Identification, cloning and developmental expression of AP-2 homologs

A 52 kd protein, AP-2, has been purified that binds to enhancer regions of SV40 and human metallothionein IIA (hMT IIA) and stimulates RNA synthesis from these promoters in vitro. Surprisingly, AP-2 also binds to two SV40 early promoter regions recognized by Sp1 and T antigen. Juxtaposed binding sites for AP-2 and Sp1 in the 21 bp repeats may facilitate productive interactions between the two factors. In contrast, sequence-specific binding of AP-2 to SV40 and hMT IIA DNA is inhibited by the viral repressor protein T antigen. Furthermore, T antigen inhibits AP-2-dependent transcriptional activation of the hMT IIA promoter in vitro. The inhibition is neither a direct nor an indirect result of T antigen binding to DNA, because the hMT IIA promoter lacks T antigen binding sites. Instead, sedimentation studies suggest that protein-protein interactions between AP-2 and T antigen block AP-2 binding to DNA. These findings suggest novel mechanisms for mediating positive and negative regulation of transcription (Mitchell, 1987).

The expression pattern of transcription factor AP-2 has been examined in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation has identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues (Mitchell, 1991).

This paper identifies a new, developmental role for transcription factor AP-2 in the activation of amphibian embryonic epidermal keratin gene expression. Keratin transcription factor KTF-1 is shown by several criteria to be identical or closely related to AP-2. KTF-1/AP-2 is tissue-specific from its first transcription in Xenopus embryos, and restricted to a small number of adult tissues, including skin. Epidermis-specific keratin transcription closely follows specification of the embryonic ectoderm in Xenopus, and is subject to regulation by growth factors and embryonic induction. In mouse basal keratinocytes, a KTF-1/AP-2-like factor is present and binds to a DNA sequence previously shown to be important in the regulation of the keratin K14 gene, which is actively expressed in these cells. Thus, the study of AP-2 and its role in the regulation of keratin gene transcription should enhance an understanding of both amphibian embryonic development and mammalian skin differentiation (Snape, 1991).

A cDNA clone has been isolated encoding the Xenopus homolog of the transcription factor AP-2 (XAP-2). The predicted amino acid sequence derived from the Xenopus cDNA shows very strong conservation with the amino acid sequence of human AP-2, suggesting that this protein is evolutionarily conserved, at least among vertebrates. This is further substantiated by the demonstration that an in vitro translation product of XAP-2 cDNA binds specifically to an AP-2 binding site from the human MT-IIA gene. Northern blot analysis of Xenopus embryo RNA revealed the existence of three major XAP-2 mRNA species that are only detectable after the midblastula transition (when embryonic transcription is activated), with peak accumulation of the transcripts occurring during gastrulation. Therefore, in contrast to other Xenopus transcription factors, XAP-2 is not maternally derived but arises exclusively from zygotic transcription. Unlike the situation in cultured human teratocarcinoma (NT2) cells, retinoic acid treatment does not induce XAP-2 mRNA in Xenopus embryos, even though the treatment has a pronounced morphogenetic effect on the embryos. These results suggest that XAP-2 may play a distinctive role during Xenopus embryogenesis (Winning, 1991).

AP-2 is a cell type-specific DNA-binding transcription factor that regulates selected target genes in vertebrate organisms. Cell type-specific expression and regulation of AP-2 has been investigated in neuroectodermal cell lineages. During retinoic acid (RA)-mediated differentiation of P19 embryonal carcinoma cells into neuroectodermal cell types that include immunohistochemically defined neurons and astrocytes, a strong induction of AP-2 transcripts and protein was observed. In contrast, AP-2 mRNA is not induced in P19 cells that undergo mesoendodermal differentiation in response to 1% dimethylsulfoxide or low concentrations of RA, respectively. The potential of both neurons and astrocytes to express AP-2 was ascertained by using cerebellar neurons and astrocytes derived from newborn mice. Unlike these types of cells, microglial cells do not express AP-2. Dibutyryl cyclic AMP further enhances levels of AP-2 transcripts in both P19 astrocytes and primary astrocytes, which also respond to agents elevating intracellular cAMP (noradrenaline, isoproterenol, forskolin). The cAMP-dependent induction of AP-2 can be blocked by inhibitors of protein kinase A. In contrast to its action in P19 cells, RA has no effect on AP-2 mRNA levels in primary astrocytes. These results indicate that AP-2 may play a role as a retinoic acid-sensitive regulator during differentiation of neurons and glia from an embryonic neural precursor. Furthermore, AP-2 may be involved in gene transcription in both mature neurons and astrocytes (Philipp, 1994).

The isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta, is described in this study. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites (consensus sequence G/CCCN3GGC) and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations reveals that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face, including the primordium of midbrain and the facial mesenchyme, differ in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta patterns of expression in the central and peripheral nervous system overlap with regions of developing sensory neurons. In adult tissues AP-2 alpha expression is found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. These analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues (Moser, 1995).

Transcription factor AP-2 has been implicated as an important regulator of gene expression during vertebrate embryogenesis. The cDNA cloning and analysis of mouse embryonic mRNA splice variants encoding four AP-2 isoforms is reported in this study. Isoform 1 is the homolog of the previously known human (HeLa) AP-2. The three new AP-2 isoforms all share the same DNA binding/dimerization domain as isoform 1 but either lack the proline-rich transcriptional activation domain encoded by exon 2 (isoform 2) or have different amino-termini encoded by two previously unknown alternative first coding exons for AP-2 (isoforms 3 and 4). All four AP-2 mRNA variants are present at significant levels between days 11.5 and 17.5 of mouse embryogenesis. Variants 1, 3, and 4 show qualitatively but not quantitatively similar restricted expression patterns in 8.5-12.5 dpc embryos examined by in situ hybridization. At mid-embryogenesis, variant 3 is the major AP-2 mRNA species in the nervous system and in total embryo RNA but is less prevalent than variants 1 and 4 in the epidermis. The four mRNAs are all induced, although unequally, during differentiation of P19 cells into neural cell types and by cAMP stimulation of primary astrocytes. Variants 1-3 are coexpressed in different ratios in HeLa cells and in three human glioblastoma cell lines. These findings reveal that transcriptional regulation by AP-2 is likely to be more complex than previously assumed given the potential for multiple AP-2 homo- and heterodimeric DNA binding forms (Meier, 1995).

