Objectives Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the bile ducts of neonates. Although BA is the leading indication for pediatric liver transplantation, the etiology remains elusive. Adducin 3 (ADD3) and X-prolyl aminopeptidase 1 (XPNPEP1) are two genes previously identified in genome-wide association studies (GWAS) as potential BA susceptibility genes. Using zebrafish, we investigated the importance of ADD3 and XPNPEP1 in functional studies. Methods To determine whether loss of either gene leads to biliary defects, we performed morpholino antisense oligonucleotide (MO) knockdown studies targeting add3a and xpnpep1 in zebrafish. Individuals were assessed for decreases in biliary function and the presence of biliary defects. Quantitative PCR (qPCR) was performed on pooled 5 days post fertilization (dpf) larvae to assess variations in transcriptional expression of genes of interest. Results While both xpnpep1 and add3a are expressed in the developing zebrafish liver, only knockdown of add3a produced intrahepatic defects and decreased biliary function. Similar results were observed in homozygous add3a mutants. Morpholino antisense oligonucleotide-mediated knockdown of add3a also showed higher mRNA expression of Hedgehog (Hh) targets. Inhibition of Hh signaling rescued biliary defects caused by add3a knockdown. Combined knockdown of add3a and glypican-1 (gpc1), another mediator of Hh activity that is also a BA susceptibility gene, resulted in more severe biliary defects than knockdown of either alone. Conclusions Our results support previous studies identifying ADD3 as a putative genetic risk factor for BA susceptibility. Our results also provide evidence that add3a may be affecting the Hedgehog pathway, an important factor in BA pathogenesis.

Biliary atresia (BA) is a progressive fibro-inflammatory disorder that is the leading indication for liver transplantation in children. Although there is evidence implicating genetic, infectious, environmental, and inflammatory causes, the etiology of BA remains unknown. We have recently reported that cholangiocytes from BA patients showed decreased DNA methylation relative to disease- and non-disease controls, supporting a potential role for DNA hypomethylation in BA etiopathogenesis. In the current study, we examined the methylation status of specific genes in human BA livers using methylation microarray technology. We found global DNA hypomethylation in BA samples as compared to disease- and non-disease controls at specific genetic loci. Hedgehog pathway members, SHH and GLI2, known to be upregulated in BA, were both hypomethylated, validating this approach as an investigative tool. Another region near the PDGFA locus was the most significantly hypomethylated in BA, suggesting potential aberrant expression. Validation assays confirmed increased transcriptional and protein expression of PDGFA in BA livers. We also show that PDGF-A protein is specifically localized to cholangiocytes in human liver samples. Injection of PDGF-AA protein dimer into zebrafish larvae caused biliary developmental and functional defects. In addition, activation of the Hedgehog pathway caused increased expression of PDGF-A in zebrafish larvae, providing a previously unrecognized link between PDGF and the Hedgehog pathway. Our findings implicate DNA hypomethylation as a specific factor in mediating overexpression of genes associated with BA and identify PDGF as a new candidate in BA pathogenesis.

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1 (Fujioka and Jaynes, 2012). Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. We localized cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function.

Apc mutations cause intestinal tumorigenesis through Tcf4 activation. However, direct techniques for studying Tcf4 activation in vivo are limited. Here, we describe the development of a Tcf4-GFP reporter mouse model for directly studying Tcf4 activation. We first developed a GFP reporter construct (Tcf4-GFP) and transfected it into SW480 cells that have constitutively activated Tcf4. Reporter activity increased 47-fold. Next, we created transgenic (Tg) mice by transducing the construct into C57BL/6J mice. Fluorescence microscopy did not detect GFP in intestinal sections, but flow cytometry showed 5% of crypt cells to be GFP(+). We then established cross-bred mice (Tg x Apc(Min/+)), which have a germline Apc mutation and sustained Tcf4 activation. Here, fluorescence microscopy showed GFP(+) cells at or near the base of normal-appearing crypts. In adenomas, in which Apc is inactivated, GFP(+) signal was even greater. Immunostaining for the Tcf4 target genes survivin (BIRC5) and cyclin D1 (CCND1) showed that their expression also paralleled GFP positivity. We conclude that GFP directly reports Tcf4 activation in vivo and tracks the predicted increases in Tcf4 activation that result from Apc inactivation, and that Apc mutation contributes to survivin and cyclin D1 overexpression through Tcf4 activation. Our Tcf4 mouse should be useful in studying the effects of chemopreventive agents on Wnt signaling and changes in proliferative crypt cell populations-including stem cells-during intestinal tumorigenesis.