Fig 2: Loss of CD166 expression does not compromise the intestinal epithelial barrier. (A) Adult WT (n = 9) and CD166–/– (n = 8) mice gavaged with FITC-dextran were evaluated for presence of fluorescence in peripheral blood. Positive control WT mice (n = 4) were injected with cytokines 24 hours before experiment. Data are presented as mean ± SEM. *P < .05 by 1-way analysis of variance. (B) Adult WT and CD166–/– mouse intestines express comparable E-cadherin (Ecad) and ZO-1 expression as determined by IHC and (C) Western blot analyses. White dashed line represents epithelial-mesenchymal boundary. Scale bars = 25 µm. Fluorescent images were captured on Leica DMR upright microscope. Experiments were repeated at least 3 times.

Fig 3: LATS2 antagonizes formation of the apical domain (A) In embryos at 16–32 cell stages, PARD6B is detectable in GFP-overexpressing and in non-injected cells, but not in Lats2-overexpressing cells (arrowheads, n = embryos).(B) At 16–32 cell stages, aPKCz is detectable in GFP-overexpressing and in non-injected cells, but not in Lats2-overexpressing cells (arrowheads, n = embryos). (C) Quantification of embryos shown in panel A (p, student’s t-test). (D) Quantification of embryos shown in panel B (p, student’s t-test). (E) At 16–32 cell stages, CDH1 is localized to the basolateral membrane in both Lats2-overexpressing and non-injected cells (n = embryos). (F) At 16–32 cell stages, Phalloidin staining demonstrates that filamentous Actin is apically enriched in Lats2-overexpressing and non-injected cells (n = embryos). (G) At 16–32 cell stages, pERM is localized to the apical membrane in both Lats2-overexpressing and non-injected cells (n = embryos).

Fig 4: Positioning and epithelialization defects in embryos with Wwtr1 and Yap1 null alleles (A) Quantification of the average number of inside cells per embryo with decreasing dose of Wwtr1 and Yap1.The number of inside cells increases as the dose of wild type Wwtr1 and Yap1 alleles is reduced (p, student’s t-test, n = embryos). (B) Quantification of the average number of outside cells per embryo with decreasing dose of Wwtr1 and Yap1. The number of outside cells decreases as the dose of wild type Wwtr1 and Yap1 alleles is reduced (p, student’s t-test, n = embryos). (C) Quantification of the average number of total cells per embryos with decreasing dose of wild type zygotic Wwtr1 and Yap1. The number of total cells decreases as the dose of wild type Wwtr1 and Yap1 is reduced (p, student’s t-test, n = embryos). (D) Quantification of the average ratio of inside to outside cells per embryo with decreasing dose of Wwtr1 and Yap1. The ratio of inside to outside cells increases as the dose of wild type Wwtr1 and Yap1 is reduced (p, student’s t-test, n = embryos). (E) Wild type embryos at E3.75 exhibit inner cell mass-specific expression of SOX2 (n = embryos). (E’) E3.75 embryos lacking maternal Wwtr1 and Yap1 and heterozygous for zygotic Wwtr1 and Yap1 cavitate and repress Sox2 in outside cells, leading to inner cell mass-specific expression of SOX2 similar to wild type embryos (n = embryos). (E’’) Embryos lacking maternal Wwtr1 and Yap1 but with only one wild type allele of Wwtr1 or Yap1 fail to cavitate and repress Sox2 in outside cells, leading to ectopic SOX2 in outside cells (arrowheads, n = embryos). (E’’’) Embryos lacking maternal and zygotic Wwtr1 and Yap1 fail to cavitate and repress Sox2 in outside cells, leading to ectopic SOX2 in outside cells (arrowheads, n = embryos). (F) Quantification of ectopic SOX2 detected in embryos such as those shown in panels E-E’’’. The percentage of outside cells with ectopic SOX2 increases as the dose of wild type Wwtr1 and Yap1 alleles is reduced (p, student’s t-test, n = embryos). (G) TUNEL analysis of embryos lacking maternal Wwtr1 and Yap1 heterozygous for zygotic Wwtr1 and Yap1 or lacking maternal and zygotic Wwtr1 and Yap1. Extensive TUNEL staining is observed in embryos lacking maternal and zygotic Wwtr1 and Yap1 indicative of cell death. Max projections of all confocal sections from a single embryo are shown (n = embryos). (H) aPKCz staining in embryos lacking maternal Wwtr1 and Yap1, either heterozygous for zygotic Wwtr1 and Yap1 or with no zygotic Wwtr1 and Yap1. aPKC is not localized to the apical membrane of embryos with no zygotic Wwtr1 and Yap1 (n = embryos). (I) ZO-1 staining in embryos lacking maternal Wwtr1 and Yap1, either heterozygous for zygotic Wwtr1 and Yap1 or with no zygotic Wwtr1 and Yap1. ZO-1 is disorganized in embryos with no zygotic Wwtr1 and Yap1, suggesting that formation of a mature epithelium depends on Wwtr1 and Yap1 (n = embryos). (J) pERM and CDH1 staining in embryos lacking maternal Wwtr1 and Yap1, either heterozygous for zygotic Wwtr1 and Yap1 or with no zygotic Wwtr1 and Yap1. pERM is localized to apical membranes and CDH1 to basolateral membranes regardless of the dose of wild type Wwtr1 and Yap1 alleles (n = embryos).

Fig 5: Gland- and Cell-Specific Expression of Salivary Proteins(A) Immunohistochemistry of glandular tissues. The SM and PAR acinar cells are highly enriched for amylase (AMY1), statherin (STATH), and lactoperoxidase (LPO) compared with the SL, consistent with these proteins being markers of serous cells. MUC7 and CRISP3 are expressed by a subset of acinar cells of the SM and SL, with little to no expression in the PAR. MUC5B is highly expressed by the SL mucous acinar cells, but not by the acinar cells of the PAR or SM, indicating it to be a marker of SL function. Scale bar, 25 µm.(B) MUC7 and amylase are expressed by distinct subtypes of serous acinar cells. The mature SM tissue section was immunolabeled for MUC7 (red), AMY (green), and ECAD (gray).(C) Gel electrophoretic separation of whole mouth saliva (WS) and glandular ductal secretions (PAR and SM/SL), followed by staining of proteins with Coomassie blue and of glycoproteins with periodic acid Schiff stain (pink bands in left panel) and probing of transfers with antibodies against MUC7, AMY, BPIFA2, CRISP3, and STATH (right panel).