Bj mutant exhibits outflow tract malalignment defects. (A,G) Echocardiography using color flow Doppler imaging showed in a normal E14.5 fetus (A), anterior positioning of blood flow from the aorta (Ao) emerging from the left ventricle (LV) and posterior positioning of blood flow from the pulmonary artery (PA) arising from the right ventricle (RV). In contrast, in the Bj mutant (G) fetus, blood flow streams from the outflow tract show parallel positioning, and this is associated with the mixing blood between the right ventricle and left ventricle, indicating aorta overriding the septum with a ventricular septal defect (VSD). Compass denotes orientation indicated by arrows; L, left; R, right; Cr, cranial; Cd, caudal. (B,H) Necropsy examination showed the aorta and pulmonary artery are abnormally positioned side by side in the mutant heart (H) compared to wild-type (B). RA, right atrium, LA, left atrium. (C,I) Further histopathology examination showed the Bj mutant aorta and pulmonary arteries are both connected to the right ventricle (RV), indicating it is a double outlet right ventricle (DORV) with a VSD (I). (D-F,J-L) The atrioventricular and semilunar valves are unaffected (J-L as compared to D-F, respectively; see arrowheads). TV, tricuspid valve; MV, mitral valve. Scale bars: 1 mm in A-H); 0.5 mm in C-L.

Bj mutants have shortened outflow tract and defects in neural crest and second heart field derivatives. (A-D) Episcopic confocal histopathology in the sagittal plane of an E10.5 heterozygous (A) and Bj mutant (B) embryo is shown, with enlarged view of the outflow tract in (C) (n=4) and (D) (n=3) indicating reduction in length of the mutant OFT versus heterozygous (+/−) littermate control (see blue line in C,D). This was confirmed with further quantitative measurements (M). (E-H) Neural crest cells in the outflow tract of E10.5 embryos are visualized via a Cx43-lacZ transgene. This showed a reduction in neural crest cells in the homozygous Bj mutant heart (F,H) as compared to the heterozygous littermate control (E,G). (I-O) Sagittal sections of E10.5 embryo immunostained with anti-Islet1 (Isl1) antibody delineating second heart field cells in the dorsal pericardial wall (DPW) of the outflow tract of a wild-type (I,K) and Bj mutant embryo (J,L), with enlarged views shown in (K) and (L). Cells in the DPW wall (yellow arrowheads, denoted by blue outline in N,O) exhibit a flat squamous epithelial morphology in the wild-type embryo (N), but a distinct cuboidal morphology was observed in the homozygous Bj mutant embryo (O). A, atrium; V, ventricle. (M) Quantitative measurement using histopathology images showed a significant decrease in the length of the OFT in the homozygous Bj mutant hearts as compared to combined wild-type and heterozygous hearts. P-values were calculated with Student's t-test; error bars show standard deviation. Scale bars: 0.5 mm in A-D; 200 µm in E-H.

Disruption of epithelial integrity in the developing outflow tract of the Bj mutant embryos. Immunostaining with antibodies against β-catenin (green) and laminin (red) (A-F), and E-cadherin (green) and PKCζ (red) (G-J), and Vangl2 (green) and Pk1 (red) (K-N) of wild-type (A,C,E,G,I,K,M) (n=3), and Bj mutant embryos (B,D,F,H,J,L,N) (n=3) showed marked disorganization of the epithelium in the transition zone (TZ) and dorsal pericardial wall (DPW) of the E10.5 Bj mutant embryo. Panels C,E and D,F are enlarged views of A and B, respectively. Panels G-J and K-N are immunostaining of the same zones of A and B, respectively. Confocal imaging showed β-catenin is distributed along the cell surface in the control and Bj mutant embryos. Laminin is localized basally (arrowhead C,E) in the TZ of the control embryo, but in the mutant embryo, it is localized apically (arrow) and basally (arrow head D,F). While no laminin is detected in the DPW of the control embryo, it is ectopically expressed in the DPW of the Bj mutant embryo (compare E versus F). E-cadherin is widely expressed at regions of cell-cell contact, both in the TZ and in the DPW (G,I) of the control embryo. However, in Bj mutant embryos, E-cadherin expression is markedly reduced or extinguished in these regions (H,J). PKC-ζ expression is restricted to the apical (arrow) membrane of cells in the TZ and DPW epithelia (G,I). This polarized distribution is lost in the Bj mutant embryo, indicating the disruption of the epithelial organization of the DPW (H,J). Arrowheads in G-J indicate E-Cad and PKC-ζ distribution in the TZ and DPW. In the control embryo, Vangl2 and Pk1 are both expressed in the TZ and DPW of the outflow tract, with extensive co-localization observed (K,M). While Vangl2 expression is retained in the TZ and DPW of the Bj mutant, Pk1 expression is reduced (L,N; n=4; P=0.044). This is associated with an increase in overall thickness of the DPW. Arrowheads in M,N indicate Pk1 expression in the DPW, asterisks in K,L indicate Pk1 expression in TZ. Scale bar: 50µm in A-N. The P values were calculated with Student's t-test.

Bj mutants show disruption of canonical Wnt signaling. (A,B) Analysis using the BAT-lacZ reporter showed increased lacZ reporter gene expression at the base of the outflow tract in the E13.5 Bj mutant heart (B) as compared to wild-type control (A). This suggests Wnt signaling is upregulated in the Bj mutant heart. Quantitative real-time PCR analysis (C) using RNA obtained from tissue isolated from the base of the OFT revealed changes in the expression of genes in both the canonical and noncanonical Wnt signaling pathway (D). Scale bar: 1 mm in A,B. The P values were calculated with Student's t-test; error bars show standard deviation.

