See the embryonic expression pattern of AP-2 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
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).
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).
Auman, H. J., et al. (2002). Transcription factor AP-2gamma is essential in the extra-embryonic lineages for early postimplantation development. Development 129: 2733-2747. 12015300
Bauer, R., McGuffin, M. E., Mattox, W. and Tainsky, M.A. (1998). Cloning and characterization of the Drosophila homologue of the AP-2 transcription factor. Oncogene 17(15): 1911-1922. 9788434
Bisgrove, D. A. and Godbout, R. (1999). Differential expression of AP-2alpha and AP-2beta in the developing chick retina: repression of R-FABP promoter activity by AP-2. Dev. Dyn. 214(3): 195-206. 10090146
Bosher, J. M., Williams, T. and Hurst, H. C. (1995). The developmentally regulated transcription factor AP-2 is involved in c-erbB-2 overexpression in human mammary carcinoma. Proc. Natl. Acad. Sci. 92(3): 744-7. 7846046
Bosher, J. M., et al. (1996). A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma. Oncogene 13: 1701-1707. 8895516
Brewer, S., Feng, W., Huang, J., Sullivan, S. and Williams, T. (2004). Wnt1-Cre-mediated deletion of AP-2alpha causes multiple neural crest-related defects. Dev. Biol. 267(1): 135-52. 14975722
Chazaud, C., et al. (1996). AP-2.2, a novel gene related to AP-2, is expressed in the forebrain, limbs and face during mouse embryogenesis. Mech. Dev. 54: 83-94. 8808408
Gaubatz, S., et al. (1995). Transcriptional activation by Myc is under negative control by the transcription factor AP-2. EMBO J. 14(7): 1508-19. 7729426
Gee, J. M., et al. (1999). Immunohistochemical analysis reveals a tumour suppressor-like role for the transcription factor AP-2 in invasive breast cancer. J. Pathol. 189: 514-520. 10629551
Huang, S., et al. (1998). Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis. EMBO J. 17: 4358-4369. 9687504
Jean, D., et al. (1998). Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells. J. Biol. Chem. 273: 16501-16508. 9632718
Kerber, B., et al. (2001). The AP-2 transcription factor is required for joint formation and cell survival in Drosophila leg development. Development 128(8): 1231-8. 11262225
Knight, R. D., Nair, S., Nelson, S. S., Afshar, A., Javidan, Y., Geisler, R., Rauch, G. J. and Schilling, T. F. (2003). Lockjaw encodes a zebrafish tfap2a required for early neural crest development. Development 130: 5755-5768. 14534133
Knight, R. D., Javidan, Y., Zhang, T., Nelson, S. and Schilling, T. F. (2005). AP2-dependent signals from the ectoderm regulate craniofacial development in the zebrafish embryo. Development. 132(13): 3127-38. 15944192
Kramer, P., et al. (2000a). Ectopic expression of luteinizing hormone-releasing hormone and peripherin in the respiratory epithelium of mice lacking transcription factor AP-2alpha. Mech. Dev. 94, 79-94. 10842061
Kramer, P., et al. (2000b). Transcription factor activator protein-2 is required for continued luteinizing hormone-releasing hormone expression in the forebrain of developing mice. Endocrinology 141: 1823-1838. 10803593
Li, B. S., et al. (2000). Molecular cloning, expression, and characterization of rat homolog of human AP-2alpha that stimulates neuropeptide Y transcription activity in response to nerve growth factor. Mol. Endocrinol. 14(6): 837-47. 10847586
LiCalsi, C., et al. (2000). AP-2 family members regulate basal and cAMP-induced expression of human chorionic gonadotropin. Nucleic Acids Res. 28(4): 1036-43. 10648798
Limesand, S. W. and Anthony, R. V. (2001). Novel activator protein-2alpha splice-variants function as transactivators of the ovine placental lactogen gene. Eur. J. Biochem. 268(8): 2390-401. 11298758
Luo, T., et al. (2002). Transcription factor AP-2 is an essential and direct regulator of epidermal development in Xenopus. Dev. Biol. 245: 136-144. 11969261
Maconochie, M., et al. (1999). Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family. Development 126: 1483-1494. 10068641
Maytin, E. V., et al. (1999). Keratin 10 gene expression during differentiation of mouse epidermis requires transcription factors C/EBP and AP-2. Dev. Biol. 216(1): 164-81. 10588870
McPherson, L. A., et al. (1997). Identification of ERF-1 as a member of the AP-2 transcription factor family. Proc. Natl. Acad. Sci. 94: 4342-4347. 9113991
