Person AD et al. (2005), Frzb modulates Wnt-9a-mediated beta-catenin sig...

XB-ART-2477

Dev Biol 2005 Feb 01;2781:35-48. doi: 10.1016/j.ydbio.2004.10.013.

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Frzb modulates Wnt-9a-mediated beta-catenin signaling during avian atrioventricular cardiac cushion development.

Person AD , Garriock RJ , Krieg PA , Runyan RB , Klewer SE .

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Normal development of the cardiac atrioventricular (AV) endocardial cushions is essential for proper ventricular septation and morphogenesis of the mature mitral and tricuspid valves. In this study, we demonstrate spatially restricted expression of both Wnt-9a (formerly Wnt-14) and the secreted Wnt antagonist Frzb in AV endocardial cushions of the developing chicken heart. Wnt-9a expression is detected only in AV canal endocardial cells, while Frzb expression is detected in both endocardial and transformed mesenchymal cells of the developing AV cardiac cushions. We present evidence that Wnt-9a promotes cell proliferation in the AV canal and overexpression of Wnt-9a in ovo results in enlarged endocardial cushions and AV inlet obstruction. Wnt-9a stimulates beta-catenin-responsive transcription in AV canal cells, duplicates the embryonic axis upon ventral injections in Xenopus embryos and appears to regulate cell proliferation by activating a Wnt/beta-catenin signaling pathway. Additional functional studies reveal that Frzb inhibits Wnt-9a-mediated cell proliferation in cardiac cushions. Together, these data argue that Wnt-9a and Frzb regulate mesenchymal cell proliferation leading to proper AV canal cushion outgrowth and remodeling in the developing avian heart.

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Species referenced: Xenopus
Genes referenced: frzb inhba not pcna wnt5a wnt8a wnt9a
GO keywords: Wnt signaling pathway

