Secondary Logo

Journal Logo

Original Articles: Hepatology and Nutrition

Intrahepatic Biliary Anomalies in a Patient With Mowat-Wilson Syndrome Uncover a Role for the Zinc Finger Homeobox Gene zfhx1b in Vertebrate Biliary Development

Cui, Shuang*; Erlichman, Jessi*; Russo, Pierre; Haber, Barbara A*; Matthews, Randolph P*

Author Information
Journal of Pediatric Gastroenterology and Nutrition: March 2011 - Volume 52 - Issue 3 - p 339-344
doi: 10.1097/MPG.0b013e3181ff2e5b


Infantile cholestatic disorders, including biliary atresia (BA), affect the biliary tree during a period of active development. The etiology of BA is unclear, but is likely a multifactorial process influenced by genetic factors that are particularly important during this period of active hepatobiliary development. In an effort to identify genes involved in hepatobiliary development associated with disease, we have examined patients with known genetic defects who also have liver anomalies consistent with BA. We previously described a patient with trifunctional protein deficiency and BA (1), and others have described patients with BA and JAGGED1 mutations (2). Such studies identify genes potentially important in the etiology of diseases such as BA, as do genome-wide association studies (3,4), and here we identify a gene potentially important in infantile biliary disease based on the appearance of cholestatic disease in a patient with a defined genetic disorder.

Patients with Mowat-Wilson syndrome (Online Mendelian Inheritance in Man #235730) demonstrate facial dysmorphism, mental retardation, and multiple congenital anomalies including Hirschsprung disease and cardiac defects. The causative gene for Mowat-Wilson syndrome is zfhx1b(5), which encodes a zinc finger homeobox transcription cofactor (ZEB2/SIP1) that interacts with SMADs (6) and regulates transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) signaling (7). TGF-β signaling appears important in biliary development because there is a complex interaction between TGF-β signaling and the activity of onecut transcription factors such as Hnf6 (8,9).

We identified a patient with Mowat-Wilson syndrome and intrahepatic biliary defects consistent with BA, and thus we hypothesized that zfhx1b may have a role in biliary development. We used the zebrafish to determine whether inhibition of zfhx1b affects biliary development. Zebrafish have been well established as a model for studying biliary development because transcription factors such as hnf6 have orthologous roles (10), and infantile biliary diseases such as Alagille syndrome (11) and arthrogryposis-renal dysfunction-cholestasis syndrome (12) have been phenocopied in zebrafish. Others have examined the role of zebrafish zfhx1b in the developing neural crest and in mediating defects in axial patterning and neural crest cell migration, similar to patients with Mowat-Wilson syndrome (13). In this study, we determined that zfhx1b was also expressed in the zebrafish liver during biliary development. Knockdown of zfhx1b using morpholino antisense oligonucleotides at lower concentrations than those used by Delalande et al (13) demonstrated impaired bile duct formation and led to a decrease in the expression of vhnf1, a transcription factor central to biliary development in mice (14,15) and zebrafish (10). These studies demonstrate ZEB2/SIP1 is essential for vertebrate biliary development and suggest an association of mutations in zfhx1b with biliary disorders.


Patient Data

Patient data were extracted from a retrospective chart review after approval was granted by the institutional review board at The Children's Hospital of Philadelphia. Histopathology slides presented were obtained during routine clinical care, as indicated, and were prepared in accordance with standard protocols.

In Situ Hybridization

There are 2 zfhx1b isoforms in zebrafish, and we chose to focus on the “b” isoform, termed zeb2b on the Sanger Centre database ( or sip1b by others (13). Antisense riboprobes for zfhx1b were synthesized essentially as described, and the remainder of the protocol was as described previously (16).