Using a differential subtractive hybridization cloning procedure, the AP-2.2 gene has been identified as a novel early retinoic acid-induced gene in murine P19 embryonal carcinoma cells. The AP-2.2 protein, which is highly related to the AP-2 transcription factor, can activate transcription when bound to an AP-2 consensus binding site. The in situ hybridization pattern of expression of AP-2.2 transcripts during mouse embryogenesis are reported in this study. At 7.5 days post-coitum, AP-2.2 transcripts are detected in the boundary region between neural plate and surface ectoderm, as well as in extra-embryonic tissues. By 8.0-8.5 gestational days, AP-2.2 transcripts appear to be expressed in premigratory and migrating neural crest cells. Over the following days, the AP-2.2 gene displays region-restricted expression in the facial mesenchyme, especially around the embryonic mouth cavity and the nasal cavities, as well as in the surface ectoderm, nasal and oral epithelia. AP-2.2 RNA is also specifically expressed in the presumptive cortical region of the forebrain vesicles. AP-2.2 transcripts are restricted to the distal mitotic area (the 'progress zone') of the limb buds and of the genital bud. AP-2.2 expression also appears to be specific for primordial germ cells in the genital ridges. Thus, the AP-2.2 gene is expressed in several embryonic areas whose development can be affected by retinoids, such as the forebrain, face and limb buds (Chazaud, 1996).

A 2.8-kb cDNA encoding a new transcription factor (AP-2.2) has been cloned from mouse P19 embryonal carcinoma cells, in which the corresponding mRNA begins to accumulate 30 min after retinoic acid (RA) addition. The predicted protein is 449 amino acids long and exhibits approximately 65% overall identity with other AP-2-related proteins (human AP-2, mouse AP-2alpha and beta). A 96-amino-acid-long sequence, which is almost fully conserved between all these proteins, corresponds to the previously characterized human AP-2 DNA binding domain. Expression of AP-2.2 in Escherichia coli generates a protein that forms a specific complex with the AP-2 recognition site GCCN3GGC. AP-2.2 activates transcription from a reporter gene containing an AP-2 DNA binding site and acts synergistically with RARalpha to activate transcription from the CRABPII gene promoter. Transcriptional activation requires the AP-2.2 amino-terminal region that contains a domain rich in proline and glutamine residues. The pattern of AP-2.2 expression in adult tissues, which is distinct from that of AP-2alpha, is essentially restricted to male and female gonads, to most if not all the squamous epithelia, and to several exocrine glands (Oulad-Abdelghani, 1996).

Embryonic facial development in chick embryos involves a sequential activation of genes that control differential growth and patterning of the beak. One such gene, the transcription factor, AP-2, has been isolated that is known to be expressed in the face of mouse embryos. The protein sequence of chick AP-2alpha is 94% homologous to human and mouse AP-2. Wholemount in situ hybridization with a probe for chick AP-2 identifies expression from primitive streak stages up to stage 28. The most striking expression patterns in the head are during neural crest cell migration when AP-2 transcripts follow closely the tracts previously mapped for neural crest cells. Later, expression in the facial mesenchyme is strongest in the frontonasal mass and lateral nasal prominences and is downregulated in the maxillary and mandibular prominences. Once limb buds are visible, high expression is seen in the distal mesenchyme but not in the apical ectodermal ridge. The expression patterns of AP-2 in stage 20 embryos suggested that the gene may be important in 'budding out' of facial prominences and limb buds. Beads soaked in retinoic acid were implanted in the right nasal pit of stage 20 embryos resulting in a specific inhibition of outgrowth of the frontonasal mass and lateral nasal prominences. AP-2 expression is completely down-regulated in the lateral nasal within 8 hr of bead application. In addition, the normal up-regulation of AP-2 in the frontonasal mass does not occur following retinoic-acid treatment. There is an increase in programmed cell death around the right nasal pit that accompanies the down-regulation of AP-2. Prominences whose morphogenesis are not affected by retinoic acid do not have altered expression patterns. The apical ectodermal ridge was removed in stage 20 limb buds it was found that AP-2 expression is partially downregulated 4 hr following ridge removal and completely downregulated 8 hr following stripping. Application of an FGF-4 soaked bead to the apex of the limb bud maintains AP-2 expression. Thus AP-2 is involved in outgrowth and could be regulated by factors such as FGFs that are present in the ectoderm of both the face and limb (Shen, 1997).

Transcription factor AP-2 has been identified as playing important roles during embryonic development of the neural tube, neural crest derivatives, skin, and urogenital tissues. A second AP-2 transcription factor, AP-2 beta, has been isolated that is 76% homologous to the previously known AP-2 alpha gene, and both genes are coexpressed in murine embryos at day 13.5 and 15.5 post coitum (pc). Specific cRNA probes have been used to study comparatively AP-2 alpha and AP-2 beta expression by in situ hybridization of murine embryonic tissue sections. Expression of both genes starts at day 8 pc in the lateral head mesenchyme and extraembryonic trophoblast. The expression pattern is identical until day 10 pc but diverges significantly during later stages of development. From day 11 forward, specific expression patterns of AP-2 alpha and AP-2 beta mRNA are observed. Specific AP-2 beta signals are detected in the midbrain, sympathetic ganglia, adrenal medulla, and cornea. Specific AP-2 alpha signals are present in the limb buds, dorsal root ganglia, tooth germs, and Moll's and Meibom's glands. In contrast, expression of both genes occurs in skin, facial mesenchyme, spinal cord, cerebellum, and renal tubular epithelia. These results indicate that both genes are expressed with different temporal and spatial patterns during embryonic development (Moser, 1997b).

Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2alpha has been localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2alpha, but variations in the predicted N-terminus were observed, and three unique AP-2alpha splice-variants were identified. Expression of AP-2alpha variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter is variant dependent. RNA transcripts for all of the ovine AP-2alpha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2alpha transcript, which shares identity to Xenopus AP-2alpha, is expressed in BeWo cells. Immunoblot analysis confirmed AP-2alpha variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2alpha splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells (Limesand, 2001).

The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. Differences in regulation of the neural crest marker AP-2 have been studied across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution (Meulemans, 2002).

