The zebrafish as a model to study polycystic liver disease

Abstract

In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2(WS25/-) mouse), we have studied hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including sec63, prkcsh, and pkd1a. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide [1 μM] or vitamin K3 [100 μM] and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against sec63, prkcsh, and pkd1a eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis.

FIG. 1.

Morpholinos against sec63, prkcsh, and pkd1a-eliminated protein expression. The figure shows a western blot of zebrafish embryos (200 μL sample buffer with 150 embryos per lane) on a 10% SDS gel (WT, wild-type embryos injected with Danio buffer; Scr, scrambled control; MO, morpholinos). A protein band was visible at the appropriate molecular weight for sec63, (77,000 kDa), prkcsh (90,000 kDa), and pkd1a (115,000 kDa).

FIG. 2.

Morpholinos induced phenotypic body changes of body curvature and kinked tail. Light microscopic images of zebrafish larvae (4 dpf) demonstrate that depletion of proteins associated with cystic liver disease led to phenotypic changes, including abnormal body curvature, while mismatched morpholinos showed no effect (magnification×30).

FIG. 3.

Morpholinos caused an increase in the cystic area of the liver. Each of the morpholinos led to a statistically significant increase in the cystic area compared with WT (magnification×60).

FIG. 4.

Morpholinos induced hepatic cystogenesis. Scanning electron micrographs show the presence of liver cysts (*) in sec63, prkcsh, and pkd1a in zebrafish larvae (4 dpf) compared with control. Left panels: WT (magnification×400; bar=50 μm); sec63 (magnification×1000; bar=25 μm); prkcsh (magnification×200; bar=100 μm); pkd1a (magnification×400; bar=50 μm). Right panels: magnification×10,000; bar=2 μm.

FIG. 5.

Exposure of embryos to pasireotide inhibited hepatic cystogenesis. Treatment of morpholino-injected embryos revealed that the liver cysts and cystic areas were significantly reduced in response to pasireotide but not vitamin K3.

FIG. 6.

Treatment with 4-phenylbutyrate (4-PBA) reduced endoplasmic reticulum (ER) stress. Morpholino-injected zebrafish larvae (4 dpf) displayed swollen and convoluted shaped ER (*) in sec63 and prkcsh but fewer in pkd1a compared with WT. After treatment with 4-PBA (0.05 mM) the ER returned to the shape and size observed in WT. Left panels: magnification×40,000; bar=0.5 μm. Right panels: magnification×100,000; bar=200 nm.