Morpholino Oligonucleotide Injection

Morpholino oligonucleotides (MOs) were designed based on sequences available from the zebrafish genome assembly and obtained from GeneTools (Philomath, OR). Morpholinos were designed to target the 5′ translational start site and the splice acceptor site for exon 7 of the zfhx1b-b gene. The sequences of the morpholinos are depicted in Table 1. For both morpholinos, 2 nL of a 1-μg/μL solution was injected, which is 10% to 25% of the amount injected in the previous study (13). Injection with either zfhx1b morpholino produced an identical phenotype. Negative controls were either standard or random-control MO, also purchased from GeneTools, giving identical results indistinguishable from vehicle control. Confirmation of knockdown by the spliceblocking morpholino is depicted in Supplemental Figure 1 (

Morpholino sequences

PED-6 Treatment, Immunostaining, and Electron Microscopy

Control and morpholino-injected larvae were soaked 2 hours in 0.1 μg/mL PED-6 at 5 days postfertilization (dpf), similar to previous studies. Larvae were sorted based on gallbladder intensity. Whole-mount keratin immunostaining of wild-type and MO-injected larvae was performed as previously described (10,11). For electron microscopy, larvae at 5 dpf were fixed, prepared, and examined as they were previously (17).

Quantitative Real-time PCR

Template complementary DNA was synthesized from RNA obtained from 5-dpf control and zfhx1b MO-injected whole larvae derived from 3 independent experiments as previously described (10). Primers for vhnf1 were previously described, and analysis was performed as described previously (10). vhnf1 expression was normalized to hprt, and was also normalized to cp (ceruloplasmin), with equivalent results.


Case Report

The patient of interest was the 3350-g product of a 37 4/7-week pregnancy. Shortly after birth, he was transferred to the neonatal intensive care unit at The Children's Hospital of Philadelphia for workup of multiple congenital anomalies, and he was noted to have the anomalies listed in Table 2. On day of life 3, he had an end-ileostomy performed and was found to have malrotation. Given the constellation of features, in particular Hirschsprung disease and cardiac defects, there was suspicion for Mowat-Wilson syndrome, which was confirmed by identification of a hemizygous deletion of the entire zfhx1b gene. The patient's cytogenetics had appeared normal, suggesting that this represented a microdeletion.

Clinical features of case patient

He was described as having been jaundiced at birth, and by day of life 34 he had a total bilirubin of 4.8 mg/dL with a conjugated fraction of 3.0 mg/dL. He was started on ursodeoxycholic acid, which initially improved his cholestasis modestly, but due to his persistent jaundice he was evaluated for obstructive jaundice. Genetic testing for Alagille syndrome was performed because the patient was noted to have posterior embryotoxon, but no mutation in JAGGED1 was uncovered. An ultrasound demonstrated a gallbladder, no extra- or intrahepatic biliary dilation, and hepatosplenomegaly. A DISIDA scan was performed and no tracer was excreted into the intestine by 24 hours. A subsequent liver biopsy demonstrated features consistent with BA, with bile plugging and duct proliferation (Fig. 1).

Liver histopathology of patient with Mowat-Wilson syndrome and cholestasis. A, Hematoxylin and eosin staining of percutaneous liver biopsy demonstrated cellular evidence of cholestasis (arrowheads) and bile duct proliferation (arrows), consistent with biliary atresia or other obstructive causes of cholestasis. B, Periodic acid-Schiff with diastase staining showing the same features as in (A), but also demonstrating portal tract expansion and fibrosis. There is some hepatocyte ballooning notable in both panels. C, Staining for cytokeratin-7 shows bile duct proliferation.

Due to the patient's deteriorating cardiorespiratory status, it was believed that intraoperative cholangiogram and possible Kasai portoenterostomy would be too risky. He developed hematemesis and his clinical course continued to worsen, and the patient expired at 4 months of age of respiratory failure. No autopsy was performed. Although a diagnosis of BA could not be confirmed, the intrahepatic biliary defects in association with a defined genetic disorder led us to hypothesize that zfhx1b may be involved in development of the intrahepatic biliary tree. Genes involved in biliary development may be important in numerous infantile hepatobiliary disorders, including BA.