AP-2 DNA-binding specificity

The AP2 transcription factors exhibit a high degree of homology in the DNA binding and dimerization domains. In this study, the binding specificity of AP2alpha and AP2gamma were methodically compared using PCR-assisted binding site selection and competitive gel shift assay and it was determined that the consensus binding site for both factors is G/C CCNN A/C/G G/A G G/C/T. The use of single site promoter constructs with either a high or low affinity site demonstrates a direct relationship between site affinity and transcriptional activation. Overexpression of AP2alpha and AP2gamma results in the activation of a low affinity binding site construct to levels comparable to those seen with a high affinity site construct at lower amounts of protein expression. Both AP2alpha and AP2gamma are able to trans-activate the cloned human estrogen receptor alpha promoter in ER-negative MDA-MB-231 cells through high affinity AP2 sites in the untranslated leader sequence. This provides a functional mechanism to explain the correlation between AP2 activity and estrogen receptor expression in breast cancer. Since there is overexpression of AP2 factors in breast cancer compared to normal breast epithelium, these results suggest that increased factor expression may activate a set of target genes containing lower affinity binding sites that would normally not be expressed in normal breast epithelium (McPherson, 1999).

AP-2 protein interactions

The Myc protein binds to and transactivates the expression of genes via E-box elements containing a central CAC(G/A)TG sequence. The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation and apoptosis. Transactivation by Myc is under negative control by the transcription factor AP-2. AP-2 inhibits transactivation by Myc via two distinct mechanisms. First, high affinity binding sites for AP-2 overlap Myc-response elements in two bona fide target genes of Myc, prothymosin-alpha and ornithine decarboxylase. On these sites, AP-2 competes for binding of either Myc/Max heterodimers or Max/Max homodimers. The second mechanism involves a specific interaction between C-terminal domains of AP-2 and the BR/HLH/LZ domain of Myc, but not Max or Mad. Binding of AP-2 to Myc does not preclude association of Myc with Max, but impairs DNA binding of the Myc/Max complex and inhibits transactivation by Myc even in the absence of an overlapping AP-2 binding site. Taken together, these data suggest that AP-2 acts as a negative regulator of transactivation by Myc (Gaubatz, 1995).

An AP2 transcription factor is required for a sleep-active neuron to induce sleep-like quiescence in C. elegans

Sleep is an essential behavior that is found in all animals that have a nervous system. Neural activity is thought to control sleep, but little is known about the identity and the function of neural circuits underlying sleep. Lethargus is a developmentally regulated period of behavioral quiescence in C. elegans larvae that has sleep-like properties. Sleep-like behavior was studied in C. elegans larvae, and it was found to requires a highly conserved AP2 transcription factor, aptf-1, which was expressed strongly in only five interneurons in the head. Expression of aptf-1 in one of these neurons, the GABAergic neuron RIS, was required for quiescence. RIS was strongly and acutely activated at the transition from wake-like to sleep-like behavior. Optogenetic activation of aptf-1-expressing neurons ectopically induced acute behavioral quiescence in an aptf-1-dependent manner. RIS ablation caused a dramatic reduction of quiescence. RIS-dependent quiescence, however, does not require GABA but requires neuropeptide signaling. It is conclude that RIS acts as a sleep-active, sleep-promoting neuron that requires aptf-1 to induce sleep-like behavior through neuropeptide signaling. Sleep-promoting GABAergic-peptidergic neurons have also been identified in vertebrate brains, suggesting that common circuit principles exist between sleep in vertebrates and sleep-like behavior in invertebrates (Turek, 2013).

Tfap2 and Sox1/2/3 cooperatively specify ectodermal fates in ascidian embryos

Epidermis and neural tissues differentiate from the ectoderm in animal embryos. While epidermal fate is thought to be induced in vertebrate embryos, embryological evidence has indicated that no intercellular interactions during early stages are required for epidermal fate in ascidian embryos. To test this hypothesis, the gene regulatory circuits were determined for epidermal and neural specification in the ascidian embryo. These circuits started with Tfap2-r.b (AP-2-like2; see Drosophila AP-2) and Sox1/2/3 (see Drosophila Dichaete), which are expressed in the ectodermal lineage immediately after zygotic genome activation. Tfap2-r.b expression was diminished in the neural lineages upon of fibroblast growth factor signaling, which is known to induce neural fate, and sustained only in the epidermal lineage. Tfap2-r.b specified the epidermal fate cooperatively with Dlx.b (see Drosophila Dll), which was activated by Sox1/2/3. This Sox1/2/3-Dlx.b circuit was also required for specification of the anterior neural fate. In the posterior neural lineage, Sox1/2/3 activated Nodal, which is required for specification of the posterior neural fate. These findings support the hypothesis that the epidermal fate is specified autonomously in ascidian embryos (Satou, 2016).

Mutation of AP-2

AP-2 transcription factors are expressed in embryonic renal tissues. AP-2beta minus mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development, expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel with the massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism it has been shown that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis, pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene (Moser, 1997a).

The homozygous disruption of the mouse AP-2 gene yields a complex and lethal phenotype that results from defective development of the neural tube, head, and body wall. The severe and pleiotropic developmental abnormalities observed in the knockout mouse suggest that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, chimeric mice composed of both wild-type and AP-2-null cells have been analyzed. The phenotypes obtained from these chimeras indicate that there is an independent requirement for AP-2 in the formation of the neural tube, body wall, and craniofacial skeleton. In addition, these studies reveal that AP-2 exerts a major influence on eye formation, which is a critical new role for AP-2 that was masked previously in the knockout mice. Furthermore, an unexpected influence of AP-2 on limb pattern formation has been uncovered; this influence is typified by major limb duplications. The range of phenotypes observed in the chimeras displays a significant overlap with those caused by teratogenic levels of retinoic acid, strongly suggesting that AP-2 is an important component of the mechanism of action of this morphogen (Nottoli, 1998).

AP-2 transcription factors are a family of retinoic acid-responsive genes, which are involved in complex morphogenetic processes. In the current study, the requirement for AP-2alpha in early morphogenesis of the eye was determined by examining the nature of the ocular defects in AP-2alpha null and chimeric mice. AP-2alpha null embryos exhibit ocular phenotypes ranging from a complete lack of eyes (anophthalmia) to defects in the developing lens involving a persistent adhesion of the lens to the overlying surface ectoderm. Two genes involved in lens development and differentiation, Pax6 and MIP26 are also misexpressed. AP-2alpha mutants exhibit defects in the optic cup consisting of transdifferentiation of the dorsal retinal pigmented epithelium into neural retina and the absence of a defined ganglion cell layer. Newly generated chimeric embryos consisting of a population of AP-2alpha-/- and AP-2alpha+/+ cells exhibit ocular defects similar to those seen in the knockout embryos. Immunolocalization of AP-2 proteins (alpha, beta, and gamma) to the normal developing eye reveal both unique and overlapping expression patterns, with AP-2alpha expressed in a number of the ocular tissues that exhibited defects in the mutants, including the developing lens where AP-2alpha is uniquely expressed. Together these findings demonstrate a requirement for AP-2alpha in early morphogenesis of the eye (West-Mays, 1999).

