Proc. Natl. Acad. Sci. USA Vol. 95, pp. 13714–13719, November 1998 Developmental Biology AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice TIMOTHY NOTTOLI*, STEPHANIE HAGOPIAN-DONALDSON*, JIAN ZHANG*, ARCHIBALD PERKINS*†, AND TREVOR WILLIAMS*‡ *Department of Molecular, Cellular, and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511; and †Department of Pathology, Yale University School of Medicine, New Haven, CT 06510 Communicated by Francis H. Ruddle, Yale University, New Haven, CT, August 28, 1998 (received for review January 15, 1998) ABSTRACT The homozygous disruption of the mouse dent developmental programs are probably regulated by AP-2. AP-2 gene yields a complex and lethal phenotype that results Therefore, we adopted an approach that enabled independent from defective development of the neural tube, head, and body developmental defects to be obtained in isolation. This strat- wall. The severe and pleiotropic developmental abnormalities egy relied on the generation of chimeric mice composed of observed in the knockout mouse suggested that AP-2 may both wild-type (wt) and AP-2-null cells; when a phenotype regulate several morphogenic pathways. To uncouple the results from the interaction of multiple independent develop- individual developmental mechanisms that are dependent on mental defects, an individual chimera will display particular AP-2, we have now analyzed chimeric mice composed of both aspects of the pathology depending on the distribution of the wild-type and AP-2-null cells. The phenotypes obtained from AP-2-null cells. A second consideration behind the generation these chimeras indicate that there is an independent require- of chimeras was to determine whether the severity of the KO ment for AP-2 in the formation of the neural tube, body wall, phenotype concealed more subtle requirements for AP-2 in and craniofacial skeleton. In addition, these studies reveal later developmental processes. In particular, we reasoned that that AP-2 exerts a major influence on eye formation, which is the exencephaly might obscure a need for AP-2 in other a critical new role for AP-2 that was masked previously in the morphogenic events shaping the head. The presence of ap- knockout mice. Furthermore, we also have uncovered an propriate populations of wt cells in a chimera might rescue unexpected influence of AP-2 on limb pattern formation; this such early defects and allow the influence of AP-2-null cells to influence is typified by major limb duplications. The range of be studied subsequently in embryogenesis. Below, we present phenotypes observed in the chimeras displays a significant the data obtained from this analysis and discuss our findings overlap with those caused by teratogenic levels of retinoic acid, in relation to the original AP-2-null phenotype. These studies strongly suggesting that AP-2 is an important component of demonstrate that AP-2 is required for at least five major the mechanism of action of this morphogen. independent morphogenic processes during mammalian em- bryogenesis and strongly support a role for AP-2 in the The transcription factor AP-2, also known as AP-2a,isa etiology of human congenital defects. retinoic acid (RA)-responsive gene that is expressed in tissues undergoing complex morphogenic changes during vertebrate MATERIALS AND METHODS embryogenesis (1–4). In both the mouse and chick, AP-2 RNA Targeting Vector Construction. and protein are first detected at the initial stages of neural crest A hygromycin-based target- cell formation, and the expression of AP-2 continues when ing vector, pHygKO (Fig. 1), was used to disrupt the remaining these cells enter their migratory phase (2, 3). At later stages of AP-2 allele in an embryonic stem (ES) cell line heterozygous embryogenesis, AP-2 expression is concentrated in the devel- for AP-2 (6). The pHygKO plasmid was identical with the neomycin cassette used previously (6), except that the neo gene oping epidermis, facial prominences, branchial arches, and the was replaced with a 6.4-kb XhoI fragment derived from the limb bud progress zone. plasmid pGKbH (a gift of Harris Jacobs, Yale University, New A functional AP-2 gene is vital for normal mammalian Haven, CT). The insertion of pGKbH sequences replaced a embryogenesis: mice that contain a homozygous disruption of critical region of the AP-2 dimerization domain with both a the AP-2 gene die perinatally and exhibit severe developmen- phosphoglycerate kinase promoteryhygromycin resistance tal defects (5, 6). The earliest gross morphological alteration gene fusion and a phosphoglycerate kinase promoterylacZ observed [embryonic day 9.0 (E9.0)] is a complete failure of gene. The pHygKO construct was linearized in vector se- cranial neural tube closure (5, 6). By E12.5, the exposed cranial quences with BspHI prior to electroporation. neural tissue expands to cover the entire head so that normal Cell Culture. ES cells were cultured by using standard cephalic morphology is