may suggest that its expression in each segment is regulated in a similar manner by upstream segmentation genes. Alternatively, each domain (ring) of dAP-2 expression could result from the combinatorial activities of multiple transcription factors, which themselves are not expressed in a repeated pattern, but instead occupy distinct and broader domains along the PD axis of the appendages. Current data provide strong evidence that the latter strategy is utilized to establish dAP-2 expression in all but the tarsal segments. It seems that the tarsus has adopted a strategy different from that of other leg segments to regulate dAP-2 expression (Ahn, 2011).

In an effort to understand molecular mechanisms controlling dAP-2 expression during leg development, the regulatory potential of dAP-2 genomic fragments was tested using transgenic reporter analyses. Multiple enhancers were successfully isolated which can independently direct reporter expression in specific leg segments and together recapitulate, almost completely, the endogenous expression pattern. It is intriguing that the relative positions of these enhancers on the chromosome are well correlated with the position of their activity along the PD axis of the leg. Importantly, the presence of segment-specific enhancers suggests that dAP-2 expression is differentially regulated in each leg segment. It is likely that each domain of dAP-2 expression in the true joints is regulated by a combination of upstream regulators involved in PD patterning using segment-specific enhancers similar to the distinct enhancers used to regulate expression of the pair-rule gene, even-skipped, in every other parasegment during embryonic segmentation. Interestingly, dAP-2 expression in the coxa is differentially regulated along the DV axis and depends on two region-specific enhancers. In addition, the EB fragment displayed relatively weaker activity in the ventral region compared to the larger E6 fragment. These data raise the possibility that DV patterning genes are also involved in dAP-2 regulation in the proximal segments. It is possible that the use of multiple region-specific enhancers is a general mechanism establishing expression of segmentation genes during leg development (Ahn, 2011).

Current data indicate that dAP-2 expression in antennal discs also requires multiple region-specific enhancers. Some of the leg enhancers showed an antennal expression pattern similar to their leg patterns with respect to the PD axis. However, there are also enhancers specific for either antennal or leg discs implying that the genes required for normal identity of the two homologous appendages might be involved in regulation of dAP-2 expression in some segments. One of the features that distinguish antennae from legs is that in antennal discs, hth expression is expanded to the intermediate region where dac expression is missing. In contrast, the expression patterns of Dll, dac and hth are very similar in the proximal and distal regions of the two appendages. It is interesting to note that the dAP-2 enhancers for the most proximal and distal regions are shared between the two appendages while the intermediate region utilizes distinct enhancers. This implies that dAP-2 expression in the intermediate region is more likely to be regulated by antennal- or leg-specific regulatory pathways. For example, although the femur and the AIII are homologous structures, the Hox-dependent proximal femur enhancer is active in the leg, but not in the antenna. Likewise, the BE enhancer is active in the proximal AIII of the Hox-free antenna, but not in the leg (Ahn, 2011).

The Hox gene Antp has been considered to be a key factor in determining leg identity since Antp mutant clones in the T2 leg cause a leg-to-antenna transformation, mainly outside of the Hth domain. Previous studies suggested that Antp performs its selector function by acting as a repressor of hth and other antennal genes in the intermediate leg. In contrast, both Antp and hth are expressed in the proximal leg, and are required for growth and segmentation of this region. Therefore, it has been proposed that the role of Antp as a repressor of hth is limited to the intermediate leg, and that both Antp and Hth contribute to proper development of the proximal leg (Ahn, 2011).

The similar loss-of-function phenotypes of hth and exd suggest that Hth and Exd act on common target genes during development of the proximal leg. In certain developmental contexts, Hth can directly bind to DNA through its homeodomain in a ternary complex including Exd and Hox proteins to regulate expression of target genes. However, it has been shown that a Hth isoform lacking the homeodomain can execute the function of Hth in PD patterning of Drosophila leg discs indicating that direct DNA binding is not necessary for its function in proximal leg discs. Since no conserved, consensus Hth binding site were found in the proximal femur enhancer of dAP-2, Hth is likely functioning in dAP-2 expression through a mechanism independent of its direct binding to DNA through its homeodomain. Instead, Hth may regulate dAP-2 expression in the proximal femur by facilitating the nuclear localization of Exd or by interacting with other transcription factors which bind DNA (Ahn, 2011).

As a cofactor of Hox proteins, Exd, and its mammalian homolog Pbx, cooperatively bind DNA with Hox proteins and regulate expression of their target genes which are involved in a variety of developmental processes in both vertebrates and Drosophila. Although previous genetic analyses have revealed essential functions of Exd and Hox proteins in leg development, it has been unclear whether these factors act together on common target genes during this process. This study has identified a conserved Exd/Hox binding site which is required for activity of the proximal femur enhancer of dAP-2. Through clonal analyses, it was demonstrated that hth, exd and Antp are necessary for dAP-2 expression in the presumptive proximal femur of leg imaginal discs. This is the first example of a direct target gene of an Exd/Hox complex in Drosophila limb development. This study also provides insight into the molecular mechanism integrating the combinatorial actions of PD patterning genes in the regulation of region-specific expression of leg segmentation genes (Ahn, 2011).

Although Antp is expressed in all three pairs of legs, most of the prothoracic (T1) and metathoracic (T3) legs with Antp mutant clones appeared to be normal, except for a rare fusion between the femur and tibia. However, Scr/Antp double mutant clones in T1 legs and Antp/Ubx double mutant clones in T3 legs generated leg defects indistinguishable from those generated by Antp mutant clones in T2 legs. It was proposed that the low penetrance of the Antp mutant phenotypes in T1 and T3 legs is due to redundancy with Scr and Ubx, which are expressed in T1 and T3 leg discs, respectively. This idea is consistent with the previous observations that Scr and Ubx both can induce antenna-to-leg transformations when ectopically expressed in antennal discs. It is proposed that Antp, Scr and Ubx can redundantly activate dAP-2 expression in the proximal femur as Exd/Hox heterodimers based on the following observations. First, dAP-2 expression in T2 leg discs, but not in T1 and T3 leg discs, requires Antp. Secondly, EMSA results demonstrate that all three Hox proteins bind strongly to the binding site in the proximal femur enhancer as Exd/Hox heterodimers. Thirdly, all three Hox proteins can activate PrF enhancer function when ectopically expressed in antennal discs (Ahn, 2011).

GENE STRUCTURE

cDNA clone length - 2181

Bases in 5' UTR - 410

Exons - 8

Bases in 3' UTR - 373

PROTEIN STRUCTURE

Amino Acids - 461 (accession CAA07279) and 465 (accession AAC64676)

Structural Domains

Drosophila AP-2 produces two different mRNAs that use different first exons and encode proteins that differ at their N termini (Bauer, 1998; Monge, 1998). The predicted Drosophila AP-2 amino acid sequence exhibits 42%-45% overall identity with the vertebrate AP-2 proteins. A sequence of 107 amino acids within the DNA binding and dimerization domain of the vertebrate AP-2 proteins is highly conserved (90%-92%) with the Drosophila AP-2 homolog. An in vitro translation product of -2 cDNA binds specifically to AP-2 consensus binding sites. Drosophila AP-2 is functionally conserved in vivo; transient transfection of a AP-2 expression plasmid activates transcription through AP-2 binding sites in both mammalian and Drosophila cell lines (Bauer, 1998).

date revised: 26 October 2011

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