The members of the AP-2 family of transcription factors play important roles during mammalian development and morphogenesis. AP-2gamma (Tcfap2c -- Mouse Genome Informatics) is a retinoic acid-responsive gene implicated in placental development and the progression of human breast cancer. AP-2gamma is present in all cells of preimplantation embryos and becomes restricted to the extra-embryonic lineages at the time of implantation. To study further the biological function of AP-2gamma, Tcfap2c-deficient mice were generated by gene disruption. The majority of Tcfap2c-/- mice failed to survive beyond 8.5 days post coitum (d.p.c.). At 7.5 d.p.c., Tcfap2c-/- mutants were typically arrested or retarded in their embryonic development in comparison to controls. Morphological and molecular analyses of mutants revealed that gastrulation can be initiated and that anterior-posterior patterning of the epiblast remains intact. However, the Tcfap2c mutants fail to establish a normal maternal-embryonic interface, and the extra-embryonic tissues are malformed. Moreover, the trophoblast-specific expression of eomesodermin and Cdx2, two genes implicated in FGF-responsive trophoblast stem cell maintenance, is significantly reduced. Chimera studies have demonstrated that AP-2gamma plays no major autonomous role in the development of the embryo proper. By contrast, the presence of AP-2gamma in the extra-embryonic membranes is required for normal development of this compartment and also for survival of the mouse embryo (Auman, 2002).

The AP-2alpha transcription factor is required for multiple aspects of vertebrate development and mice lacking the AP-2alpha gene (tcfap2a) die at birth from severe defects affecting the head and trunk. Several of the defects associated with the tcfap2a-null mutation affect neural crest cell (NCC) derivatives including the craniofacial skeleton, cranial ganglia, and heart outflow tract. Consequently, there is considerable interest in the role of AP-2alpha in neural crest cell function in development and evolution. In addition, the expression of the AP-2alpha gene is utilized as a marker for premigratory and migratory neural crest cells in many vertebrate species. This study specifically addresses how the presence of AP-2alpha in neural crest cells affects development by creating a conditional (floxed) version of tcfap2a which has subsequently been intercrossed with mice expressing Cre recombinase under the control of Wnt1 cis-regulatory sequences. Neural crest-specific disruption of tcfap2a results in frequent perinatal lethality associated with neural tube closure defects and cleft secondary palate. A small but significant fraction of mutant mice can survive into adulthood, but have retarded craniofacial growth, abnormal middle ear development, and defects in pigmentation. The phenotypes obtained confirm that AP-2alpha directs important aspects of neural crest cell function. At the same time, neurocristopathies affecting the head and heart, that might be expected based on the phenotype of the AP-2alpha-null mouse, were not detected. These results have important implications for the evolution and function of the AP-2 gene family in both the neural crest and the vertebrate embryo (Brewer, 2004).

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2alpha is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2alpha mutants die before melanogenesis. Zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2alpha (ap-2alpha MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2alpha MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2alpha works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2alpha MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2alpha regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, reduction of other neural crest derivatives is documented in ap-2alpha MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2alpha regulates multiple steps of melanophore development, and is required for development of other neuronal and non-neuronal neural crest derivatives (O'Brien, 2004).

The neural crest generates multiple cell types during embryogenesis but the mechanisms regulating neural crest cell diversification are incompletely understood. Previous studies using mutant zebrafish indicated that foxd3 and tfap2a (transcription factor AP-2alpha, a sequence-specific DNA-binding protein expressed in neural crest lineages with the highest levels of expression corresponding to early neural crest cells) function early and differentially in the development of neural crest sublineages. This study shows that the simultaneous loss of foxd3 and tfap2a function in zebrafish foxd3zdf10;tfap2alow double mutant embryos globally prevents the specification of developmentally distinct neural crest sublineages. By contrast, neural crest induction occurs independently of foxd3 and tfap2a function. The failure of neural crest cell diversification in double mutants is accompanied by the absence of neural crest sox10 and sox9a/b gene expression; forced expression of sox10 and sox9a/b differentially rescues neural crest sublineage specification and derivative differentiation. These results demonstrate the functional necessity for foxd3 and tfap2a for neural crest sublineage specification and that this requirement is mediated by the synergistic regulation of the expression of SoxE family genes. These results identify a genetic regulatory pathway functionally discrete from the process of neural crest induction that is required for the initiation of neural crest cell diversification during embryonic development (Arduini, 2008).

AP-2 transcriptional regulation

Mice lacking transcription factor AP-2alpha exhibit defects in the formation of the head, body wall, heart, neural tube, eye, and limbs, reflecting important sites of AP-2alpha expression in the developing embryo. AP-2alpha is also expressed in the postnatal mammary gland and has been linked to tumor progression and defects in growth regulation in the breast. A transgenic mouse approach has been used to identify tissue-specific cis-acting sequences associated with expression of the human AP-2alpha gene. The analysis indicates that multiple elements located throughout the gene contribute to expression in the trigeminal ganglia, spinal cord, mammary gland, and epidermis. A discrete cis-element located within the fifth intron is required for expression in the face and limbs, and a permanent line of AP-2alpha::lacZ transgenic mice has been derived to assess expression of this latter enhancer throughout morphogenesis. This transgene was introduced into an AP-2alpha-null mouse background and subtle alterations of its expression were detected within the progress zone and apical ectodermal ridge of the forelimbs. Similar changes in lacZ expression were observed within the zeugopod, and these correlated with defects in radius condensation in AP-2alpha-knockout mice. Taken together, these findings indicate that cell:cell communication within the forelimb is altered in the absence of AP-2alpha and reveal novel regulatory potential for AP-2alpha in limb development (Zhang, 2003).

AP-2 transcriptional targets

Overexpression of the c-erbB-2/HER2 protooncogene in breast carcinoma is controlled not only by the degree of amplification of the gene but also at the level of gene transcription. Thus, whether or not the gene is amplified, the activity of the c-erbB-2 promoter is enhanced in overexpressing cells through the binding of an additional transcription factor, OB2-1, whose activity is increased in these lines. OB2-1 is identical to the developmentally regulated transcription factor AP-2. Functional assays confirm that AP-2 is able to regulate c-erbB-2 expression in mammary-derived cell lines. Furthermore, although AP-2 is barely detectable in cells with the low c-erbB-2 expression phenotype, protein levels are clearly elevated in a panel of c-erbB-2-overexpressing lines. These findings demonstrate an important role for this transcription factor in human cancer (Bosher, 1995).