Developmental Expression of zfhx1b in Zebrafish Liver

To have a role in biliary development, we would expect zfhx1b to be expressed in the developing liver during biliary growth and remodeling. zfhx1b expression has been reported in the developing human and mouse brain, as well as in mouse liver at E13.5 and E16.5 (18), during mouse biliary growth and remodeling (14). Others have examined expression of zfhx1b in the developing zebrafish nervous system, with results consistent with mammalian expression studies (13). This group reported 2 zfhx1b isoforms in zebrafish; we focused on the “b” isoform (termed sip1b or zeb2b) because it has higher homology to human zfhx1b (Supplemental Figure 2, We examined zebrafish zfhx1b gene expression at 3 dpf, at which point the bile ducts are beginning to form (10,11). Figure 2 demonstrates that zebrafish zfhx1b was expressed in the developing liver at 3 dpf. As evidenced from Figure 2A, there was also neural expression, consistent with previous results, although our studies demonstrate less detail because of darker staining that also allowed us to observe liver expression that was only faintly visible in the previous study. These results are consistent with a role of zfhx1b in vertebrate biliary development.

Liver expression of zfhx1b in zebrafish larvae. A, In situ hybridization of zfhx1b in a 3-day postfertilization (dpf) zebrafish larva, demonstrating liver expression (white arrow). There is also expression in the developing brain (black arrowhead) and intestine (white arrowhead). Original magnification ×100. B, Higher magnification (original ×400) of similar larva, showing liver expression (white arrow). C, No-probe control of similar stage larva showing lack of signal after prolonged development similar to A and B.

Knockdown of zfhx1b Leads to Defects in Biliary Development

To assay the function of zfhx1b in zebrafish biliary development, we performed gene knockdown experiments using antisense MOs. Morpholinos were designed to target the 5′ translational start site or the splice acceptor site for exon 7 of the zfhx1b gene. Previous studies used similar experiments to determine that knockdown of zfhx1b produces a neural phenotype consistent with defects seen in Mowat-Wilson syndrome (13). Knockdown of zfhx1b at lower concentrations did not affect the overall appearance of larvae at 5 dpf (Fig. 3). However, gallbladder fluorescence following the ingestion of the quenched fluorescent lipid, PED-6 (19), was reduced in zfhx1b MO-injected larvae (Fig. 3). PED-6 functions as a marker of lipid absorption and biliary function; abnormal gallbladder uptake of PED-6 is consistent with defects in intrahepatic biliary development. These results suggest that lack of zfhx1b expression may lead to defects in intrahepatic biliary development. Figure 3 also demonstrates that knockdown of zfhx1b led to abnormal intrahepatic biliary anatomy. Duct length appears reduced in the morphant, and there were fewer terminal ductules, consistent with an effect on biliary development. Thus, zfhx1b appears to have a role in vertebrate intrahepatic biliary development.

Biliary defects in zebrafish larvae injected with morpholino antisense oligonucleotides directed against zfhx1b. A and B, Live views from the left side of 5-dpf larvae demonstrating similarity in overall appearance between control (A, cont) and zfhx1b morphant (B). C and D, Right-sided views of 5-dpf larvae incubated with the fluorescent lipid PED-6, demonstrating intense uptake into the gallbladder in control (C), and decreased uptake into the gallbladder of the zfhx1b morphant (D). Gallbladders are noted with white arrows. i, intestine. E and F, Confocal projections of cytokeratin immunostaining of whole-mount livers from control (E) and zfhx1b morphants (F). Ducts in (F) are sparser and shorter compared to the control (E), as more clearly seen in the schematics (G, H). Panels shown are representative of 10 larvae examined with similar appearance.

