p53-Mediated Biliary Defects Caused by Knockdown of cirh1a, the Zebrafish Homolog of the Gene Responsible for North American Indian Childhood Cirrhosis

the Zebrafish Homolog of the Gene Responsible for North American Indian Childhood Cirrhosis. PLoS ONE 8(10): e77670. doi:10.1371/journal.pone.0077670 p53-Mediated Biliary Defects Caused by Knockdown of cirh1a, the Zebrafish Homolog of the Gene Responsible for North American Indian Childhood Cirrhosis Benjamin J. Wilkins 0 Kristin Lorent 0 Randolph P. Matthews 0 Michael Pack 0 Steven R Ellis, University of Louisville, United States of America 0 1 Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America, 2 Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America, 3 Department of Pediatrics, Children's Hospital of Philadelphia , Philadelphia, Pennsylvania , United States of America North American Indian Childhood Cirrhosis (NAIC) is a rare, autosomal recessive, progressive cholestatic disease of infancy affecting the Cree-Ojibway first Nations of Quebec. All NAIC patients are homozygous for a missense mutation (R565W) in CIRH1A, the human homolog of the yeast nucleolar protein Utp4. Utp4 is part of the t-Utp subcomplex of the small subunit (SSU) processome, a ribonucleoprotein complex required for ribosomal RNA processing and small subunit assembly. NAIC has thus been proposed to be a primary ribosomal disorder (ribosomopathy); however, investigation of the pathophysiologic mechanism of this disease has been hindered by lack of an animal model. Here, using a morpholino oligonucleotide (MO)-based loss-of-function strategy, we have generated a model of NAIC in the zebrafish, Danio rerio. Zebrafish Cirhin shows substantial homology to the human homolog, and cirh1a mRNA is expressed in developing hepatocytes and biliary epithelial cells. Injection of two independent MOs directed against cirh1a at the one-cell stage causes defects in canalicular and biliary morphology in 5 dpf larvae. In addition, 5 dpf Cirhin-deficient larvae have dose-dependent defects in hepatobiliary function, as assayed by the metabolism of an ingested fluorescent lipid reporter. Previous yeast and in vitro studies have shown that defects in ribosome biogenesis cause stabilization and nuclear accumulation of p53, which in turn causes p53mediated cell cycle arrest and/or apoptosis. Thus, the nucleolus appears to function as a cellular stress sensor in some cell types. In accordance with this hypothesis, transcriptional targets of p53 are upregulated in Cirhin-deficient zebrafish embryos, and defects in biliary function seen in Cirhin-deficient larvae are completely abrogated by mutation of tp53. Our data provide the first in vivo evidence of a role for Cirhin in biliary development, and support the hypothesis that congenital defects affecting ribosome biogenesis can activate a cellular stress response mediated by p53. - Funding: The study was supported by National Institutes of Health grant R01DK09211 (M.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Infantile cholestasis and/or jaundice results from disorders that disrupt hepatobiliary development, inborn errors of metabolism, toxin exposure and infectious or immune-mediated diseases [1]. While the most common cause of infantile cholestasis, extrahepatic biliary atresia, has no definitive etiology, several less common heritable cholestatic disorders are caused by single gene defects [2]. The genes affected in these disorders, which collectively have been referred to as cholangiopathies, encode signaling molecules necessary for bile duct development, such as Alagille syndrome [3,4], or proteins necessary for the secretion or modification of bile by hepatocytes or biliary epithelial cells, as seen in progressive familial intrahepatic cholestasis [PFIC 1-3], cystic fibrosis, and arthrogryposis-renal dysfunction-cholestasis syndrome. In addition to aiding in the diagnosis of these disorders, identification of these disease genes has led to greater understanding of normal mechanisms that direct biliary development and hepatobiliary function in neonates. North American Indian Childhood Cirrhosis (NAIC, OMIM 604901) is a rare, autosomal recessive cholestatic disease of infancy that affects the Cree-Ojibway First Nations in Quebec [5,6]. NAIC presents as neonatal jaundice that resolves spontaneously by age 1 year, but affected individuals have persistent direct hyperbilirubinemia that almost uniformly progresses to portal hypertension and biliary cirrhosis. Liver biopsy at the time of diagnosis typically shows bile duct proliferation with luminal bile plugs and portal fibrosis, findings that are nearly identical to extrahepatic biliary atresia (BA) and consistent with biliary epithelial cell injury. Like patients with BA, nearly all reported NAIC patients develop biliary fibrosis with secondary portal hypertension and liver dysfunction. In a case series reporting 30 patients, 47% had died and 23% had undergone liver transplantation in the first two decades of life; all but one of the remaining living patients had compensated cirrhosis, with the oldest of these patients aged 26 years [6]. All known NAIC patients are homozygous for an identical missense mutation in the CIRH1A gene located on chromosome 16 (16q22), likely due to founder effect in a relatively small and historically isolated community [7]. The encoded 686 amino-acid protein, CIRHIN, contains multiple WD40 repeats, thus suggesting it could act as a scaffold within the ribosomal SSU processome (discussed below). The CIRH1A NAIC mutation encodes a single amino acid substitution, Arg565Trp (R565W), located C-terminal to the WD40 repeats in a novel domain with no known homologues. Unique among proteins mutated in infantile cholangiopathies, CIRHIN has been localized to the nucleolus of human cells [8]. The yeast homolog of CIRHIN, Utp4, is a member of the small subunit (SSU) processome, a ribonucleoprotein complex that is required for processing of pre-ribosomal RNA and assembly of the mature small subunit [911]. Other studies in human cells have suggested that CIRHIN is a trans-acting factor at NFkappa-B-responsive enhancers [12]. Mutations in ribosomal proteins or proteins required for ribosome biogenesis underlie human diseases that collectively have been termed ribosomopathies [13,14]. The bestcharacterized of these, Diamond-Blackfan anemia (DBA), is a rare disorder that causes short stature, red cell aplasia (presenting as congenital macrocytic anemia) and an increased risk of malignancy later in life [15]. DBA and the other proposed ribosomopathies (Shwachman-Diamond syndrome, 5qsyndrome, dyskeratosis congenita, and cartilage-hair hypoplasia) share hematologic cytopenias and cancer predisposition among other disparate findings. One exception is Treacher-Collins syndrom (...truncated)