The proto-oncogene c-erbB-2 is overexpressed in 25%-30% of breast cancers through increased transcription and amplification of the gene. OB2-1, which upregulates c-erbB-2 transcription, is closely related to the developmentally regulated transcription factor, AP-2. Further analysis of affinity purified OB2-1 has now shown that it is in fact a combination of proteins from three AP-2-related genes, the previously described AP-2alpha gene and two new human family members, AP-2beta and AP-2gamma whose cloning and characterization are described here. All three AP-2 proteins show a high degree of homology and are capable of binding to the c-erbB-2 promoter as homo- or hetero-dimers. The three proteins can also activate a c-erbB-2 reporter construct, but AP-2alpha and AP-2gamma are 3-4 times more active in this regard than AP-2beta. In addition both AP-2alpha and AP-2gamma were expressed at elevated levels in the majority of c-erbB-2 overexpressing mammary tumor lines examined (Bosher, 1996).

Expression of the tyrosine kinase receptor, c-KIT, progressively decreases during local tumor growth and invasion of human melanomas. Enforced c-KIT expression in highly metastatic cells inhibits tumor growth and metastasis in nude mice. Furthermore, SCF (the ligand for c-KIT) induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. Loss of c-KIT expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The c-KIT promoter contains three binding sites for AP-2 and EMSA gels have demonstrated that AP-2 protein binds directly to the c-KIT promoter. Transfection of wild-type AP-2 into c-KIT-negative A375SM melanoma cells activates a c-KIT promoter-driven luciferase reporter gene, while expression of a dominant-negative AP-2B in c-KIT-positive Mel-501 cells inhibits its activation. Endogenous c-KIT mRNA and expression of proteins are upregulated in AP-2-transfected cells, but not in control cells. In addition, re-expression of AP-2 in A375SM cells suppresses their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of c-KIT is highly regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly through c-KIT transactivation and SCF-induced apoptosis. Therefore, loss of AP-2 expression might be a crucial event in the development of malignant melanoma (Huang, 1998).

Hoxa2 is expressed in cranial neural crest cells that migrate into the second branchial arch and is essential for proper patterning of neural-crest-derived structures in this region. Transgenic analysis was used to begin to address the regulatory mechanisms that underlie neural-crest-specific expression of Hoxa2. By performing a deletion analysis on an enhancer from the Hoxa2 gene that is capable of mediating expression in neural crest cells in a manner similar to the endogenous gene, it has been demonstrated that multiple cis-acting elements are required for neural-crest-specific activity. One of these elements consists of a sequence that binds to the three transcription factor AP-2 family members. Mutation or deletion of this site in the Hoxa2 enhancer abrogates reporter expression in cranial neural crest cells but not in the hindbrain. In both cell culture co-transfection assays and transgenic embryos, AP-2 family members are able to trans-activate reporter expression, showing that this enhancer functions as an AP-2-responsive element in vivo. Reporter expression is not abolished in an AP-2(alpha) null mutant embryos, suggesting redundancy with other AP-2 family members for activation of the Hoxa2 enhancer. Other cis-elements identified in this study critical for neural-crest-specific expression include an element that influences levels of expression and a conserved sequence, which when multimerized directs expression in a broad subset of neural crest cells. These elements work together to co-ordinate and restrict neural crest expression to the second branchial arch and more posterior regions. These findings have identified the cis-components that allow Hoxa2 to be regulated independently in rhombomeres and cranial neural crest cells (Maconochie, 1999).

The ERF-1 transcription factor is involved in the regulation of estrogen receptor (ER) gene transcription in hormonally responsive breast and endometrial carcinomas. ERF-1 activates ER gene transcription by binding to the imperfect palindrome CCCTGCGGGG within the promoter of the ER gene. ERF-1 protein was purified from the ER-positive breast carcinoma cell line, MCF7, utilizing ion exchange and DNA affinity chromatography. Peptide sequence analysis was used to isolate a 2.7 kb cDNA clone from an MCF7 cDNA library. This cDNA encodes a protein of 48 kDa previously identified as the AP2gamma transcription factor. By gel-shift analysis, in vitro synthesized ERF-1 comigrates with MCF7 native ERF-1 complex and demonstrates identical sequence binding specificity as native ERF-1. In addition, AP2 polyclonal antisera supershifts both in vitro synthesized and native ERF-1 complexes. These results show that ERF-1 is a member of the AP2 family of developmentally regulated transcription factors. Given the central role of ER expression in breast carcinoma biology, ERF-1 is likely to regulate expression of a set of genes characteristic of the hormonally-responsive breast cancer phenotype (McPherson, 1997).

MCAM/MUC18 is a cell-surface glycoprotein of 113 kDa, originally identified as a melanoma antigen, whose expression is associated with tumor progression and the development of metastatic potential. Enforced expression of MCAM/MUC18 in primary cutaneous melanoma leads to increased tumor growth and metastatic potential in nude mice. The mechanism for up-regulation of MCAM/MUC18 during melanoma progression is unknown. Up-regulation of MCAM/MUC18 expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The MCAM/MUC18 promoter contains four binding sites for AP-2, and electrophoretic mobility shift assay gels demonstrate that the AP-2 protein binds directly to the MCAM/MUC18 promoter. Transfection of AP-2 into highly metastatic A375SM melanoma cells (AP-2-negative and MCAM/MUC18-positive) inhibits MCAM/MUC18 promoter-driven chloramphenicol acetyltransferase reporter gene in a dose-dependent manner. MCAM/MUC18 mRNA and protein expression are down-regulated in AP-2-transfected but not in control cells. In addition, re-expression of AP-2 in A375SM cells inhibits their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of MCAM/MUC18 is regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly by down-regulating MCAM/MUC18 gene expression. Since AP-2 also regulates other genes that are involved in the progression of human melanoma such as c-KIT, E-cadherin, MMP-2, and p21(WAF-1), it is proposed that loss of AP-2 is a crucial event in the development of malignant melanoma (Jean, 1998).

