Cirrhosis remains one of the central challenges in clinical hepatology. It is the common final outcome of a variety of diseases, including chronic alcohol intake, steatohepatitis, viral hepatitides, and hereditary metabolic conditions. Decompensated cirrhosis can be life-threatening—and this clinical scenario is incurable, except by liver transplantation. A cardinal feature in the fibrogenic response to any noxious stimulus is the deposition of extracellular matrix by activated hepatic stellate cells (HSCs). Initially described in 1876 by von Kupffer as liver Sternzellen (“star-shaped cells”) and then by Ito as vitamin-A storing cells, HSCs have since been well characterized, and much is known about their molecular and cellular biology.1 However, the exact developmental origin of HSCs remains unknown, and until recently we have lacked the capabilities to observe stellate cell activation in vivo. Moreover, we have been unable to discover novel chemical and genetic factors that regulate stellate cell development and activation. In the current issue of HEPATOLOGY, Yin et al.2 describe a novel cell population in the zebrafish liver that exhibits all the hallmarks of HSCs in mammals, from their morphology, to their capacity to store fat and vitamin A, to their expression profile and activation in response to injury. How can this newly discovered cell type in the zebrafish help us understand HSC regulation and improve patient outcomes?
Over the past two decades, zebrafish have been established as an excellent model to study early development and organogenesis. The embryos are transparent, allowing for direct visualization of in vivo processes, and they develop rapidly, so that they harbor differentiated hepatocytes by 3 days of life.3, 4 Zebrafish are equally amenable to forward genetic and chemical genetic screening approaches. Despite several hundreds of millions of years of divergent evolution, the zebrafish gastrointestinal tract and liver are remarkably similar to those of mammals, both in their cellular organization and in the molecular signals governing organ development, growth, regeneration, and malignant transformation.5, 6 Using transgenic zebrafish lines, as well as by in situ hybridization and immunocytological methods, hepatocytes, biliary epithelial cells and endothelial cells can be identified with specific markers.3, 7
Yin et al. describe the discovery of HSCs as a novel cell type in the zebrafish liver. Their study made use of recently generated transgenic reporter fish, which highlight the expression of the bHLH transcription factor hand2 (heart and neural crest derivates expressed transcript 2). This gene, expressed early in the lateral plate mesoderm, had been described previously by the authors as an essential factor for gut looping and laterality during early endoderm development.8 The authors demonstrate the morphological similarity to mammalian HSCs, with a star-shaped appearance and cellular processes that lie in close proximity to endothelial cells, expressing desmin and glial fibrillary acidic protein. The authors further elucidate the developmental origin of this cell type in the mesoderm, which had been shown via lineage-tracing experiments to be the source of HSCs in mouse liver.9 More importantly, Yin et al. demonstrate in the zebrafish a conserved HSC response to alcohol injury: HSC number and gene expression is enhanced after alcohol exposure, consistent with findings in mammalian organisms. In addition, the authors demonstrate that alterations in HSC formation and activation in response to modulation of signaling pathways by chemical exposure can be documented in vivo.
Several aspects of the Yin et al. study highlight the classic strengths of the zebrafish as a developmental model. This elegant study make use of fluorescent transgenic reporter fish and genetic mutants or targeted knockdowns, which allow the direct in vivo assessment of the impact of genetic modulations within the context of the entire developing embryo. Hand2-expressing cells invade the liver to take up residence next to endothelial cells. However, despite their physical proximity, endothelial cells are not required for HSC development; in other words, absence of all endothelial cells in the zebrafish mutant cloche led to a smaller liver bud, but the number of hand2-positive cells in these mutant livers remained the same. Although not required for HSC development, endothelial cells appeared to play an important role in the proper positioning of HSCs within the liver. These aspects of the paper illustrate the advantages of the zebrafish model, such as to dissect genetic pathways and determine cellular requirements during organogenesis.