Cell polarity and myocardialization defects in the OFT of Bj mutants. (A-F) Phalloidin (red) and MF20 (green) staining of paraffin sections of wild-type (A,C,E,) (n=3) and Bj mutant (B,D,F,) (n=3) heart. In the wild-type heart, phalloidin staining showed actin filament alignment with the direction of cell invasion into the OFT cushion (A,C), but in the Bj mutant, this pattern of actin alignment is not observed (B,D). Arrows in A indicate the two prongs of cardiomyocytes invading the cardiac septum. Examination of the striated banding pattern from the MF20 immunostain showed the developing myofilaments are closely aligned and oriented towards the direction of myocardialization in the wild-type embryo (arrows, E), but in the Bj mutant, the myofilaments are sparse and are largely oriented perpendicular to the direction of myocardialization and septum formation (arrows, F). (G) Quantitation of myofilaments showed wild-type (n=20) cardiomyocytes are polarized in the direction of cell invasion; in Bj mutant hearts (n=20), the cell orientation is not properly aligned (P=0.0149). Quantitative analysis showed cardiomyocytes (MF20 positive) in this conotruncal region of the heart is significantly reduced compared to controls (+/+, +/m 70%; m/m 22%: P=0.0025). Scale bar: 20 µm. The P values were calculated with a two-sample Wilcoxon rank-sum test.

Bj mutants show stereocilia patterning defects and biliary duct (BD) malformations. (A-D) Immunostaining with phalloidin (green) and Vangl2 (red) of the cochlea of near-term wild-type (A,C) and Bj mutant (B,D) embryo. The phalloidin staining delineated the normal chevron-shaped stereocilia in the outer hair cells (OHCs), showing malalignment in the Bj mutant (boxed B) compared to wild-type (boxed A). In addition, Vangl2 staining revealed a subtle misalignment of supporting cells connecting the inner hair cells (IHCs) in the Bj mutant (B), better seen in the enlarged views shown in panels C and D (see arrowheads). (E-J) Shown are bile ducts from a wild-type newborn mouse (E) (n=6) as compared to that of a Bj mutant (F) (n=6), which is noticeably shorter. Quantitative analysis showed a significant decrease in both the length and width of the Bj mutant biliary duct (G). Examination of hematoxylin and eosin stained sections of newborn wild-type (H) (n=4) and Bj mutant (I) biliary ducts suggest a decrease in the mucosal folds of the mutant duct (I) (n=5). This was confirmed with quantitative analysis, which showed a significant decrease in mucosal folds in the mutant duct (J). Error bars show standard deviation. (K-P) Immunostaining with antibodies against β-catenin (red) and E-cadherin (green) showed β-catenin expression is markedly reduced in the Bj mutant biliary duct (N,P), but no change was observed for E-Cadherin (O,P,). Scale bars: 100 µm in A-E); 30 µm (K-P). The P values were calculated with a two-sample Wilcoxon rank-sum test. GB, gallbladder; BD, bile duct.

Wound closure assay show defect in cell polarity and polarized cell migration in Bj mutant MEFs. (A-G). Analysis of cell polarity and directional cell migration using a MEF wound closure assay. Wildtype MEFs (A) are aligned with the direction of wound closure (white arrows), but Bj mutant MEFs (B) exhibit randomized pattern of cell migration. Cell orientation is delineated by examining the Golgi position after 24 h observed by Golga2 (green) immunostaining (C,D). Golgi orientation is indicated by a white line drawn from the cell nucleus (white circle) through the center of the Golgi. (E-G) Quantitative analysis of Golgi orientation relative to the direction of cell migration showed Golgi orientation is polarized along the direction of cell migration in wild-type (n=100), but not Bj mutant MEFs (n=150) (E). Tracings of the migratory path of MEFs after 8 h showed straight migration paths for wild-type MEFs (n=28), but tortuous migration paths with increased velocity for mutant MEFs (n=27) (F,G) V, speed of cell locomotion; D, directionality of cell movement. (H-K) Phalloidin (green) and Pk1 (red) antibody staining of wild-type (H,J) and Bj mutant (I,K) MEFs migrating into a wound gap after 10 h. This showed well formed actin stress fibers aligned with the direction of cell migration in the wild-type MEFs (H,J), but in the Bj mutants MEFs, the actin cytoskeleton exhibited a cortical basket formation (I,K). Interestingly, wild-type MEFs showed Pk1 localization in the cytoplasm (J), but in Bj mutant MEFs, Pk1 is localized to the nucleus (K). The P values were calculated with a two-sample Wilcoxon rank-sum test. Scale bars: 50 µm in A-D; 30 µm in H-K.

Cilia defects in Bj mutant tracheae. (A) Quantitative analysis shows a reduction in cilia beat frequency of Bj mutant tracheal airway cilia. Error bars show standard deviation. (B,C) Transmission electron microscopy (TEM) of motile cilia in both mutant (C) and control (B) trachea show normal planar polarity based on the orientation of basal feet (see boxed insets) on individual cilia towards the proximal (oral) direction. D, dorsal; P, proximal. (D-I). Scanning electron microscopy of Bj mutant trachea (bottom G,H) shows that compared to control (D,E) nonciliated and multiciliated cells have large apical membrane bulges (arrows G,H), which in the multiciliated cells incorporate the axonemes. (I) Some mutant ciliary axonemes also have blebbed surface (boxed I′). (J-L) TEM of Bj mutant cilia show some abnormal axonemal microtubules (K,L), membrane blebs and compound axonemes compared to control (J). (M-P) Enlarged views of (J-L) show the absence of a microtubule doublet in the mutant axoneme (N), blebbed membrane (O), and a compound axoneme (P) compared to the control (M). Compound axonemes are likely contained within the membrane blebs observed by SEM (G,H).