The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated products. One of these products, keratin K10, is critical to epidermal integrity, because mutations in k10 lead to abnormal blistering. For the normal expression of k10, differentiation-associated transcription factors C/EBPalpha, C/EBPbeta, and AP-2 are well positioned to play an important role. Regulation of the k10 gene has been examined in keratinocytes in the skin of normal mice and in transgenic mice carrying targeted deletions of c/ebpbeta and ap-2alpha. In cultured cells, C/EBPalpha and C/EBPbeta are each capable of activating the k10 promoter via three binding sites, identified by site-directed mutagenesis. In a given epidermal cell in vivo, however, the selection of C/EBPalpha versus C/EBPbeta for k10 regulation is determined via a third transcription factor, AP-2. This novel regulatory scheme involves: (1) unique gradients of expression for each transcription factor, i.e., C/EBPbeta and AP-2 are most abundant in the lower epidermis, C/EBPalpha in the upper; (2) C/EBP-binding sites in the ap-2alpha gene promoter, through which C/EBPbeta stimulates ap-2alpha; and (3) AP-2 binding sites in the c/ebpalpha promoter, through which AP-2 represses c/ebpalpha. Promoter-analysis and gene-expression data presented herein support a regulatory model in which C/EBPbeta activates and maintains AP-2 expression in basal keratinocytes, whereas AP-2 represses C/EBPalpha in those cells. In response to differentiation signals, loss of AP-2 expression leads to derepression of the c/ebpalpha promoter and activation of k10 as cells migrate upward (Maytin, 1999).

Expression of the D(1A) dopamine receptor in brain is restricted to specific neuronal populations. To investigate the mechanism of this selective expression, a silencer upstream of the human D(1A) gene was localized and its binding transcription factor was identified in the D(1A)-negative neural cell line Neuro2a. Using deletion CAT analysis, this silencer was narrowed to the region between nucleotides -561 and -532 relative to the CAP site. This 30-bp region, designated D1AS1, contains a sequence homologous to the AP-2 binding site and binds to a factor that also interacts with the AP-2 consensus sequence. In gel supershift assays, this factor is recognized by anti-AP-2beta antibody. Co-transfection of Neuro2a cells with an AP-2beta expression vector represses the basal CAT activity of D(1A) promoter-reporter plasmids in a D1AS1-dependent manner. RT-PCR analysis indicates that, among AP-2 family members, Neuro2a cells express only AP-2beta. Furthermore, co-transfection of these cells with decoy oligonucleotides corresponding to the D1AS1 sequence de-represses the D(1A) gene promoter. Unlike in Neuro2a cells, AP-2beta can not repress the D(1A) promoter in the D(1A)-positive neural cell line, NS20Y. In addition, the expression of AP-2beta in different brain regions does not inversely correlate with that of D(1A) dopamine receptor. These observations taken together indicate that AP-2beta is a repressive transcription factor that acts on the D1AS1 silencer of the D(1A) dopamine receptor gene via some cell-specific mechanism(s) in Neuro2a (Takeuchi, 1999).

Retinal fatty acid binding protein (R-FABP) is the avian counterpart of murine brain FABP implicated in glial cell differentiation and neuronal cell migration. R-FABP is highly expressed in the undifferentiated retina and brain of chick embryos. The AP-2 transcription factor binds to a consensus AP-2 binding site in the R-FABP promoter region. Based on the expression pattern of AP-2 in the developing retina and on mutational analysis of the AP-2 binding site in DNA transfection experiments, it is proposed that AP-2 could be involved in the down-regulation of R-FABP transcription. Two members of the AP-2 family expressed in the chick retina, AP-2alpha and AP-2beta, are described. R-FABP mRNA and the AP-2 factors are expressed in mutually exclusive patterns in the differentiating retina: whereas AP-2alpha and AP-2beta are selectively expressed either in amacrine, or in amacrine and horizontal cells, respectively, R-FABP mRNA is found in Muller glial cells and/or bipolar cells. Furthermore, a decrease in R-FABP-dependent expression is obtained upon cotransfection of primary retinal cultures with AP-2 expression vectors and a CAT reporter construct. The early and cell-specific expression of AP-2alpha and AP-2beta in the developing retina suggest a role for this transcription factor family in the early steps of amacrine and horizontal cell differentiation. Repression of the R-FABP gene in these cells may be an important component of their developmental program (Bisgrove, 1999).

Neuropeptide Y (NPY) plays an important role in the central regulation of neuronal activity, endocrine and sexual behavior, and food intake. Although transcription activity of the NPY gene in PC12 cells is regulated by a number of agents such as nerve growth factor (NGF), the mechanism responsible for the NGF-elicited increase in the transcription of the NPY gene remains to be explored. In this study, a nuclear protein was isolated and characterized that is bound to NGF-response elements (NGFRE) which lie between nucleotide -87 and -33 of the rat NPY promoter gene. This nuclear protein is identical to the rat homolog of human transcription factor AP-2alpha. Rat AP-2a promotes efficient NPY transcription activity in response to NGF. Direct evidence is provided that the mice lacking transcription factor AP-2alpha exhibit reduced expression of NPY mRNA compared with wild-type mice, further supporting the hypothesis that AP-2alpha is an important transcription factor in regulating NPY transcription activity (Li, 2000).

The AP-2 family of transcriptional regulator proteins has three members: alpha, beta and gamma. AP-2alpha and gamma are expressed in placenta and in the human trophoblast cell line JEG-3. AP-2 has been shown to regulate expression of the placental human chorionic gonadotropin (hCG) alpha- and beta-subunit genes, however, previous work did not distinguish between the family members. Tryptic peptides of the AP-2 protein complexes purified from JEG-3 cells by oligo-affinity chromatography using the hCGalpha AP-2 site match the amino acid sequence of AP-2gamma. The fact that AP-2gamma is present at significant levels and binds the hCGalpha trophoblast-specific element suggests that AP-2gamma is at least part of the binding complex in vivo and plays a role in regulating hCG expression. Mutation of each of four AP-2 binding sites within the hCGbeta promoter decreases expression in transfection assays, demonstrating that all four sites are required for maximal expression in JEG-3 cells. Furthermore, differences in regulation of the family members are found: AP-2alpha mRNA levels increase in response to cAMP while AP-2gamma mRNA levels do not. The demonstrated importance of the AP-2 sites in controlling hCGalpha and beta expression and the likely involvement of more than one family member suggest that a balance in AP-2 proteins is involved in coordinate regulation of these genes. Moreover, many placenta-restricted genes are regulated by AP-2 proteins, thus members of this family may play an important overall role in placenta-specific expression (LiCalsi, 2000).