vhnf1 Is Downregulated in zfhx1b Morphants

We were intrigued that the zfhx1b morphants appeared similar to hnf6 morphants (10), suggesting possible mechanistic overlap. As mentioned above, hnf6 and its downstream target vhnf1 play important roles in biliary development in mammals and zebrafish. Thus, we examined the expression level of hnf6 and vhnf1 in zfhx1b morphants. Quantitative PCR studies demonstrate that expression of vhnf1 normalized to hprt was decreased 2-fold in zfhx1b morphants (Fig. 4; similar results were obtained normalizing to liver-specific cp, not shown). In situ hybridization studies also showed decreased vhnf1 expression in zfhx1b morphant livers (Fig. 4). Interestingly, though, the expression level of hnf6 was unchanged in zfhx1b morphants (data not shown). These data support a role for zfhx1b in biliary development, and suggest that zeb2/sip1 may act either directly or indirectly in modulating vhnf1 expression.

Decreased expression of vhnf1 in zfhx1b morphants. A, Real-time quantitative PCR from cDNA derived from RNA isolated from whole control (cont) or zfhx1b morphant larvae. Error bars represent standard deviation from 4 separate larvae in control and morphant conditions, P < 0.03. B–E, In situ hybridizations of 4-dpf control (B, D) and zfhx1b (C, E) morphant larvae, probed for vhnf1 (B, C) or ceruloplasmin (cp; D, E). Liver expression is noted by the white circles, whereas intestinal staining is noted by the white arrow and pronephric duct staining by the white arrowheads.


Mowat-Wilson syndrome involves neurological defects in both the central nervous system and the gut, and is caused by the mutation of zfhx1b. We observed biliary defects suggestive of BA in a patient with confirmed Mowat-Wilson syndrome. This suggested to us that zfhx1b could be involved in biliary development or in mediating biliary damage. Here, we present data using zebrafish that support a role for zfhx1b in biliary development. In addition, we present evidence demonstrating that zfhx1b knockdown results in decreased expression of liver vhnf1, suggesting that zfhx1b may affect biliary development by modulating vhnf1.

Hepatobiliary Disease in Mowat-Wilson Syndrome

In a recent review of patients with Mowat-Wilson syndrome, none of the 171 patients reported had hepatobiliary disease (20). This suggests that our patient may have had some other risk factor for hepatobiliary disease, such as an additional mutation or exposure to an infectious agent or environmental toxin. A deletion of zfhx1b, such as that seen in our patient and in 19% of patients with Mowat-Wilson syndrome (20), suggests the possibility of deletion of a neighboring gene, although such larger deletions would be expected to have been detected by the cytogenetic analysis. The region of chromosome 2 surrounding zfhx1b is relatively gene poor, with only 2 genes, gtdc1 and arhgap15, within 1.5 Mb on both sides ( These genes, which encode a glycosyl transferase and ρ GTPase-activating protein, have not been reported to be involved in disease formation; in fact, a recent report documents a Mowat-Wilson patient with typical clinical features with deletion of these genes as well as zfhx1b(21). Thus, deletion of an additional neighboring gene seems unlikely, and no clear genotype–phenotype correlations can be drawn.

Infantile cholestatic conditions such as BA are relatively uncommon, and thus simply based on the number of patients we cannot yet determine whether patients with Mowat-Wilson syndrome are at greater risk. Given our results demonstrating a role for zfhx1b in vertebrate hepatobiliary development, however, a greater risk for infantile hepatobiliary disease in Mowat-Wilson syndrome seems plausible.

zeb2/sip1 May Act on vhnf1 to Mediate Biliary Developmental Processes

zeb2/sip1, encoded by the zfhx1b gene, is a transcriptional cofactor that modulates SMAD activity. This protein binds directly to DNA and inhibits interactions with other cofactors of SMAD5, effectively inhibiting SMAD-dependent gene expression (6,7). TGF and BMP signaling operates upstream of SMAD-mediated gene expression, and zeb2/sip1 acts as a transcriptional inhibitor of TGF and BMP signaling. Both Hnf1b (vhnf1) expression and TGF/BMP signaling are activated in biliary differentiation, including increased expression of Smad5(22), supporting a potential importance of zeb2/sip1 in this process as well.