In recent years, zebrafish have advanced from serving as a primary developmental model to providing insight into disease processes relevant to human health and enabling translational opportunities. For example, zebrafish larvae have a highly conserved response to alcohol exposure and develop alcoholic liver disease with steatosis that requires sterol regulatory element binding protein activation.2 This powerful model was exploited by Yin et al. to demonstrate HSC activation in vivo, thereby showing convincingly that the hand2-labeled cells not only look like HSCs, but also behave characteristically in response to injury: acute alcohol exposure caused HSC proliferation and morphological changes with a loss of cytoplasmic processes and a more elongated cell shape. Moreover, the authors observed deposition of the matrix proteins laminin and type IV collagen, which is indicative of an early fibrotic response. This functional characterization of the injury response in a whole-embryo context should greatly enhance our understanding of the complex molecular and cellular mechanisms involved in repair and early fibrogenesis.
Other work has provided functional insight into the hepatocyte function and response to injury that further illustrates the ability to delineate liver physiology and model liver disease in zebrafish; the use of fluorescently labeled lipids has enabled the direct in vivo observation of hepatic metabolic activity.10, 11 In our own studies,12 zebrafish proved to be susceptible to toxic injury from acetaminophen, developing elevated liver enzymes, necrosis, and death. Importantly, the damage could be reversed by the clinical antidote N-acetylcysteine. Furthermore, techniques to perform partial hepatectomies and assess liver regeneration in zebrafish have been developed, and these models have enabled investigators to identify novel regulatory pathways.5, 13, 14 Zebrafish have also increasingly been used to model cancer.15 Liver tumors, generated after exposure to carcinogens, exhibit a stage-specific expression profile comparable to human HCC.6 These examples illustrate the increasing relevance and impact of the zebrafish to model liver disease.
The study by Yin et al. also highlights the feasibility of performing chemical screens. Due to the small size and high number of zebrafish embryos and larvae, thousands of animals can be exposed to chemicals in a single experiment. Here, the authors tested 338 compounds for their capacities to modulate HSC numbers. The power of this system notwithstanding, the necessity to use a different transgenic cell line remains: for the screen, the authors used another transgenic line, labeling wt1b-positive cells. The expression of wt1b overlaps with hand2 in the liver but has not been explicitly characterized in the report. This minor shortcoming underscores the remaining challenges of using in vivo fluorescent reporters for screening purposes. This screen identified two retinoid receptor agonists with opposite effects on HSC formation, confirming the reported importance for retinoic acid in HSC formation. Taken together, these results demonstrate the potential to identify novel compounds that affect HSC number and activity in the zebrafish with direct therapeutic implications. What this model still needs to prove, however, is the identification of novel signaling pathways affecting HSC formation and biology that have not been elucidated in other systems.
Zebrafish have recently made the jump from the fish tank to the bedside, demonstrating our ability to discover novel therapeutics: a chemical genetic screening approach identified prostaglandin E2 as a novel regulator of hematopoietic stem cells.16 This research inspired translational work17 that led to a recently completed clinical phase 1 trial, which demonstrated the use of prostaglandin E2 treatment of umbilical cord blood stem cells prior to transplantation into patients with leukemia and lymphoma (NCT00890500).18 Similarly, current work in a zebrafish melanoma model will result in a soon-to-be-opened clinical trial19 (L. Zon, personal communication). Our work using (NCT01611675) the acetaminophen model combined with chemical genetic screening has also fostered the discovery of novel therapeutics and considerations for a clinical trial.12 Each of these examples underscores the growing relevance of the zebrafish in translational medicine.
The study by Yin et al. represents a major step toward the use of the zebrafish model for many aspects of hepatology research: it opens the door for further studies into HSC activation and physiology in a tractable in vivo model. It will enable the discovery of novel regulators and therapeutics in forward genetic and chemical genetic screens. Because the responses to injury, regeneration, and fibrosis are common to many forms of liver damage, the identification of HSCs in the zebrafish may help enable our ultimate goal of reversing cirrhosis. In a challenging research arena, the zebrafish may indeed swim ahead and lead the way!