LHRH is the neuropeptide responsible for reproductive function. Prenatally, LHRH expression begins when neurons are in the olfactory pit and continues as these cells migrate into the brain. Thus, LHRH neurons maintain neuropeptide expression through very distinct environments. The regulatory interactions that control onset and continued expression of the LHRH phenotype are unknown. To begin to address this question primary LHRH neurons were removed from nasal explants at different ages. A complementary DNA (cDNA) subtraction screen was performed comparing a 3.5-days in vitro LHRH neuron [approximately embryonic day 15 (E15) in vivo] to two 10.5-days in vitro LHRH neurons (approximately postnatal day 1 in vivo). The transcription factor activator protein-2 (AP-2alpha) is differentially expressed and is present in the developmentally younger LHRH neuron. In vivo analysis reveals that LHRH neurons express AP-2 as they migrated across the cribriform plate and into the forebrain beginning on E13.5, but that coexpression of LHRH and AP-2 is no longer detected in postnatal day 1 animals. This suggested a regulatory role for AP-2 in LHRH neurons. Analysis of animals lacking AP-2alpha reveals a dramatic decrease in forebrain LHRH neurons between E13.5 and E14.5, correlating with normal onset of AP-2 expression in LHRH neurons as they enter the central nervous system. Nasal cells robustly expressing LHRH are still present on E 14.5. The continued presence of forebrain LHRH cells is proposed based on a second marker, galanin, and lack of increased apoptotic/necrotic cells in this region. A decrease in LHRH messenger RNA in forebrain neurons indicates regulation of LHRH occurs at the transcriptional or posttranscriptional level in mutant animals. These results indicate a developmentally restricted involvement of the transcription factor AP-2 in LHRH expression once the LHRH neurons have migrated into the forebrain, but before establishment of an adult-like distribution (Kramer, 2000b).

Manganese superoxide dismutase (Mn-SOD) is a primary antioxidant enzyme whose expression is essential for life in oxygen. Mn-SOD has tumor suppressor activity in a wide variety of tumors and transformed cell systems. Mn-SOD expression is inversely correlated with expression of AP-2 transcription factors in normal human fibroblasts and their SV-40 transformed counterparts. Thus it was hypothesized that AP-2 may down-regulate Mn-SOD expression. To examine the functional role of AP-2 on Mn-SOD promoter transactivation AP-2-deficient HepG2 cells were cotransfected with a human Mn-SOD promoter-reporter construct and expression vectors encoding each of the three known AP-2 family members. AP-2 significantly represses Mn-SOD promoter activity, and this repression was both Mn-SOD promoter and AP-2-specific. The three AP-2 proteins appear to play distinct roles in Mn-SOD gene regulation. Moreover, although all three AP-2 proteins can repress the Mn-SOD promoter, AP-2alpha and AP-2gamma are more active in this regard than AP-2beta. Transcriptional repression by AP-2 is not a general effect in this system, because another AP-2-responsive gene, c-erbB-3, is transactivated by AP-2. Repression of Mn-SOD by AP-2 is dependent on DNA binding, and expression of AP-2B, a dominant negative incapable of DNA binding, relieves the repression on Mn-SOD promoter and reactivates Mn-SOD expression in the AP-2 abundant SV40-transformed fibroblast cell line MRC-5VA. These results indicate that AP-2-mediated transcriptional repression contributes to the constitutively low expression of Mn-SOD in SV40-transformed fibroblasts and suggest a mechanism for Mn-SOD down-regulation in cancer (Zhu, 2001).

AP-2 and lens development

AP-2alpha is a developmentally important transcription factor that has been implicated in the regulation of cell growth, programmed cell death, and differentiation. To investigate the specific function of AP-2alpha in differentiation of the lens, AP-2alpha was expressed in the differentiating lens fiber cells under control of the alphaA-crystallin promoter. Normally, AP-2alpha is selectively expressed in lens epithelial cells and expression terminates at the lens equator, where epithelial cells terminally differentiate into fiber cells. Ectopic expression of the AP-2alpha gene in the fiber cell compartment results in bilateral cataracts and microphthalmia in mice by 2 weeks of age. Histological evaluation of embryonic and adult transgenic lenses revealed a significant reduction in lens size and anterior shifting of the transitional zone. Two aspects of fiber cell differentiation were also blocked, including the migration of newly formed fiber cells and an inhibition in fiber cell denucleation. Correlated with these defects was expanded expression of E-cadherin in the lens transitional zone and reduced expression of the fiber cell-specific protein MIP (major intrinsic protein). Together, these data demonstrate that AP-2alpha acts as a negative regulator of terminal fiber cell differentiation through the regulation of genes involved in cell adhesion and migration (West-Mays, 2002).

AP-2 and craniofacial development

During closure of the neural tube in the mouse, transcription factor AP-2 is expressed in ectoderm and in neural-crest cells migrating from the cranial neural folds. Cranial neural crest cells provide patterning information for craniofacial morphogenesis, generate most of the skull bones, and together with placodal ectoderm, form the cranial ganglia. To study the role of AP-2 during embryogenesis, a targeted mutagenesis of the AP-2 gene was undertaken in the mouse. AP-2 minus mice die perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face, skull, sensory organs and cranial ganglia. Failure of cranial closure between 9 and 9.5 days postcoitum coincides with increased apoptosis in the midbrain, anterior hindbrain and proximal mesenchyme of the first branchial arch, but does not involve loss of expression of twist or Pax-3, two other regulatory genes known to be required for cranial closure (Schorle, 1996).

The retinoic acid-inducible transcription factor AP-2 is expressed in epithelial and neural crest cell lineages during murine development. AP-2 can regulate neural and epithelial gene transcription, and is associated with overexpression of c-erbB-2 in human breast-cancer cell lines. To ascertain the importance of AP-2 for normal development, mice containing a homozygous disruption of the AP-2 gene have been derived. These AP-2-null mice have multiple congenital defects and die at birth. In particular, the AP-2 knockout mice exhibit anencephaly, craniofacial defects and thoraco-abdominoschisis. Skeletal defects occur in the head and trunk region, where many bones are deformed or absent. Analysis of these mice earlier in embryogenesis indicates a failure of cranial neural-tube closure and defects in cranial ganglia development. AP-2 is a fundamental regulator of mammalian craniofacial development (Zhang, 1996).

Char syndrome is an autosomal dominant trait characterized by patent ductus arteriosus, facial dysmorphism and hand anomalies. Using a positional candidacy strategy, TFAP2B (AP-2beta), encoding a transcription factor expressed in neural crest cells, has been mapped to the Char syndrome critical region and missense mutations have been identified altering conserved residues in two affected families. Mutant TFAP2B proteins dimerized properly in vitro, but showed abnormal binding to TFAP2 target sequence. Dimerization of both mutants with normal TFAP2B adversely affects transactivation, demonstrating a dominant-negative mechanism. This work shows that TFAP2B has a role in ductal, facial and limb development and suggests that Char syndrome results from derangement of neural-crest-cell derivatives (Satoda, 2000).

