MOLECULAR AND CELLULAR BIOLOGY, Jan. 2006, p. 77–87 Vol. 26, No. 1 0270-7306/06/$08.00ϩ0 doi:10.1128/MCB.26.1.77–87.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Defects in Yolk Sac Vasculogenesis, Chorioallantoic Fusion, and Embryonic Axis Elongation in Mice with Targeted Disruption of Yap65 Elizabeth M. Morin-Kensicki,1,2†* Brian N. Boone,1† Michael Howell,1 Jaclyn R. Stonebraker,3 Jeremy Teed,1 James G. Alb,1 Terry R. Magnuson,2 Wanda O’Neal,3 and Sharon L. Milgram1,3 Department of Cell and Developmental Biology, CB7090, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-70901; Department of Genetics, CB7264, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-72642; and Cystic Fibrosis/Pulmonary Research and Treatment Center, CB7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-72483 Received 6 May 2005/Returned for modification 10 July 2005/Accepted 8 October 2005 YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yaptm1Smil allele (Yap؊/؊) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostain- ing, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in .Yap؊/؊ embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap؊/؊ embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithe- lium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation. Yes-associated protein (YAP65; referred to as YAP through- anchoring and/or targeting of c-Yes to position the kinase to out) is a modular adapter protein first identified as a binding respond to specific extracellular cues or to phosphorylate spe- partner for the product of the proto-oncogene c-Yes (57). YAP cific cellular substrates. Cytosolic YAP also may modulate contains multiple protein interaction domains, including a pro- growth factor receptor signaling. For example, YAP associates line-rich amino terminus, a 14-3-3 binding site (3), WW domains with the inhibitory Smad7 to attenuate transforming growth (32, 57), SH3 binding motifs (12, 57), a coiled-coil, and a consen- factor ␤ signaling (14) and may affect signaling via the ErbB-4 sus PDZ binding motif at the extreme COOH terminus (43). receptor (32, 45). In the nucleus, YAP may function as a YAP mRNA is broadly distributed, and expressed sequence tags coregulator, modulating the activity of several transcription have been identified in cDNA libraries from many different spe- factors. In this manner, YAP interacts with RUNX family cies and from many tissues, cell lines, and tumor samples. Al- members (64, 69), which impact hematopoiesis and osteogen- though the cellular and subcellular localization of the YAP pro- esis, as well as TEAD family members (39, 60), which are tein has been less well characterized, it is expressed in multiple implicated in muscle cell and neural crest cell differentiation. cell types and can be localized to both cytoplasmic and nuclear Furthermore, we recently found that the proline-rich amino compartments (3, 26, 32, 45, 60, 64, 69). This broad distribution terminus of YAP associates in the nucleus with heterogeneous and the modular structure of YAP together suggest multiple nuclear ribonucleoprotein U (26), a protein involved in mRNA cellular functions. The identification of YAP-interacting proteins has provided processing and the control of gene expression. Finally, several insight into potential roles for YAP in cell signaling within pieces of data argue that regulated localization of YAP may both cytosol and nucleus. YAP cytosolic interactions may im- impact apoptosis and cell cycle progression. Indeed, in the pact cell signaling pathways by several possible mechanisms. nucleus the YAP WW domain associates directly with the p53 ␣ ␤ ␣ For example, YAP binds the SH3 domain of c-Yes (57) and gene family members p73 , p73 , and p63 and enhances the can also associate with cytoplasmic PDZ proteins via its transcription of proapoptotic Bax and Mdm2 reporter con- COOH terminus (43). Therefore, YAP may play a role in the structs and endogenous Bax (55). In addition, phosphorylation by Akt stimulates YAP interaction with cytosolic 14-3-3 and attenuates p73-mediated apoptosis (55). YAP may also asso- * Corresponding author. Mailing address: 510 Taylor Hall, CB7090, ciate with p53BP2 (12), a protein known to inhibit the activity Department of Cell and Developmental Biology, University of North of the p53 tumor suppressor. Thus, YAP likely exists in cell Carolina at Chapel Hill, Chapel Hill, NC 27599-7090. Phone: (919) 966-0389. Fax: (919) 966-1856. E-mail: [email protected]. type- and compartment-specific protein complexes that define † E.M.M.