disrupted. The exencephaly is accom- procedures (7) on a layer of g-irradiated feeder fibroblasts in panied by major alterations in the craniofacial skeleton, such DMEM (Mediatech, Herndon, VA) containing 15% fetal that many bones of the achordal skeleton are missing or bovine serum (HyClone) and supplemented with 1,000 deformed. In the trunk region, the absence of AP-2 disrupts units/ml leukemia inhibitory factor (Life Technologies, Gaith- two additional morphogenic events. First, the ventral body wall ersburg, MD). Following electroporation, clones were selected fails to close, resulting in thoracoabdominoschisis. Second, in the presence of 200 mg/ml G418 (Life Technologies), 100 many AP-2-null mice have forelimb phocomelia, which is units/ml hygromycin (Calbiochem), and 2 mM ganciclovir characterized by loss of the radius. (Syntex, Palo Alto, CA) on a layer of feeder-free extracellular Given the severe and pleiotropic abnormalities displayed by matrix (8). ES cell clones that had undergone a second the knockout (KO) mice, we reasoned that multiple indepen- homologous recombination were identified by Southern blot- ting (Fig. 1). Clones that contained a nonhomologous insertion The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: KO, knockout; wt, wild type; RA, retinoic acid; RAR, accordance with 18 U.S.C. §1734 solely to indicate this fact. retinoic acid receptor; ES, embryonic stem; b-gal, b-galactosidase; E, © 1998 by The National Academy of Sciences 0027-8424y98y9513714-6$2.00y0 embryonic day. PNAS is available online at www.pnas.org. ‡To whom reprint requests should be addressed. 13714 Downloaded by guest on September 28, 2021 Developmental Biology: Nottoli et al. Proc. Natl. Acad. Sci. USA 95 (1998) 13715 the cell types responsible for a particular defect, we neverthe- less found that populations of AP-2-null cells were always associated with the site of pathology in a given chimera (for example, Fig. 2; and data not shown). Glucose-6-phosphate isomerase analysis of adult mouse tissues and whole-mount b-gal staining of 0.2% paraformal- dehyde-fixed embryos were performed by standard procedures (7). For cryosections, embryos were fixed in 0.2% paraformal- dehyde and embedded in OCT medium (Tissue-Tek). Subse- quently, sections were stained for b-gal activity (7) and coun- terstained with nuclear fast red. For paraffin sections, animals were fixed in 4% paraformaldehyde prior to embedding and then stained with hematoxylin and eosin. Skeletal staining with alcian blue and alizarin red was as described previously (9). RESULTS AND DISCUSSION Generation of Chimeras. To identify the individual devel- opmental programs affected by the absence of a functional AP-2 gene, chimeric mice derived from either AP-21y2 or AP-22y2 ES cells were examined between E9.5 and birth for any overt developmental defects. Of the 60 mice generated from the heterozygous 1y2 ES cells, only two showed any defects, and these defects were both minor herniations asso- ciated with the umbilicus (data not shown). In contrast, 88 of FIG. 1. Targeting of both alleles of the AP-2 gene. (A) Targeting 2y2 strategy. The wt AP-2 gene (Top) and original neo-targeted disruption 240 embryos generated from AP-2 cells showed gross (6) (Bottom) are shown along with the pHygKO vector and the developmental abnormalities ('37%; Table 1). The extent of recombinant allele derived from this targeting construct (Middle). the pathology in the chimeras varied from undetectable to an Exons 5, 6, and 7 are indicated by black boxes; TK, thymidine kinase almost complete recapitulation of the AP-2-null phenotype gene; restriction enzyme sites (R, EcoRI; S, SacI; H, HindIII; P, PstI; (compare Fig. 2 A and B). Overall, the developmental defects B, BglII) and locations of 59 and 39 external probes are also indicated. (B)(Left) Southern blot of EcoRI-digested DNA from heterozygous observed in the chimeras could be grouped into five major ES cell line (1y2) and AP-2-null line TN63 (2y2) probed with 59 categories affecting critical morphogenic events: formation of fragment. In TN63, the remaining 12-kb wt fragment is converted to the body wall, face, neural tube, eye, and limbs (Figs. 2–5). The 7 kb by homologous recombination. (Right) Southern blot of SacI- individual pattern formation defects were frequently seen in digested DNA from heterozygous ES cell lines (1y2) and AP-2-null various combinations, but each of these five categories of lines TN91 and TN95 (2y2) by using the 39 probe. The 14-kb congenital abnormality also were observed in isolation (Table neo-disrupted allele is constant; the 7.5-kb wt fragment is converted to 1), demonstrating that AP-2 is independently required for each 8.5 kb by homologous recombination. of these developmental processes. Below, we discuss these of the hyg gene elsewhere in the genome and still retained one individual developmental abnormalities and their relationship wt allele of AP-2 were selected as heterozygous controls.