176 - The role of WNT/PCP signaling during cardiac development

Publication Number: 176.6728

Kevin Carthy, Indiana University School of Medicine, Carmel, IN, United States; Alyx D. Posorske, Indiana University School of Medicine, Indianapolis, IN, United States; James Zwierzynski, Stanford University, Stanford, CA, United States; Rebecca Lauren Judith. Douglas, Indiana University, Indianapolis, IN, United States; Catherine Guilfoy, Indiana University School of Medicine, Indianapolis, IN, United States; Emma Kennedy, Marian University College of Osteopathic Medicine, Indianapolis, IN, United States; Nina J. Jain, University of California, Davis, Carmel, IN, United States; Ashleigh McMullan, Indiana University School of Medicine, Indianapolis, IN, United States; Caelan Rathke, Indiana University School of Medicine, Shelbyville, IN, United States; Ellen Voskoboynik, Indiana University School of Medicine, Indianapolis, IN, United States; Laura Haneline, Riley Hospital for Children at Indiana University Health, Indianapolis, IN, United States; Stephanie M.. Ware, Indiana University School of Medicine, Indianapolis, IN, United States; Weinian Shou, Indiana University School of Medicine, Indianapolis, IN, United States; Matthew Durbin, Indiana University School of Medicine Riley Hospital for Children, Indianapolis, IN, United States;Ellen Trautman, Indiana University School of Medicine, Carmel, IN, United States

Background: Congenital heart disease (CHD) is the leading cause of death due to birth defects, however, underlying etiologies are poorly understood. Gene-gene interactions within the fundamental Noncanonical WNT/Planar Cell Polarity (WNT/PCP) signaling pathway are important for cardiac development and may have a role in CHD. A recent report updated the principal logic of WNT/PCP, by demonstrating that during neural tube development, central component DVL2 is bimodally regulated by VANGL2, due to DVL2’s ability to bind either VANGL2, or its effector DAAM1, but not both.

Objective: To test the hypothesis that bimodal signaling is also present in cardiac development with implications for CHD.

Design/Methods: We utilized loss-of-function murine models for DVL2, VANGL2, and DAAM1, (Dvl2+/∆, Vangl2+/∆ and Daam1+/gt.) We crossed loss-of-function mice to generate litters haploinsufficient for one, two, or all three proteins and performed histologic analysis of embryonic hearts for cardiac defects. We also performed a complementary analysis of the transcriptome, using bulk RNA sequencing, as well as on the proteome using tandem-mass tag mass spectroscopy, of the global and phosphoproteome. Finally, we performed in vitro functional analysis using both CRISPR-Cas9 derived knockout models and siRNA knockdown models, for VANGL2 and DVL2.

Results: Analysis indicates that singly heterozygous null, Dvl2, Vangl2 and Daam1 hearts had a similar, low frequency of membranous VSDs, that did not significantly differ from the wild type littermates by chi-square analysis. However, the triple compound heterozygous null embryos, (Dvl2+/∆;Vangl2+/∆;Daam1+/gt ) had an increased frequency of VSDs (7 of 29, frequency 0.24, p< 0.05). Interestingly, homozygous null Dvl2 embryos that also had a compound heterozygous null allele in Vangl2 had a rescue of their defects, with significantly decreased VSD frequency, mirroring findings in neural tube defects. Analysis of the transcriptome and proteome revealed mechanistic insight, including significant alterations in pathways such as cell migration and cell adhesion. In vitro functional analysis validated these findings, demonstrating cell migration defects which were impacted by the interaction between DVL2 and VANGL2.

Conclusion(s): Our data highlight genetic and functional interactions within the fundamental WNT/PCP pathway, important to cardiac development. These insights have implications for CHD inheritance, pathogenesis and therapeutic development in humans.