HFD enhances HCC progression and modulates the immune response in the liver microenvironment. HFD induced changes in macrophage polarization with increased numbers of TNFa-positive macrophages in the liver. HFD reduces T cell infiltration to liver area in NASHassociated HCC larvae. Ablation of macrophages reduces disease progression in NASH-associated HCC larvae, but not in HCC alone. Metformin specifically affects the progression induced by diet in NASH-associated HCC in zebrafish.
Background and Aims: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. Approach and Results:Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver threedimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly
Abbreviations used in this paper: NAFLD, Nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; HCC, hepatocellular carcinoma; HCD, high-cholesterol diet; TME, tumor microenvironment; TNF, tumor necrosis factor; dpf, days post-fertilization; ND, normal diet; MTZ, metronidazole; NTR, nitroreductase. AbstractDiabetes and obesity have been associated with nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) and increased incidence of hepatocellular carcinoma (HCC). Here we use optically transparent zebrafish to visualize liver inflammation and disease progression in a NAFLD/NASH-HCC model. We combined a high-cholesterol diet (HCD) with a transgenic zebrafish HCC model induced by hepatocyte-specific activated βcatenin and found that diet induced an increase in liver size and enhanced angiogenesis and neutrophil infiltration in the liver. Although macrophage number was not affected by diet, HCD induced changes in macrophage morphology and polarization with an increase in liver associated TNFα-positive macrophages. Treatment with metformin altered macrophage polarization and reduced liver size in NAFLD/NASH-associated HCC larvae. Moreover, ablation of macrophages limited progression in NAFLD/NASH-associated HCC larvae but not in HCC alone. These findings suggest that HCD alters macrophage polarization and exacerbates the liver inflammatory microenvironment and cancer progression in a zebrafish model of NAFLD/NASH-associated HCC.
Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.
The extent to which differentiated cells, while remaining in their native microenvironment, can be reprogrammed to assume a different identity will reveal fundamental insight into cellular plasticity and impact regenerative medicine. To investigate in vivo cell lineage potential, we leveraged the zebrafish as a practical vertebrate platform to determine factors and mechanisms necessary to induce differentiated cells of one germ layer to adopt the lineage of another. We discovered that ectopic co-expression of Sox32 and Oct4 in several non-endoderm lineages, including skeletal muscle, can specifically trigger an early endoderm genetic program in a cell-autonomous manner.Gene expression, live imaging, and functional studies reveal that the endoderm-induced muscle cells lose muscle gene expression and morphology, while specifically gaining endoderm organogenesis markers, such as the pancreatic specification genes, hhex and ptf1a, via a mechanism resembling normal development. Endoderm induction by a pluripotent defective form of Oct4, endoderm markers appearing prior to loss of muscle cell morphology, a lack of dependence on cell division, and a lack of mesoderm, ectoderm, dedifferentiation, and pluripotency gene activation, together, suggests that reprogramming is endoderm specific and occurs via direct lineage conversion. Our work demonstrates that within a vertebrate animal, stably differentiated cells can be induced to directly adopt the identity of a completely unrelated cell lineage, while remaining in a distinct microenvironment, suggesting that differentiated cells in vivo may be more amenable to lineage conversion than previously appreciated. This discovery of possibly unlimited lineage potential of differentiated cells in vivo challenges our understanding of cell lineage restriction and may pave the way towards a vast new in vivo supply of replacement cells for degenerative diseases such as diabetes.
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