Importantly, the effect of zeb2/sip1 on vhnf1 may not involve TGF, but may result from direct interaction of zeb2/sip1 on the vhnf1 promoter or indirectly through other signaling pathways. Our data suggest that inhibition of zeb2/sip1 leads to decreased vhnf1 expression and to defects in biliary development; future studies could address the importance of this pathway in mammalian systems.

Potential Importance of zeb2/sip1 Dysfunction in Patients With Biliary Atresia

We presented an association in a patient with a known mutation in zfhx1b and the development of intrahepatic findings consistent with BA. Demonstration of biliary anomalies in a model system after zfhx1b knockdown suggests that the zfhx1b mutation in our patient may have at least been contributory to his biliary phenotype. Although BA is clearly not a simple genetic disorder, it is likely that the trigger—infectious, environmental toxin, or some other insult—acts on a genetically susceptible individual. The exclusive occurrence of BA in infancy, during biliary growth and remodeling, suggests that developmental factors may play a key role in determining this genetic susceptibility. Broad genome-wide association studies may uncover other susceptibility genes, as will continued identification of patients with known syndromes and BA and other biliary anomalies. A potential importance of zeb2/sip1 in biliary development and BA in particular is appealing because of the importance of TGF-β signaling in both biliary development and liver fibrosis. As stated above, TGF signaling is activated during in vitro biliary differentiation (22), and is part of a complex regulatory loop with Hnf6 and related transcription factors (8,9), which also regulate Hnf1 (vHnf1) expression (14). TGF signaling plays a well-known role in mediating fibrosis, as well as in epithelial-to-mesenchymal transition in the liver (23).

Importantly, overexpression of zeb2/sip1 also leads to epithelial-to-mesenchymal transition (24), whereas inhibition of the related zeb1 leads to mesenchymal-epithelial transition (25). We demonstrate here that inhibition of zfhx1b in zebrafish leads to developmental biliary defects. Thus, genetic polymorphisms in zfhx1b could alter zeb2/sip1 activity and therefore affect biliary development and hepatic fibrogenesis, making the patient more susceptible to biliary anomalies and a clinical appearance consistent with BA.

Our studies here identify a gene with a possible role in biliary development and disease, identified as a candidate because of the occurrence of hepatobiliary disease in a patient with a defined genetic disorder. We were able to perform these studies because use of a facile model organism such as zebrafish allowed relatively rapid examination of the potential importance of this candidate gene in a developmental process that was easily assayed. These studies underscore the use of zebrafish in determining the importance of genes in development and disease.


The authors thank Steven EauClaire for expert technical assistance.


