SHROOM3 is downstream of the planar cell polarity pathway and loss-of-function results in congenital heart defects

Abstract

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3, in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3^gt/gt) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3^gt/gt mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3^gt/gt hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3's novel role in cardiac development and CHD.

Keywords: Cardiac development; Congenital heart disease; Non-canonical wnt signaling pathway; Planar cell polarity pathway; Wnt.

Fig. 1.

Shroom3 is expressed in the heart during cardiac development Spatiotemporal expression of Shroom3 during cardiac development by WISH and β-galactosidase expression via X-gal staining from heterozygous Shroom3^+/gt embryos, in whole mount and in section. WISH (A) and whole-mount X-gal staining (B, E, and F) at E9.5–12.5 (with magnification of the heart (C, D, G and H). Sections of E10.5 hearts (I–L). Shroom3 expression pattern is consistent by WISH (A and C) and by β-galactosidase staining (B and D). Shroom3 expression is present as previously reported in the neural tube, gut, mandibular arch and limbs (A, B, E and F). In whole mount E9.5 embryos, Shroom3 expression is detected within the facial region of the head, lateral mesoderm and splanchnic mesoderm just dorsal to the cardiac outflow tract (A–D). At E10.5 and E12.5 Shroom3 is detected in the four chambers of the heart (E–H) and in the OFT. In sections from X-gal staining, expression is evident in the OFT (I with magnification in J, and Shroom3 OFT expression indicated by an arrow in J), in both atria and in both ventricles (K and L). Shroom3 expression is detected in the population of cells forming the aortic sac and in the splanchnic mesoderm (I and J). (as, aortic sac; ht, heart; ivs, intraventricular septum; lv, left ventricle; oft, outflow tract; rv, right ventricle).

Fig. 2.

Shroom3 is expressed within second heart field cells and cardiac neural crest cells Shroom3 is expressed in cardiac neural crest cells and second heart field cells as indicated by overlapping β-galactosidase expression via X-gal staining and immunofluorescence by immunohistochemistry analysis. Serial sections of E9.5 Shroom3^+/gt embryos (A and B) demonstrate overlapping expression of Shroom3 by X-gal staining (A) and immunofluorescence for cardiac neural crest cell marker AP-2α (B) within the developing aortic sac. Serial transverse sections of E10.5 Shroom3^+/gt embryos at the level of the developing outflow tract demonstrates overlapping expression of Shroom3 by X-gal staining (C) and immunofluorescence for second heart field marker ISL1 (D). (oft, outflow tract.)

Fig. 3.

Shroom3 is expressed within cardiomyocytes Shroom3 is expressed in cardiomyocytes, indicated by β-galactosidase expression via X-gal staining and immunofluorescence by immunohistochemistry analysis. E12.5 Shroom3^+/gt embryo serial sections (A–D) demonstrate Shroom3 expression detected by X-gal staining (A and B) in cardiomyocytes of the left ventricle and intraventricular septum, which also demonstrate immunofluorescence for cardiac troponin (C and D). E11.5 Shroom3^+/gt embryo, left ventricular wall cryosections (E–G), demonstrate colocalization of immunofluorescence for X-gal (E) and immunofluorescence for cardiac troponin (F and G).

Fig. 4.

Shroom3^gt/gt mice exhibit cardiac defects Transverse sections of hearts stained with H&E in Shroom3^+/+ littermate controls (A and D) and Shroom3^gt/gt embryos with indicated heart defects (B, C, E and F). Frequency of identified heart defects (G) and left ventricular measurements (H). Shroom3^+/+ littermate controls have normal cardiac structures (A and D). Shroom3^gt/gt embryos display CHDs (63%; p=0.0001 [B, C, E and F]). Shroom3^gt/gt mice have ventricular septal defects (VSD)(48%; p=0.0004), with membranous VSD (indicated by an asterisk in B) and muscular VSD (indicated by an arrowhead in C); and double outlet right ventricle, in which both the aorta (E) and pulmonary artery arise from the right ventricle (19%; p=0.04). In contrast Shroom3^+/+ littermate controls (A and D) have a complete ventricular septum and the aorta arises from the left ventricle and the pulmonary artery from the right ventricle. A single Shroom3^gt/gt embryo with double inlet left ventricle (F), with both the left atrium and mitral valve and right atrium and tricuspid valve emptying into the left ventricle, was identified. Shroom3^gt/gt mice exhibit ventricular thinning in the left ventricular compact myocardium. Mean lv wall thickness measurements in (H) with measurements represented by a black line in panel (A) and (B) (125.0±43μm, 82.0±37μm; p=0.008) shown. Measurements of the left ventricle were made by capturing images of transverse sections of E14.5 hearts from Shroom3^gt/gt and Shroom3^+/+ littermate control embryos, then selecting a matching plane of section which included a 4-chamber view of the heart. The compact myocardium was measured at the lowest point of the left ventricular wall where it meets the ventricular septum. We compared Shroom3^gt/gt hearts and Shroom3^+/+ littermate control hearts using Fisher’s two-sided t-test for statistical analysis. A difference in denominator between groups reflects histologic section availability due to technical determinants. (ao, aorta; DILV, double inlet left ventricle; DORV, double outlet right ventricle; la, left atrium; lv, left ventricle; mv, mitral valve; pa, pulmonary artery; ra, right atrium; rv, right ventricle; tv, tricuspid valve; ivs, intraventricular septum.)