McPherson, L. A. and Weigel, R. J. (1999). AP2alpha and AP2gamma: a comparison of binding site specificity and trans-activation of the estrogen receptor promoter and single site promoter constructs. Nucleic Acids Res. 27(20): 4040-9. 10497269
Meier, P., et al. (1995). Alternative mRNAs encode multiple isoforms of transcription factor AP-2 during murine embryogenesis. Dev. Biol. 169: 1-14. 7750631
Meulemans, D. and Bronner-Fraser, M. (2002). Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns. Development 129: 4953-4962. 12397104
Mitchell, P. J., Wang, C. and Tjian, R. (1987). Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell 50: 847-861. 3040262
Mitchell, P. J., Timmons, P. M., Hebert, J. M., Rigby, P. W. J. and Tjian, R. (1991). Transcription factor AP-2 is expressed in neural crest lineages during mouse embryogenesis. Genes Dev. 5: 105-119. 1989904
Monge, I. and Mitchell, P. J. (1998). DAP-2, the Drosophila homolog of transcription factor AP-2. Mech. Dev. 76: 191-195. 9867351
Monge, I., et al. (2001). Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development. Development 128(8): 1239-52. 11262226
Moser, M., et al. (1995). Cloning and characterization of a second AP-2 transcription factor: AP- 2 beta. Development 121: 2779-2788. 7555706
Moser, M., et al. (1997a). Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta. Genes Dev. 11: 1938-1948. 9271117
Moser, M., et al. (1997b). Comparative analysis of AP-2 alpha and AP-2 beta gene expression during murine embryogenesis. Dev. Dyn. 208: 115-124. 8989526
Nottoli, T., et al. (1998). AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice. Proc. Natl. Acad. Sci. 95: 13714-13719. 9811866
O'Brien, E. K., et al. (2004). Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebrafish. Dev. Biol. 265(1): 246-61. 14697367
Oulad-Abdelghani, M., et al. (1996). AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during differentiation of P19 embryonal carcinoma cells. Exp. Cell Res. 225: 338-347. 8660922
Philipp, J., et al. (1994). Cell type-specific regulation of expression of transcription factor AP-2 in neuroectodermal cells. Dev. Biol. 165(2): 602-14. 7958425
Satoda, M., et al. (2000). Mutations in TFAP2B cause Char syndrome, a familial form of patent ductus ateriosus. Nat. Genet. 25: 42-46. 10802654
Schorle, H., et al. (1996). Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381: 235-238. 8622765
Shen, H., Wilke, T., Ashique, A. M., Narvey, M., Zerucha, T., Savino, E., Williams, T. and Richman, J. M. (1997). Chicken transcription factor AP-2: cloning, expression and its role in outgrowth of facial prominences and limb buds. Dev. Biol. 188: 248-266. 9268573
Snape, A. M., Winning, R. S. and Sargent, T. D. (1991). Transcription factor AP-2 is tissue-specific in Xenopus and is closely related or identical to keratin transcription factor 1 (KTF-1). Development 113: 283-293. 1722450
Takeuchi, S., et al. (1999). AP-2beta represses D(1A) dopamine receptor gene transcription in neuro2a cells. Brain Res. Mol. Brain Res. 74(1-2): 208-16. 10640692
West-Mays, J. A., et al. (1999). AP-2alpha transcription factor is required for early morphogenesis of the lens vesicle. Dev. Biol. 206(1): 46-62. 9918694
West-Mays, J. A., et al. (2002). Ectopic expression of AP-2alpha transcription factor in the lens disrupts fiber cell differentiation Dev. Biol. 245: 13-27. 11969252
Winning, R. S., Shea, L. J., Marcus, S. J. and Sargent, T. D. (1991). Developmental regulation of transcription factor AP-2 during Xenopus laevis embryogenesis. Nucleic Acids Res. 19: 3709-3714. 1852613
Zhang, J., et al. (1996). Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. Nature 381: 238-241. 8622766
Zhang, J., et al. (2003). Overexpression of transcription factor AP-2alpha suppresses mammary gland growth and morphogenesis. Dev. Bio. 256: 127-145. 12654297
Zhang, J. and Williams, T. (2003). Identification and regulation of tissue-specific cis-acting elements associated with the human AP-2alpha gene. Dev. Dyn. 228(2): 194-207. 14517991
Zhu, C. H., et al. (2001). A family of AP-2 proteins down-regulate manganese superoxide dismutase expression. J. Biol. Chem. 276(17): 14407-14413. 11278550
date revised: 15 October 2005
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