1. Matthews RP, Russo P, Berry GT, et al. Biliary atresia associated with a fatty acid oxidation defect. J Pediatr Gastroenterol Nutr 2002; 35:624–628.
2. Kohsaka T, Yuan ZR, Guo SX, et al. The significance of human jagged 1 mutations detected in severe cases of extrahepatic biliary atresia. Hepatology 2002; 36(4 Pt 1):904–912.
3. Garcia-Barcelo MM, Yeung MY, Miao XP, et al. Genome-wide association study identifies a susceptibility locus for biliary atresia on 10q24.2. Hum Mol Genet 2010;19:2917–25.
4. Leyva-Vega M, Gerfen J, Thiel BD, et al. Genomic alterations in biliary atresia suggest region of potential disease susceptibility in 2q37.3. Am J Med Genet A 2010; 152A:886–895.
5. Zweier C, Albrecht B, Mitulla B, et al. “Mowat-Wilson” syndrome with and without Hirschsprung disease is a distinct, recognizable multiple congenital anomalies-mental retardation syndrome caused by mutations in the zinc finger homeo box 1B gene. Am J Med Genet 2002; 108:177–181.
6. Postigo AA, Depp JL, Taylor JJ, et al. Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. EMBO J 2003; 22:2453–2462.
7. Postigo AA. Opposing functions of ZEB proteins in the regulation of the TGFbeta/BMP signaling pathway. EMBO J 2003; 22:2443–2452.
8. Clotman F, Jacquemin P, Plumb-Rudewiez N, et al. Control of liver cell fate decision by a gradient of TGF beta signaling modulated by Onecut transcription factors. Genes Dev 2005; 19:1849–1854.
9. Plumb-Rudewiez N, Clotman F, Strick-Marchand H, et al. Transcription factor HNF- 6/OC-1 inhibits the stimulation of the HNF-3alpha/Foxa1 gene by TGF-beta in mouse liver. Hepatology 2004; 40:1266–1274.
10. Matthews RP, Lorent K, Russo P, et al. The zebrafish onecut gene hnf-6 functions in an evolutionarily conserved genetic pathway that regulates vertebrate biliary development. Dev Biol 2004; 274:245–259.
11. Lorent K, Yeo SY, Oda T, et al. Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy. Development 2004; 131:5753–5766.
12. Matthews RP, Plumb-Rudewiez N, Lorent K, et al. Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6. Development 2005; 132:5295–5306.
13. Delalande JM, Guyote ME, Smith CM, et al. Zebrafish sip1a and sip1b are essential for normal axial and neural patterning. Dev Dyn 2008; 237:1060–1069.
14. Clotman F, Lannoy VJ, Reber M, et al. The onecut transcription factor HNF6 is required for normal development of the biliary tract. Development 2002; 129:1819–1828.
15. Coffinier C, Gresh L, Fiette L, et al. Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta. Development 2002; 129:1829–1838.
16. Wallace KN, Pack M. Unique and conserved aspects of gut development in zebrafish. Dev Biol 2003; 255:12–29.
17. Matthews RP, Lorent K, Manoral-Mobias R, et al. TNF{alpha}-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafish s-adenosylhomocysteine hydrolase. Development 2009; 136:865–875.
18. Bassez G, Camand OJ, Cacheux V, et al. Pleiotropic and diverse expression of ZFHX1B gene transcripts during mouse and human development supports the various clinical manifestations of the “Mowat-Wilson” syndrome. Neurobiol Dis 2004; 15:240–250.
19. Farber SA, Pack M, Ho SY, et al. Genetic analysis of digestive physiology using fluorescent phospholipid reporters. Science 2001; 292:1385–1388.
20. Garavelli L, Mainardi PC. Mowat-Wilson syndrome. Orphanet J Rare Dis 2007; 2:42.
21. Smigiel R, Szafranska A, Czyzewska M, et al. Severe clinical course of Hirschsprung disease in a Mowat-Wilson syndrome patient. J Appl Genet 2010; 51:111–113.
22. Ader T, Norel R, Levoci L, et al. Transcriptional profiling implicates TGFbeta/BMP and Notch signaling pathways in ductular differentiation of fetal murine hepatoblasts. Mech Dev 2006; 123:177–194.
23. Dooley S, Hamzavi J, Ciuclan L, et al. Hepatocyte-specific Smad7 expression attenuates TGF-beta-mediated fibrogenesis and protects against liver damage. Gastroenterology 2008; 135:642–659.
24. Ohashi S, Natsuizaka M, Wong GS, et al. Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-tomesenchymal transition through ZEB transcription factors. Cancer Res 2010; 70:4174–4184.
25. Liu Y, El-Naggar S, Darling DS, et al. Zeb1 links epithelial-mesenchymal transition and cellular senescence. Development 2008; 135:579–588.

biliary atresia; cholestatic liver disease; Hirschsprung disease; liver development; zebrafish; zfhx1b

Supplemental Digital Content

Copyright 2011 by ESPGHAN and NASPGHAN