Fig. 5.

Shroom3 interacts with and is downstream of key planar cell polarity component Dishevelled2 and SHROOM3 loss impacts planar cell polarity components A schematic of PCP signaling (A), a Co-IP immunoblot demonstrating SHROOM and DVL2 physically interact within the heart (B), Dvl2 transcriptionally regulates Shroom3 demonstrated by gene expression analysis (C), X-gal (D–E) and WISH analysis (F–I) and H&E stained transverse heart sections demonstrating Shroom3 and Dvl2 genetic interaction (J–O). PCP signaling activation by immunofluorescence in cell culture (P–V), gene expression analysis (W) and proteins by immunoblot analysis (X) in embryo hearts. (A) Planar cell polarity pathway terminal components including, key cytoplasmic component DVL2, and terminal effectors, RAC1, JNK, RHOA and ROCK1, which phosphorylates MLC and MYPT1, driving Actomyosin cytoskeletal rearrangement and subsequent changes in cell shape and movement. (B) SHROOM3 and DVL2 physically interact in the heart, demonstrated by Co-IP immunoblot, with DVL2 detected within P7 mouse heart lysate, pulled down using SHROOM3 antibody bound to agarose beads. (C) Expression analysis of Shroom3 by RT-qPCR analysis in Dvl2^+/+, Dvl2^+/− and Dvl2^−/− hearts at E12.5 (n=5 of each genotype). In Dvl2^−/− mouse hearts, there is a 60% reduction in Shroom3 expression (p=0.03.) Analysis of Shroom3 tissue specific expression pattern within the heart demonstrated a global reduction in Shroom3 expression, demonstrated by comparing Dvl^+/+ and littermate Dvl2^−/−embryos by both X-gal staining (D and E) and WISH for Shroom3 (F–I). Shroom3 interacts genetically with Dvl2 during cardiac development, demonstrated in crosses of Shroom3^+/gt and Dvl2^+/− mice, with histologic analysis of H&E stained transverse heart sections of embryos after ventricular septation at E14.5. OFT and ventricular septum in heterozygous Shroom3^+/gt(J and K) and Dvl2^+/−(L and M) embryos are phenotypically normal, whereas a littermate compound heterozygous Shroom3^+/gt;Dvl2^+/− embryo has PCP associated cardiac defects DORV (N) and membranous VSD (O, asterisk) (n=76). (P–V) Mouse embryonic fibroblasts (MEF) were subjected to a scratch wound, treated with Wnt5a recombinant protein and 24 hours later analyzed by immunofluorescence. We imaged the scratch wound area, capturing cells having moved into the wound area, and quantified total cells, positive for phalloidin (P), as well as cells with positive immunofluorescence for activated MYPT1 within the cytoplasm (Q), in the entirety of each 1mm² visual field and calculated a ratio of MYPT1 positive cells over phalloidin positive cells (V). Compared with Shroom3^+/+ MEFs, Shroom3^gt/gt MEFS have a significantly reduced ratio of activated MYPT1 positive cells (0.73 versus 0.33, standard deviation 0.11 and 0.19, p=0.02; averages of 4 different scratch wound areas with an average of 18 cells per area from (n=4) Shroom3^+/+ and (n=3) Shroom3^gt/gt littermate embryos). (W) Expression analysis of PCP signaling pathway genes by RT-qPCR of Shroom3^gt/gt mouse hearts and Shroom3^+/+ littermate control hearts at E12.5 (n=3–5 of each genotype). In Shroom3^gt/gt mouse hearts, there is a statistically significant reduction in the expression of multiple PCP components (Daam1, JNK, Rho, and a trend towards decreased expression of Rock1 and Rac1). P-values are calculated directly from mean dCq values and error bars represent standard deviation calculated from mean dCq values as a ratio of mean to total exponential values. (X) Immunoblot analysis of whole heart lysates from E12.5 and E14.5 Shroom3^+/+ and Shroom3^gt/gt littermates. Activation of PCP represented by activated, phosphorylated, PCP central component DVL2 as well as terminal components MYPT1 and MLC, as compared to total protein. Shroom3^gt/gt hearts exhibit observable reductions in PCP proteins and terminal components including phosphorylated MYPT1, phosphorylated MLC (pMLC), and phosphorylated DVL2 (pDVL2). (ao, aorta; co-immunoprecipitation assay, CoIP; ivs, intraventricular septum; la, left atrium; lv, left ventricle; oft, outflow tract; ra, right atrium; rv, right ventricle).

Fig. 6.

