Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

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.

Clinically relevant orthotopic xenograft models of patient-derived glioblastoma in zebrafish.

An accurate prediction of the intracranial infiltration tendency and drug response of individual glioblastoma (GBM) cells is essential for personalized prognosis and treatment for this disease. However, the clinical utility of mouse patient-derived orthotopic xenograft (PDOX) models remains limited given current technical constraints, including difficulty in generating sufficient sample numbers from small tissue samples and a long latency period for results. To overcome these issues, we established zebrafish GBM xenografts of diverse origin, which can tolerate intracranial engraftment and maintain their unique histological features. Subsequent single-cell RNA-sequencing (scRNA-seq) analysis confirmed significant transcriptional identity to that of invading GBM microtumors observed in the proportionally larger brains of model animals and humans. Endothelial scRNA-seq confirmed that the zebrafish blood-brain barrier is homologous to the mammalian blood-brain barrier. Finally, we established a rapid and efficient zebrafish PDOX (zPDOX) model, which can predict long-term outcomes of GBM patients within 20 days. The zPDOX model provides a novel avenue for precision medicine of GBM, especially for the evaluation of intracranial infiltration tendency and prediction of individual drug sensitivity.

Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafish model of Alagille Syndrome.

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 three-dimensional 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 preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Lineage analysis reveals an endodermal contribution to the vertebrate pituitary.

Vertebrate sensory organs arise from epithelial thickenings called placodes. Along with neural crest cells, cranial placodes are considered ectodermal novelties that drove evolution of the vertebrate head. The anterior-most placode generates the endocrine lobe [adenohypophysis (ADH)] of the pituitary, a master gland controlling growth, metabolism, and reproduction. In addition to known ectodermal contributions, we use lineage tracing and time-lapse imaging in zebrafish to identify an endodermal contribution to the ADH. Single-cell RNA sequencing of the adult pituitary reveals similar competency of endodermal and ectodermal epithelia to generate all endocrine cell types. Further, endoderm can generate a rudimentary ADH-like structure in the near absence of ectodermal contributions. The fish condition supports the vertebrate pituitary arising through interactions of an ancestral endoderm-derived proto-pituitary with newly evolved placodal ectoderm.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish.

Liver duct paucity is characteristic of children born with Alagille Syndrome (ALGS), a disease associated with JAGGED1 mutations. Here, we report that zebrafish embryos with compound homozygous mutations in two Notch ligand genes, jagged1b (jag1b) and jagged2b (jag2b) exhibit a complete loss of canonical Notch activity and duct cells within the liver and exocrine pancreas, whereas hepatocyte and acinar pancreas development is not affected. Further, animal chimera studies demonstrate that wild-type endoderm cells within the liver and pancreas can rescue Notch activity and duct lineage specification in adjacent cells lacking jag1b and jag2b expression. We conclude that these two Notch ligands are directly and solely responsible for all duct lineage specification in these organs in zebrafish. Our study uncovers genes required for lineage specification of the intrahepatopancreatic duct cells, challenges the role of duct cells as progenitors, and suggests a genetic mechanism for ALGS ductal paucity.The hepatopancreatic duct cells connect liver hepatocytes and pancreatic acinar cells to the intestine, but the mechanism for their lineage specification is unclear. Here, the authors reveal that Notch ligands Jagged1b and Jagged2b induce duct cell lineage in the liver and pancreas of the zebrafish.