The intrahepatic biliary system is a network of tubes within the liver that transports bile away from hepatocytes. Formation of this system requires specification of BECs and subsequent remodeling of the BECs to form a functional three-dimensional tubular network. Defective formation of the network or its canalicular system disrupts bile flow and causes cholestatic disease. The mechanisms that underlie cholestatic disease due to mutations in genes encoding tight junction proteins have not been identified (Carlton et al., 2004
). Here, we present evidence that a tight junction protein, Cldn15lb-a, plays a role in the remodeling of the biliary network. In zebrafish cldn15lbfh290
mutants, BECs do not remodel properly and they remain clustered or in close proximity to each other, resulting in a malformed network with larger tubules. In addition, hepatocytes are not properly polarized and bile canaliculus formation is disrupted. This and previous studies, taken together, identify components of tight junctions to be important to establish a functional biliary network for effective bile flow (Baala et al., 2002
; Hadj-Rabia et al., 2004
; Son et al., 2009
; Yeh et al., 2010
is a novel claudin gene that is alternatively spliced to generate two isoforms expressed in a tissue- and cell-specific manner. The two isoforms differ in their first extracellular loop whose amino acid composition determines barrier properties (Van Itallie and Anderson, 2006
). As such, the two variants may convey different barrier properties and functions. Even though Cldn15lb is phylogenetically similar to mammalian Cldn15, based on amino acid sequence, it is also closely related to mammalian Cldn10. Both Cldn15lb-a and human Cldn10 are expressed in hepatocytes and BECs (Nemeth et al., 2009
). Mouse Cldn10 is weakly expressed in tight junctions of hepatocytes (Van Itallie et al., 2006
). On the other hand, expression of Cldn15lb-b in the gut is more similar to that of mammalian Cldn15. Interestingly, mammalian Cldn10
has also been described to be alternatively spliced in the first exon similarly to cldn15lb
(Van Itallie et al., 2006
; Gunzel et al., 2009
). Mammalian Cldn10
has six transcript variants that are differentially expressed in various tissues and exhibit different functions (Gunzel et al., 2009
). Three of these six transcripts also lack the fourth exon and the resulting proteins localize intracellularly suggesting that they may play a novel role within the cell (Gunzel et al., 2009
transcripts lacking the fourth exon are not present in the zebrafish EST database at this time.
Expression of this novel gene in cultured MDCK cells showed that it is expressed in tight junctions. However, we could not identify defects in any of the three types of tight junctions in the liver based on immunostaining. Furthermore, tight junctions between hepatocytes observed in electron micrographs also appear to be normal. Unfortunately, we were not able to identify tight junctions between BECs or between BECs and hepatocytes in the electron micrographs. Such analyses may reveal tight junction abnormalities that cannot be detected by immunostaining. These tight junctions may indeed have functional defects that are too subtle or minor to detect but could account for the observed delay in PED-6 processing in the mutants. Alternatively, it is possible that tight junctions are not significantly affected in the mutants due to redundancy within the Claudin family. Cldn-1, -2, -3, -4, -7, -8, and -10 are all expressed in the mammalian liver (Nemeth et al., 2009
), and the other isoform of Cldn15lb is expressed at low levels in the liver starting at 72 hpf. Furthermore, Cldn2 was shown to be upregulated in patients with Claudin-1
mutations (Hadj-Rabia et al., 2004
). Thus, other members of the Claudin family could compensate for the lack of Cldn15lb-a function.
Since we observed GFP-tagged Cldn15lb in the lateral and basal membranes of MDCK cells, Cldn15lb might have yet other functions that could account for the mutant phenotypes. Our immunostaining in zebrafish also localizes Cldn15lb to the entire membrane of hepatocytes and BECs. Cldn15lb is initially expressed in both hepatocytes and BECs and is subsequently restricted to BECs. This expression profile is reminiscent of that of the adhesion molecules Cadherin and Alcam (Sakaguchi et al., 2008
; Curado et al., 2010
; data not shown). Since Claudins were first identified as calcium-independent adhesion molecules (Kubota et al., 1999
) and members of the JAM family of tight junction proteins have been shown to co-localize with ZO-1 at adherens-like cell-cell contacts in fibroblasts (Morris et al., 2006
), it is possible that Cldn15lb-a functions as an adhesion molecule during liver development.
