Yap and Taz show widespread expression by RT-PCR across a series of mouse embryo developmental stages. PCR amplification of gene-specific products from cDNAs derived from mouse embryo total RNA obtained at E3.5, E6.5, E7.5, E10.5, E12, E13.5, E16.5, and E18.5 with (+) or without (−) RT. Yap expression was found at all stages examined from E3.5 blastocyst to E18.5 perinatal, in contrast to Taz expression, which was detected at all examined stages except E3.5 blastocyst.

Dynamic but broad Yap expression in wild-type mouse embryos visualized by whole mount in situ hybridization. Yap mRNA is broadly distributed in trophectoderm and epiblast derivatives from E6.5 to E8.5 and yet shows stage- and region-specific domains of relatively stronger expression. Control sense probes gave little background at any stage (D, G, and I). Embryos are shown with anterior to the left when known and proximal to the top. (A to D) Embryos at E6.5 showed expression in extraembryonic ectoderm (arrows in panels A and B) with high levels of Yap expression in epiblast becoming restricted toward proximal epiblast (above arrows in panel C) and lowest expression levels in visceral endoderm (arrowheads in panels A and B). (E to G) Embryos at E7.5 show highest expression in extraembryonic mesoderm and in a mid proximodistal domain in a ring of extraembryonic ectoderm (black arrows in panels E and F) that was proximal to the region forming the chorion (white arrows in panels E and F). Again, lowest expression levels were in visceral endoderm (arrowheads in panels E and F). (H and I) A broad Yap expression domain was found in embryos at E8.5 with highest expression in distal tip allantois (arrowhead in panel H). Asterisks mark regions damaged at dissection. Stage-specific scale bars, 50 μm.

Targeted disruption of the Yap gene. (A) The Yap genomic locus was altered by using a targeting vector that replaced approximately 1 kb of genomic sequence including the Yap ATG start site (*) and most of exon 1 with a floxed reverse orientation MC1-neo construct. The 3′ PGK-tk cassette allowed negative selection. A 5′ genomic probe (large dark bar) that recognized ∼6-kb native locus and ∼5-kb targeted BamHI fragments revealed proper ES cell targeting and allowed genotyping by Southern transfer. A primer trio (small bars) with a shared 5′ primer (YF) and distinct 3′ primers, one in a region deleted after targeting (YR) and the other specific for MC1-neo sequence (NR) allowed PCR genotyping. (B) Southern blot of ES cell DNA showing proper targeting of two ES cell lines. (C) PCR genotype of E8.5 embryos showing wild type, heterozygous, and homozygous mutant genotypes. (D) Western blot with affinity-purified rabbit polyclonal antibody to C-terminal YAP protein on E8.5 whole embryo lyses, indicating undetectable levels of YAP in homozygous mutant embryos.

Morphological phenotype of Yap^tm1Smil homozygous embryos from E7.5 to E9.5. Wild-type (A, D, E, and H) and homozygous mutant (B, C, F, G, and I) embryos from Yap^tm1Smil heterozygous intercross litters were analyzed by light microscopy for gross morphological defects. E7.5 and E8.5 embryos are with anterior to the left and proximal to the top unless otherwise noted; E9.5 embryos are shown in a left-lateral view. A comparison of wild-type (A) and homozygous mutant embryos at E7.5 revealed variability in the mutant phenotype ranging from overtly normal (B) to highly abnormal (C). At E8.5 compared to wild-type siblings (D and E), homozygous mutant embryos showed excessive folding of the anterior epithelium (e) and failure of chorioallantoic fusion despite elongation of the allantois (al). In embryos viewed from left lateral (D and F) the normal distal point (dp)—the bend point of the unturned body axis for both wild-type and homozygous mutant embryos—is indicated for comparison and illustration of caudal dysgenesis observed in mutant embryos. Comparison from a dorsal view with anterior to the left, of wild-type (E) and mutant (G) embryos clearly shows development of an abnormally short and wide axis with YAP deficiency. By E9.5, compared to wild-type siblings (H), homozygous mutant embryos (I) were much smaller and showed a stereotyped morphology that included failure of ventral closure and turning and thus retention of the distal point (dp) of earlier stages, a bulbous unfused allantois (al), and a distinctly rippled yolk sac (ys). Scale bars: 130 μm (A to C), 325 μm (D to G), and 400 μm (H and I).

