AP-2alpha transcription factor is required for early morphogenesis of the lens vesicle.

AP-2 transcription factors are a family of retinoic acid-responsive genes, which are involved in complex morphogenetic processes. In the current study, we determine the requirement for AP-2alpha in early morphogenesis of the eye by examining the nature of the ocular defects in AP-2alpha null and chimeric mice. AP-2alpha null embryos exhibited 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 were also misexpressed. AP-2alpha mutants also exhibited 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 revealed 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.

AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice.

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 suggested that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, we have now analyzed chimeric mice composed of both wild-type and AP-2-null cells. 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, we also have uncovered an unexpected influence of AP-2 on limb pattern formation; 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.

The HPV16 E5 protein: expression, detection, and stable complex formation with transmembrane proteins in COS cells.

The human papillomavirus-16 (HPV16) E5 gene is able to induce stable growth transformation and transient mitogenic stimulation in a variety of cultured cell systems. To characterize the biochemical properties of the hydrophobic HPV16 E5 transforming protein, we have constructed vectors expressing the wild-type HPV16 E5 gene and have generated antipeptide antisera. The 10-kDa E5 protein was readily detectable in transfected COS monkey cells by using these antisera either for immunoprecipitation of metabolically labeled cells or for immunoblotting. Coimmunoprecipitation analysis of cells coexpressing the viral protein and various growth factor receptors demonstrated stable complex formation between the E5 protein and the epidermal growth factor receptor, platelet-derived growth factor beta receptor, colony stimulating factor-1 receptor, and p185neu. The E5 protein also formed a stable complex with the vesicular stomatitis virus glycoprotein. These experiments indicated that the HPV16 E5 protein was able to participate in complex formation with a variety of transmembrane proteins, a property which may contribute to the biological activities of the viral protein. In addition, the expression vectors and antibodies described here will be useful reagents in examining various aspects of HPV16 E5 expression and function.

The E2 transactivator of bovine papillomavirus type 1 is expressed from multiple promoters.

The E2 proteins of bovine papillomavirus type 1 (BPV-1) are a family of site-specific DNA-binding proteins which regulate viral transcription by repression and activation. Repressors E2-TR and E8/E2 are expressed from promoters P5 (P3080) and P3 (P890), respectively. Previous reports have provided evidence that the transcript for the 48-kilodalton transactivator is initiated from a promoter proximal to the open reading frame encoding this protein (P2440 or P4). Our studies extend these findings and show that the E2 transactivation gene is expressed from multiple promoters. We have described the isolation of a cDNA (N15-2) which represents an RNA species expressed from the P3 promoter. The major exon of this species was produced by splicing to an acceptor located at nucleotide 2558 and contained the complete E2 open reading frame. The acceptor is probably utilized by yet another more abundant mRNA expressed from the P2 promoter (A. Stenlund, J. Zabielski, H. Ahola, J. Moreno-Lopez, and U. Pettersson, J. Mol. Biol. 182:541-554, 1985). Linked to a surrogate promoter, the N15-2 cDNA can transactivate an E2-responsive reporter gene. BPV-1 plasmids containing mutations either in the 2558 splice acceptor or in the P4 promoter showed significantly reduced transforming ability and reduced ability to transactivate an E2-responsive reporter, while a double mutant was inactive in both assays. The transformation defect was complemented by an E2 expression vector, and the BPV genome absolutely required the E2 protein to transactivate in the second assay. Thus, these genetic experiments show that alternate modes of E2 expression contribute to the E2 mRNA pool. Direct analysis of cytoplasmic RNA from transformed cultured cells proves that transcripts containing the 2558 acceptor exon are approximately as abundant as the P4 type E2 mRNAs. Furthermore, analysis of the E2 proteins present in various cell lines harboring specific BPV-1 mutants, including the 2558 acceptor mutant, proves that alternate modes of E2 expression exist. The ability of the E2 activator and repressors to each be independently expressed from multiple E2-responsive promoters probably adds to the resiliency of the latent virus as a plasmid and may be important for its homeostasis within the cell in different environmental or developmental situations.