Shroom3^gt/gt hearts have disrupted cardiomyocyte polarity, organization and morphology Cardiomyocyte polarity, organization and morphology measured with immunofluorescence analysis within the OFT and lv of E10.5, E11.5 and E14.5 hearts. Immunofluorescence analysis of adherens-junction component β-catenin in the OFT of E10.5 Shroom3^+/+ (A–B) and littermate Shroom3^gt/gt embryos (C–D), with further magnification of boxed sections (A and C) in (B and D), showing abnormal structure of the OFT membrane. In the Shroom3^+/+ OFT membrane (B), cells are organized and aligned, with β-catenin localized to adheres junctions (white arrowheads). The Shroom3^gt/gt OFT membrane (D) cells are disorganized with loss of β-catenin localization at the adherens junctions (white arrowheads). Immunofluorescence analysis of basolateral membrane marker SCRIB and cardiomyocyte marker sarcomeric α-actinin (α-actinin) in the lv wall of E11.5 Shroom3^+/+ (E–F) and littermate Shroom3^gt/gt embryos (G–H), with further magnification of boxed sections (E and G) in (F and H), showing abnormal structure of the lv wall. The lv wall of Shroom3^+/+ mice is compact and organized with well aligned cells and Scribble localized at the basolateral membrane of the epicardium (F, arrowhead). Whereas Shroom3^gt/gt lv wall cardiomyocytes are disorganized and less compact with more space between cells, and without evident SCRIB at the ventricle epicardium basolateral membrane, though still evident in the basolateral membrane within the unaffected atria (G, arrow). F-actin and pMLC immunofluorescence analysis of hearts from E14.5 Shroom3^+/+ (I) and littermate Shroom3^gt/gt embryos (L), with further magnification of boxed sections showing abnormal structure of the membranous ventricular septum (J and M) and lv wall (K and N). In the Shroom3^+/+ ventricular septum (J), lamellipodia and filopodia are visible, indicated with white arrows. The Shroom3^gt/gt septum (M) cardiomyocytes are rounded, lacking lamellipodia and filopodia, and a membranous VSD is evident, indicated by an asterisk. The left ventricular wall of Shroom3^+/+ mice is comprised of mature, elongated cardiomyocytes (K). We quantified cardiomyocyte area (O), roundness (P) and disorganization (Q) in Shroom3^gt/gt and Shroom3^+/+ littermate control embryos. We demonstrate that, compared to Shroom3^+/+ littermate controls, Shroom3^gt/gt embryo cardiomyocytes are the same size (56.1 μm² vs 57.1 μm², standard deviation= 8.8 and 11.9, p=0.9), but are more round (roundness 2.0 versus 2.1 (roundness=4*area in μm²/(π*major axis²), standard deviation= 0.04 and 0.04, p=0.04) and less compact-more disorganized (lacunarity 0.33 versus 0.42, standard deviation= 0.04 and 0.03, p=0.04). (ao, aorta; ivs, intraventricular septum; la, left atrium; lv, left ventricle; oft, outflow tract; ra, right atrium; rv, right ventricle).

Fig. 7.

Shroom3^gt/gt hearts have increased left ventricular cardiomyocyte proliferation Immunofluorescence analysis of proliferation in E9.5 (A and B) and E14.5 embryos (C and D) with quantification (E–H). Immunofluorescence analysis of proliferation marker pHH3, in E9.5 Shroom^+/+ embryos (A) and littermate Shroom3^gt/gt. embryos (B), demonstrating increased proliferation in Shroom3^gt/gt embryos (B). Immunofloresence analysis of pHH3 and cTnT to identify cardiomyocytes in the left ventricle, in matching transverse sections of E14.5 Shroom^+/+ mice (C) and littermate Shroom3^gt/gt embryos (D), again demonstrating increased proliferation in Shroom3^gt/gt embryos (D). We quantified all positive cardiomyocytes (positive for pHH3 and cTnT) in the entire 1mm² visual field (E–H). Shroom3^gt/gt embryos have significantly increased cardiomyocyte proliferation within the left ventricle (E) (12 versus 18 pHH3 positive cardiomyocytes per mm², standard deviation=1.3 and 1.2, p=0.003). Shroom3^gt/gt mice have increased proliferation localized to the ventricular trabeculae (1.9 versus 5.8 pHH3 positive cardiomyocytes per mm², standard deviation=0.8 and 1.0, p=0.002), whereas cardiomyocyte proliferation was equal within the left ventricle wall and ventricular septum (lv wall; 6.3 versus 6.6 pHH3 positive cardiomyocytes per mm², standard deviation=0.6 and 3.0, p=0.8) (septum; 4.3 versus 5.4 pHH3 positive cardiomyocytes per mm², standard deviation=0.8 and 1.0, p=0.47) (ht, heart; ivs, intraventricular septum; lv, left ventricle).

Fig. 8

Proposed location of SHROOM3 within the Wnt Signaling/ Planar Cell Polarity (PCP) pathway A schematic of the noncanonical Wnt signaling/PCP pathway is shown with a proposed location of SHROOM3, based on these data and previous studies, in which SHROOM3 binds DVL2 and ROCK1 and functions with PCP’s terminal actomyosin effectors.