Wild-type and homozygous mutant Yap^tm1Smil embryos sectioned in utero for histological analysis. Representative sections from wild-type (A to H) and homozygous mutant (A′ to H′) embryos stained with hematoxylin and eosin are shown. Unstained sections for each embryo were genotyped by PCR. By E7.5 wild-type embryos (A and B) developed chorion (#), amnion (large arrowhead) and mesoderm including the allantoic bud (*) and yolk sac mesoderm (ysm), organized into early blood islands adjacent to yolk sac visceral endoderm (ve). Homozygous mutant embryos at E7.5 (A′ and B′) showed a developed chorion (#) but also variable developmental perturbations that included a pronounced constriction at the embryonic-extraembryonic boundary (large arrows) and disorganized yolk sac mesoderm (small arrow; B′). Sections at E8.5 (C to H′) show proper tissue-specific development of amnion (C and C′, arrowhead), chorion (C and C′, #), neurectoderm (D and D′), somites (E and E′), heart (F and F′), and allantois (G and G′) in both wild-type and homozygous mutant embryos. Compared to wild-type embryos, mutant embryos showed abnormal development of posterior epiblast (C and C′, small arrowhead to arrow), excessive folds of neurectoderm extending into the amniotic cavity (D and D′) and poorly defined blood islands in the yolk sac (H and H′). bi, blood island; ht, heart; s, somite; ne, neurectoderm. Scale bar: 240 μm (C and C′), 120 μm (A, A′, D, and D′), 60 μm (E, E′, G, and G′), and 30 μm (B, B′, F, F′, H, and H′).

Analysis of early endothelial and erythroblast markers in Yap^tm1Smil homozygous mutant embryos. Whole-mount immunohistochemical marking of PECAM1 protein (A to F) and in situ visualization of alpha globin expression (G to K). Endothelial cells marked by the PECAM1 antigen were present in both wild-type (A to C) and homozygous mutant (D to F) embryos late in the eighth day of development. Flat-mount images show endothelial cells organized into a primitive vascular plexus in wild-type (A) but not mutant (D) yolk sacs, where instead they showed an abundant but disordered distribution. In contrast, endothelial cells formed blood vessels in both the allantois (B and E) and the embryo proper (C and F) in both wild-type and mutant embryos. Erythroblasts expressing alpha globin were present in the yolk sac region of both wild-type (G and H) and homozygous mutant (I to K) embryos in the eighth day of development. In contrast to the organized early yolk sac ring of expression (G) or later intravessel distribution (H) of erythroblasts in wild-type embryos, alpha globin-expressing cells in mutant embryos were abundant but disordered (I to K). A higher magnification image of yolk sac is shown above each embryo image in G through K. Scale bar: 40 μm (A, B, D, E), 160 μm (C), 80 μm (F), and 250 and 100 μm (lower and upper, respectively [G to K]).

Whole-mount in situ analyses of Yap65^tm1Smil homozygous mutant embryos with postgastrulation markers of body axis development. Anteroposterior pattern was revealed by restricted domains for Fgf8 (A to C) at E9.5. Homozygous mutant embryos showed one or two stripes of Fgf8 expression (B and C, arrows) consistent with the restricted neurectoderm expression domains in wild-type sibling embryos. The streak/tailbud region is marked by both Fgf8 (A to C, asterisk) and Brachyury (T; D to F, asterisk) in both wild-type (A and D) and homozygous mutant (B, C, E, and F) embryos. Mutant embryos show an abnormally discontinuous (E, arrows) and broad (F) Brachyury expression domain in midline cells compared to wild-type embryos. Scale bar: 325 μm (A to C, F to H), 290 μm (D), and 145 μm (E).