-K. and B.N.B contributed equally to this study. its function throughout development and in the adult organ- 77 78 MORIN-KENSICKI ET AL. MOL.CELL.BIOL. ism, and yet specific in vivo requirements for YAP remain Nonetheless, to assess the potential impact on our further studies of having also undefined. disrupted this locus, we generated PCR primers in predicted loc434366 exons 2 and YAP shows significant similarity to the product of the re- 3 (sequence below) and examined possible expression by reverse transcription-PCR (RT-PCR). This primer pair generates a 433-nt amplicon from reverse-transcribed lated gene, Taz (30). With amino acid identity approaching RNA sequence but covers 3.2 kb of genomic sequence. We examined expression of 50%, YAP and TAZ may share common protein partners, and loc434366 in a mouse kidney cDNA library (Stratagene) and in wild-type embryos at yet distinctions have been described (8, 21, 25, 26, 30, 37, 47) specific stages ranging from blastocyst (embryonic day 3.5 [E3.5]) stage to late The extent to which these proteins show unique or overlapping gestation (E18.5) and found expression in kidney but no detectable expression in embryos until after E7.5 (data not shown). function in vivo remains unclear. Thus, the potential for re- Genotyping. ES cells, mice, and tissue scrapings from paraffin sections were dundancy between these proteins emphasizes the probable genotyped by either Southern blot or PCR-based methods. We generated a complexity of YAP function and highlights the need for un- 542-nt Southern probe by PCR amplification from subcloned genomic sequences derstanding in vivo requirements for YAP. 5Ј to the Yap targeting construct using the primer pair 5Ј-GCTGCCATTTCA Ј Ј Ј Although an integrated view of YAP protein interactions ACTTTCTAC-3 and 5 -CAGCAGTCTATCGCTTTGTG-3 . This probe rec- ognizes an approximately 6-kb fragment from genomic DNA and an approxi- and function is lacking, the data suggest that YAP is an adap- mately 5-kb fragment from the targeted allele after enzymatic digestion with tor protein that modulates multiple signal transduction path- BamHI. To genotype by PCR, we used a primer trio that included a shared 5Ј ways in many cell types. These pathways have been explored in primer from sequence upstream of the Yap gene (YF, 5Ј-GAAGCTGTGGCA biochemical assays and in cell culture model systems, but little CAAAGA-3Ј),a3Ј primer in sequence deleted in the targeted allele (YR, 5Ј-ATGCAAAGGCCACACTGT-3Ј)anda3Ј primer internal to the neo insert is known regarding YAP function in the intact organism. To (NR, 5Ј-CGACGTTAACGGTACCAA-3Ј). investigate in vivo requirements for YAP, we generated mice Mouse colony maintenance. The transmitted Yaptm1Smil allele was maintained carrying a targeted disruption in the Yap gene. The embryonic on a mixed 129SvEv/C57BL/6J background. Crossing to an outbred (CD1; lethal phenotype of homozygous mutant mice indicates that Charles River) background to obtain larger litters for embryo analyses did not YAP is essential for embryogenesis and that, in fundamental perceptibly alter the phenotype of homozygous mutant embryos. Litters for phenotype characterization were generated by natural matings and staged by developmental events, TAZ does not compensate for YAP. assigning the first half-day of embryonic development (E0.5) as noon the day of These results demonstrate for the first time a critical role for vaginal plug observation. YAP in early embryonic development. RT-PCR. Gene expression was assessed in wild-type embryos from blastocyst stage (E3.5) to late prenatal stage (E18.5) by RT-PCR. Positive controls for these reactions were heart or kidney cDNA libraries (Stratagene). Whole-embryo total MATERIALS AND METHODS RNA was extracted by using the TRIzol reagent (Invitrogen) according to the Generation of the Yap tm1Smil allele. A 507-nucleotide (nt) probe generated by manufacturer’s protocol with modifications for small tissue volume in embryos at PCR amplification from a mouse lung cDNA library (primer pair 5Ј-AGTTTC ՅE10.5 as follows. (i) Embryos were dissected and rinsed several times in TGTCTCAGTTGGGACG-3Ј [nt 13 to 34 of accession X80508] and 5Ј-CAT ice-cold phosphate-buffered saline and then transferred with a minimal volume GCTGTGGAGTGAGAGGCTC-3Ј [nt 520 to 500 of X80508]) was used to to 40 ␮l of TRIzol. (ii) Embryos were homogenized by pulling through a 22- screen a 129SvEv mouse genomic library packaged in Lambda Gem-ll with 13- to gauge 1 needle, and then the TRIzol volume was brought to 100 ␮l.