Supplementary Materials Supplemental Material supp_32_21-22_1443__index. remain to be resolved. Here, we provide genetic and molecular evidence that vertebrate BCL9 and Pygo proteins contribute as tissue-specific mediators of -catenin SW044248 in the development of specific structures and organs, in particular during heart formation. In zebrafish mutants for the and genes or upon selective chemical inhibition of the BCL9C-catenin interaction, we uncovered that disrupting the -cateninCBCL9CPygo complex causes limited developmental phenotypes, including heart defects. In mice, both constitutive SW044248 and heart-specific conditional loss of or or the simultaneous impairment of the BCL9/9LC-catenin and BCL9/9LCPYGO2 interactions leads to heart malformations, which include defects in chamber septation and outflow tract (OFT) and valve formation. These data reveal that, in vertebrates, the Wnt-dependent function of the BCL9CPygo module is restricted to select processes. Transcriptome analyses established that, in the developing heart and pharyngeal structures, the -cateninCBCL9CPygo complex regulates the expression of tissue-specific groups of genes. In addition, genome-wide MADH9 chromatin-binding profiling revealed that -catenin and PYGO co-occupy putative at and mutations in (Christiansen et al. 2004; Brunet et al. 2009; Tomita-Mitchell et al. 2012; Dolcetti et al. 2013). Results BCL9 and Pygo perturbations cause developmental heart defects in zebrafish and mice To investigate the contribution of BCL9/9L proteins to vertebrate heart development based on their repeated association with CHD, we applied maximized CRISPRCCas9-mediated mutagenesis in zebrafish SW044248 embryos to generate crispants (Fig. 1ACC; Burger et al. 2016): We targeted both BCL9 family genes and with individual single-guide RNAs (sgRNAs) by injection of Cas9 ribonucleoprotein complexes into one-cell stage zebrafish embryos and observed highly penetrant cardiac phenotypes following somatic mutagenesis of (Fig. 1B,C). We established mutant alleles for both and and as well as homozygous zebrafish and their maternal-zygotic mutant offspring (MZdisplayed unaltered expression of early cardiac markers (lead to cardiac defects in zebrafish. (as a potential regulator of heart morphogenesis. (crispants have heart-looping defects, as visible in gene locus and generation of the germline allele. A sgRNA was designed to target the coding exon 6 between HD1 and HD2 of SW044248 the zebrafish gene. The locus is represented as per annotation allele. In the isolated allele, black boxes mark coding exons (CDS), white boxes mark UTRs, blue boxes represent the CDSs that contribute to HD1, and purple boxes represent the CDSs that contribute to HD2. (germline allele with a 29-base-pair (bp) deletion. The shows genomic reference (features an out-of-frame deletion introducing a frameshift followed by 157 novel amino acids terminated by two consecutive stop codons, thus disconnecting HD1 from HD2. The black box indicates the exact position of the sgRNA sequence, the gray-shaded box indicates the and embryos and their wild-type-looking siblings (lateral views; anterior is to the left). Mutant embryos demonstrated heart-looping problems and cardiac edema (asterisks). Furthermore, mutant embryos didn’t inflate their swim bladders (arrows), presumably because of failing in gasping atmosphere due to craniofacial malformations (dark arrowheads). (embryos (ventral sights; anterior can be to the very best; imaged after viable heart-stopping BDM treatment). and depict maximum-intensity projections, and show close-ups of the dotted square in and depict optical sections at the atrioCventricular canal level. Compared with siblings that form correctly looped hearts with atrioCventricular canal valves and a bulbus arteriosus (BA; heart outlined with red dotted line; = 4; embryos show heart-looping defects (= 8; (= 16) compared with.