The vertebrate transcription factor activator protein-2 (AP-2alpha) is involved in craniofacial morphogenesis. In the nasal placode AP-2alpha expression delineates presumptive respiratory epithelia from olfactory epithelia, with AP-2alpha expression restricted to the anterior region of the respiratory epithelium (absent from the olfactory epithelium) at later stages. To address the role AP-2alpha plays in differentiation of cell groups in the nasal placode, the spatiotemporal expression pattern of four markers normally associated with olfactory epithelial structures was analyzed in mice lacking AP-2alpha. These markers are the intermediate filament protein peripherin, the neuropeptide luteinizing hormone-releasing hormone (LHRH), the neural cell adhesion molecule (NCAM) and the olfactory transcription factor Olf-1. Development of cells expressing these markers is similar in both genotypes until embryonic day 12.5 (E12.5), indicating that the main olfactory epithelium and olfactory pit formation is normal. At E13.5 in mutant mice, ectopic LHRH neurons and peripherin axons were detected in respiratory epithelial areas, areas devoid of Olf-1 and NCAM staining. Over the next few days, an increase in total nasal LHRH neurons occurred. The increase in nasal LHRH neurons could be accounted for by LHRH neurons arising and migrating out of respiratory epithelial regions on peripherin-positive fibers. These results indicate that AP-2alpha is not essential for the separation of the olfactory and respiratory epithelium from the nasal placode and is consistent with AP-2alpha preventing recapitulation of developmental programs within the respiratory epithelium that lead to expression of LHRH and peripherin phenotypes (Kramer, 2000a).

The neural crest is a uniquely vertebrate cell type that gives rise to much of the craniofacial skeleton, pigment cells and peripheral nervous system, yet its specification and diversification during embryogenesis are poorly understood. Zebrafish homozygous for the lockjaw (low) mutation show defects in all of these derivatives. low (allelic with montblanc) encodes a zebrafish tfap2a (AP2a), one of a small family of transcription factors implicated in epidermal and neural crest development. A point mutation in low truncates the DNA binding and dimerization domains of tfap2a, causing a loss of function. Consistent with this, injection of antisense morpholino oligonucleotides directed against splice sites in tfap2a into wild-type embryos produces a phenotype identical to low. Analysis of early ectodermal markers has revealed that neural crest specification and migration are disrupted in low mutant embryos. TUNEL labeling of dying cells in mutants revealed a transient period of apoptosis in crest cells prior to and during their migration. In the cranial neural crest, gene expression in the mandibular arch is unaffected in low mutants, in contrast to the hyoid arch, which shows severe reductions in dlx2 and hoxa2 expression. Mosaic analysis, using cell transplantation, has demonstrated that neural crest defects in low are cell autonomous and secondarily cause disruptions in surrounding mesoderm. These studies demonstrate that low is required for early steps in neural crest development and suggest that tfap2a is essential for the survival of a subset of neural crest derivatives (Knight, 2003).

AP2 transcription factors regulate many aspects of embryonic development. Studies of AP2a (Tfap2a) function in mice and zebrafish have demonstrated a role in patterning mesenchymal cells of neural crest origin that form the craniofacial skeleton, while the mammalian Tfap2b is required in both the facial skeleton and kidney. This study shows essential functions for zebrafish tfap2a and tfap2b in development of the facial ectoderm, and for signals from this epithelium that induce skeletogenesis in neural crest cells (NCCs). Zebrafish embryos deficient for both tfap2a and tfap2b show defects in epidermal cell survival and lack NCC-derived cartilages. Cartilage defects arise after NCC migration during skeletal differentiation, and they can be rescued by transplantation of wild-type ectoderm. A model is proposed in which AP2 proteins play two distinct roles in cranial NCCs: an early cell-autonomous function in cell specification and survival, and a later non-autonomous function regulating ectodermal signals that induce skeletogenesis (Knight, 2005).

AP-2 and ectodermal development

Expression of the Xenopus homolog of the mammalian transcription factor AP-2ß (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo (Luo, 2002).

AP-2 and mammary gland development

AP-2 transcription factors are key regulators of mouse embryonic development. Aberrant expression of these genes has also been linked to the progression of human breast cancer. The role of the AP-2 gene family in the postnatal maturation of the mouse mammary gland has been investigated. Analysis of AP-2 RNA and protein levels demonstrates that these genes are expressed in the mammary glands of virgin and pregnant mice. Subsequently, AP-2 expression declines during lactation and then is reactivated during involution. The AP-2alpha and AP-2gamma proteins are localized in the ductal epithelium, as well as in the terminal end buds, suggesting that they may influence growth of the ductal network. This hypothesis was tested by targeting AP-2alpha expression to the mouse mammary gland using the MMTV promoter. These studies indicate that overexpression of AP-2alpha inhibits mammary gland growth and morphogenesis, and this coincides with a rise in parathyroid hormone-related protein expression. Alveolar budding is severely curtailed in transgenic virgin mice, while lobuloalveolar development and functional differentiation are inhibited during pregnancy and lactation, respectively. These studies strongly support a role for the AP-2 proteins in regulating the proliferation and differentiation of mammary gland epithelial cells in both mouse and human (Zhang, 2003).

AP-2 and cancer

This paper describes the generation and characterization of a monoclonal antibody specific for two members of the AP-2 family of transcription factors, AP-2alpha and AP-2beta, and its subsequent application to archival primary breast tumor material. Nuclear localization of AP-2 was found in all expressing cases, but in general levels of immunostaining were low, with only 17 per cent of the 86 tumors examined showing very high expression levels. Nevertheless, data analysis of the whole patient series allows the identification of significant relationships between levels of AP-2 and other important breast markers. Thus, expression of AP-2alpha/beta has been found to correlate significantly with expression of both Estrogen receptor and the universal cell-cycle inhibitor p21(cip), but is inversely related to levels of the proto-oncogene ErbB2. AP-2-positive tumors also showed a low rate of proliferation, with significantly reduced mitotic count and a lower tumor grade. There is no significant relationship with clinical parameters, but samples with adjacent normal tissue indicate that loss of the AP-2 marker is associated with disease progression from normal breast through to invasive disease. This was confirmed by examining separate series of pure normal and pure DCIS samples, both of which expressed significantly higher levels of AP-2 than the invasive tumors. Overall, these findings implicate AP-2alpha/beta as having a role akin to that of a tumor suppressor in breast cancer (Gee, 1999).

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

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