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	Fig. 1. Expression of Wnt-9a in the AV canal cushion endocardium. Whole-mount in situ hybridizations demonstrating Wnt-9a expression in the AV canal cardiac cushions of chicken hearts at stage 16 (A), stage 20 (B), and stage 24 (C). Section in situ hybridizations of stage 16 (D), stage 20 (E), and stage 24 (F) show Wnt-9a expression localized to the endocardial cell layer (arrows) of the AV canal cardiac cushions. Abbreviations: A = atrium; V = ventricle; M = myocardium; E = endocardium.
	Fig. 2. Production and characterization of RCAS Wnt-9a and Wnt-9aD288. (A) Alignment of mWnt-1 and XWnt-8 with chicken Wnt-9a nucleotide sequences. C-terminal truncations of both mWnt-1 and XWnt-8 after the SPXXC motif (box) create Wnts that function as dominant-negative inhibitors (Hoppler et al., 1996). A similar 288 amino acid avian Wnt-9a construct (Wnt-9aD288) was generated by truncating the C-terminal 66 amino acids after the SPSFC domain. (B) Western blot analysis of DF-1 cell lysates infected with RCAS Wnt-9a and Wnt-9aD288 viruses detect proteins of ~45 and ~38 kDa, respectively. Wnt-9a is not detected in DF-1 cells infected with RCAS control virus.
	Fig. 3. Wnt-9a and Wnt-9aD288 affect cell proliferation and apoptosis in AV canal cultures. (A) Infections of AV canal cultures with RCAS Wnt-9a results in a significant increase in migratory mesenchymal cells compared to RCAS control infected AV canal cultures. Infection of AV canal cultures with RCAS Wnt-9aD288 results in a decrease in migratory mesenchymal cells compared to RCAS control infected AV canal cultures. (B) Real-time PCR data showing infections of AV canal cultures with RCAS Wnt-9a and RCAS Wnt-9aD288 resulting in a 6.3- and 7.7-fold expression increase respectively over endogenous Wnt-9a in RCAS controls. (C) AV canal cultures infected with RCAS Wnt-9a show an increase in BRDU incorporation compared to RCAS control infected AV canal cultures. RCAS Wnt-9aD288-infected cultures did not show differences in BRDU incorporation compared to controls. (D) Wnt-9a overexpression in AV canal explants resulted in decreased TUNEL staining compared to RCAS controls. Overexpression of Wnt-9aD288 results in an increase in TUNEL staining compared to controls (D). All experiments consisted of at least 16 AV canal explants and were performed in triplicate.
	Fig. 4. Wnt-9a activates h-catenin in AV canals and causes axis duplications in Xenopus embryos. (A) AV canal explants infected with RCAS Wnt-9a activate the h-catenin responsive TOPFLASH reporter but not the FOPFLASH reporter containing mutated TCF binding sites. Infection of AV canal explants with RCAS control virus does not activate the TOPFLASH or FOPFLASH reporter. (B) Wnt-9a mRNA (50 pg) injected into a ventral blastomere of four-cell Xenopus embryos causes duplication of the embryonic axis. Co-injecting 50 pg of Wnt-9a with 200 pg (C) or 500 pg (D) of Wnt-9aD288 rescues axis duplications. Injection of 50 pg of XWnt-8 also results in axis duplications (E) that are not rescued with co-injection with 200 pg of Wnt-9aD288 (F). Coinjection of Xwnt-8 with 500 pg of Wnt-9aD288 partially rescues XWnt-8-induced twinning as two cement glands are still evident (G). Uninjected embryos showing Xnot-1 expression in the mesoderm overlying the dorsal blastopore lip (H). Stage 11 embryos injected with 50 pg of Wnt-9a mRNA show ectopic Xnot-1 expression consistent with secondary axis formation (I). This ectopic Xnot-1 expression is no longer detected in embryos co-injected with Wnt-9a (50 pg) and Wnt-9aD288 (500 pg) (J). Uninjected control embryos at stage 15 undergoing normal gastrulation (K). Ventral injection of XWnt-5a inhibits convergent extension creating gastrulation defects (L). These gastrulation defects caused by XWnt-5a injections are not rescued by co-injection with 500 pg of Wnt-9aD288 (M). Abbreviation: C = cement gland.
	Fig. 5. Frzb is expressed in the endocardial and mesenchymal cells of the AV canal cushions. Whole-mount in situ hybridizations show Frzb expression in the AV canal cushions at HH stage 16 (A), stage 20 (B), and stage 24 (C). In situ hybridization analysis of sections reveals Frzb is expressed in the endocardium and transformed mesenchyme of AV canal cushions at stage 16 (D), stage 20 (E), and stage 24 (F). Frzb expression is absent from the myocardium at all stages examined. Abbreviations: A = atrium; V = ventricle; E = endocardium; M = myocardium.
	Fig. 6. Frzb is a dose-dependent negative regulator of Wnt-9a-mediated cell proliferation. (A) Addition of recombinant FRP-3, the mouse orthologue of chicken Frzb, results in a decrease in mesenchymal cell number in AV canal cultures. (B) Treatments of AV canal explants with two distinct Frzb antisense oligodeoxynucleotides result in an increase in mesenchymal cell number compared to controls (Lipofectamine alone and control oligo treatments). (C) Infection of AV canal cultures with RCAS Wnt-9a results in an increase in mesenchymal cell number while treating RCAS Wnt-9ainfected cultures with FRP-3 at 10 ng/ml reduces mesenchymal levels to those seen in RCAS control infected cultures. FRP-3 (30 ng/ml) further decreases mesenchymal cell number in RCAS Wnt-9a-infected cultures (C).
	Fig. 7. Wnt-9a and Wnt-9aD288 affect cell proliferation, apoptosis, and cushion morphology in vivo. (A–F) Analysis of stage 18 AV endocardial cushions following Wnt-9a perturbations. (A–C) RCAS infection (anti-p27) is shown in red and PCNA immunostaining is shown in green. Co-expressing cells are yellow. Wnt-9a-infected AV cushions show a pronounced increase in PCNA staining in both infected endocardial and mesenchymal cells compared to control and Wnt-9aD288-infected hearts. Arrows indicate the endocardium while arrowheads point to the myocardium of the AV canal junction. (D–F) TUNEL staining (seen as red) is increased in cushion mesenchymal cells of Wnt-9aD288-infected hearts compared to control and Wnt-9a-infected hearts. The endocardial cell layer is marked by a white dotted line. (G–L) Heart valve remodeling defects following Wnt-9a overexpression. (K) Wnt-9a-infected hearts at stage 26 display an elongated AV inlet and an enlarged atrium with blood pooling. (L) Wnt-9a-treated AV cushions are enlarged, hyperplastic, and display multiple AV cushion projections into the lumen (arrowheads). RCAS Wnt-9aD288-infected hearts, at stage 33, display looping abnormalities and a single AV inlet with hypoplastic AV cushions (M, N). Viral infection is detected with anti-GAG immunostaining shown in red (H, J, L, N). Abbreviations: A = atrium; V = ventricle; C = conus arteriosus; RA = right ventricle; LA = left ventricle; RA = right atrium; LA = left atrium.