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).

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. Drosphila 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 April 2001

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