It is also possible that Cldn15lb functions to maintain hepatocyte polarity and that the absence of Cldn15lb-a function gives rise to defective bile canaliculi. Similar to what is observed in cldn15lbfh290
mutant larvae, bile canaliculi in Cldn2
-deficient mice and Cldn2
-deficient hepatic cell lines display abnormal morphology (Son et al., 2009
; Yeh et al., 2010
). During the development of bile canaliculi, apical pumps and transporters involved in bile secretion, such as BSEP and MRP2, are delivered to each canaliculus via a process called apical membrane biogenesis. In hepatocytes where Claudin expression is disrupted, the apical domain is not well-defined, and thus, targeted delivery of apical proteins and apical membrane biogenesis is likely to be affected. In support of this hypothesis, disruption of Rab11a-mediated protein trafficking causes bile canaliculi malformation (Wakabayashi et al., 2005
; Cullinane et al., 2010a
). In cldn15lbfh290
mutants where hepatocyte polarity is disrupted, trafficking of BSEP and other apical proteins is also likely to be affected.
Polarization of hepatocytes has been associated with biliary duct development and biliary physiology (Sakaguchi et al., 2008
; Cullinane et al., 2010a
). Patients with arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome have mutations in genes encoding proteins involved in membrane trafficking used to establish hepatocyte polarity. Disruption of these proteins in mouse and zebrafish results in reduced levels of hepatic E-cadherin, abnormal duct development, and disrupted bile flow (Matthews et al., 2005
; Cullinane et al., 2010a
). Our study provides additional support for the hypothesis that hepatocyte polarity and biliary duct remodeling are linked, and raise the possibility that Claudins and other tight junction proteins influence this remodeling process. First, the remodeling defect in cldn15lbfh290
mutants appears to occur between 80 and 100 hpf when Cldn15lb-a is still expressed in hepatocytes. Second, apical lumen remodeling was shown to be defective in hepatocytes with defective adherens junctions (Theard et al., 2007
). Since Claudin-containing tight junctions in hepatocytes are important to define their apical domain, one hypothesis is that BECs receive signals from a polarized epithelium to direct the remodeling process. In the absence of a polarized hepatic epithelium, BECs would not receive such signals and thus, the remodeling of the biliary network would be affected.
Although we were not able to fully characterize the hepatic lesions we observed in adult cldn15lbfh290
mutants, it is interesting to speculate on the etiology of these lesions, particularly given the hepatobiliary abnormalities we observed during development. The central necrosis and pigment deposition raise the possibility that the lesions may represent granulomas, which can be caused by infections, including mycobacterial or fungal infections, or non-infectious entities including stress-induced sarcoidosis, primary biliary cirrhosis, or foreign body reaction (Lamps, 2008
; Swaim et al., 2006
). In this case, it is possible that loss of Cldn15lb-a results in damage to the hepatobiliary system that renders the mutants more susceptible to infection or other cause of granuloma formation. Alternatively, it is possible that the epithelioid cells at the periphery of at least some of the lesions represent squamous metaplasia of the biliary epithelium. In this case, the lesions may represent a response to abnormal bile flow and/or bile accumulation, which could be caused by leakage of bile through an abnormal biliary epithelium, by accumulation of bile due to partial obstruction of an abnormally-developed biliary network, or by some other biliary abnormality present in the cldn15lbfh290
mutants. Regardless of the identity of these lesions, it is important to note that lesions in the mutant livers are significantly larger than those found in only one of the wildtype livers. Clearly, loss of Cldn15lb-a is associated with hepatic abnormalities in adult fish which may be due to a chronic, possibly subtle, defect with effects that accumulate over time resulting in more abundant and larger lesions. Additional experiments will be necessary to determine whether the adult phenotype is a direct or indirect result of the developmental defects, and how the loss of Cldn15lb-a might cause the exacerbated adult phenotype.
Targeted deletion of Hnf1b
has been shown to cause a loss of organization of biliary ductal structures (Coffinier et al., 2002
), and our microarray analysis suggests that Hnf1b may do so in part by regulating the expression of cldn15lb
to mediate biliary remodeling during morphogenesis. Our study of the liver defects in the cldn15lbfh290
mutants raises the intriguing possibility that cholestasis in patients with mutations in genes encoding tight junction proteins may arise in response to abnormal bile canaliculi and/or biliary duct architecture due to defects in remodeling. Therefore, understanding the cellular mechanisms by which Claudins and other tight junction proteins regulate the development of the intrahepatic biliary network should also bring novel insights into our understanding of cholestasis.