Exploiting zebrafish model by transgenic technology for the study of gastrointestinal diseases

Abstract

Chapter1.Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, transgenic phenotypes of zebrafish over-expressing either Indian Hh or Sonic Hh were investigated. This investigation was done along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially observed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry.The results showed that over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induced morphologic changes in the developing pancreas without derangement in acinar differentiation. The transgenic zebrafish showed progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular were activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands also induced matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, did not cause pancreatic tumors. On treatment with inhibitors, the embryonic phenotypes were found to be reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis was only prevented by HPI-4.This study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells in vivo. Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These experiments showed that the transgenic models can be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.Chapter2.Chronic inflammation is an important process leading to carcinogenesis. Therefore targeting and controlling inflammation can be a promising cancer therapy. Inflammation is often caused by a variety of inflammatory cytokines. Interleukin-6 (IL-6), a pleiotrophic cytokine, is not only involved in the regulation of regenerative process, but also in immune and inflammatory responses in various carcinogenesis including liver cancer. Yet, the nature of its involvement in tumorigenesis is still unclear. Although animal models have been widely used to elucidate the nature of IL-6''s involvement in hepatic tumorigenesis, to date, no animal model revealing direct consequence of the inflammatory IL-6 expression and hepatic tumorigenesis have been suggested. In this study, an in vivo hepatic tumorigenesis model using transgenic zebrafish was generated to demonstrate IL-6 driven tumorigenesis. Transgene expression under the regulation of LFABP promoter driving hIL6 gene was found starting from embryonic day 4 and persisted until adulthood. Interestingly, the aberrant hIL6 gene expression, specifically expressed in the zebrafish liver, caused a chronic inflammation accompanying infiltration of inflammatory cells of myeloid and B cell lineage. The inflammation induced cell damage and compensatory proliferation. This eventually resulted in the generation of precancerous dysplastic lesions including clear cell foci, large cell changes, and eosinophilic and also basophilic bodies. Hepatocellular carcinoma developed in a subset of the transgenics was finally observed. Molecular analyses revealed up-regulation of majority of the components involved in the IL-6 driven pathway during inflammatory process as evidenced by quantitative RT-PCR and Western blot, supporting that the aberrant high level of IL-6 production in the liver causes hepatic tumorigenesis. Among the downstream pathway from IL-6, while PI3K/Akt pathway activation was confined to inflammatory cells, activation of Jak/Stat3 pathway was noted in inflammed hepatocytes as well as in cells at dysplatic foci, suggesting Jak/Stat3 pathway played dominant role in the hepatic tumorigenesis. Conclusion: As far as we know, the current transgenic zebrafish most closely mimic hepatic carcinogenesis in human induced by chronic B or C viral infection at both cellular and molecular level. We provide a straightforward evidence of relationship between chronic inflammation and tumorigenesis, and reinforces the pivotal role of IL-6 in inflammation-associated hepatocellular carcinoma. Chapter3.In an effort to completion of zebrafish model platform of gastrointestinal expression, transgenic zebrafish has been established by using Cre-Loxp system (Tg(IFABP-CreERT2pA-Cmcl2-mcherry)). In this system, tamoxifen-dependent transcription factor CreERT2 was expressed under the control of the regulatory element of zebrafish intestinal fatty acid binding protein (IFABP) gene. Transgene CreERT2 expression was confirmed by ISH for Cre in 4 dpf embryos. This platform line will be crossed with transgenic zebrafish in which transgene is expressed by recombinational excision of Loxp-stop-Loxp sequence by CreERT2 such as (Tg(UBB-Loxp-mCherry-Loxp-GFPKrasG12D). This strategy is used to induce expression of the transgene spcifically to intestinal organ and the established platform will be a valuable model for the study of human intestinal diseases.