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1.
Cell Rep ; 39(1): 110596, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35385752

RESUMO

Upon extensive hepatocyte loss or impaired hepatocyte proliferation, liver regeneration occurs via biliary epithelial cell (BEC) transdifferentiation, which includes dedifferentiation of BECs into bipotential progenitor cells (BP-PCs) and then redifferentiation of BP-PCs to nascent hepatocytes and BECs. This BEC-driven liver regeneration involves reactivation of hepatoblast markers, but the underpinning mechanisms and their effects on liver regeneration remain largely unknown. Using a zebrafish extensive hepatocyte ablation model, we perform an N-ethyl-N-nitrosourea (ENU) forward genetic screen and identify a liver regeneration mutant, liver logan (lvl), in which the telomere maintenance 2 (tel2) gene is mutated. During liver regeneration, the tel2 mutation specifically inhibits transcriptional activation of a hepatoblast marker, hematopoietically expressed homeobox (hhex), in BEC-derived cells, which blocks BP-PC redifferentiation. Mechanistic studies show that Tel2 associates with the hhex promoter region and promotes hhex transcription. Our results reveal roles of Tel2 in the BP-PC redifferentiation process of liver regeneration by activating hhex.


Assuntos
Sistema Biliar , Regeneração Hepática , Animais , Hepatócitos , Fígado , Regeneração Hepática/fisiologia , Proteínas Repressoras , Células-Tronco , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética
2.
Nat Protoc ; 15(10): 3361-3379, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32908315

RESUMO

RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and skin removal are used to permeate tissues and preserve antigen epitopes, making this protocol applicable to both whole-mount embryos and larvae. Off-target hybridization and FISH background are reduced by using PCR-amplified DNA templates and stringent buffers. This protocol simultaneously detects multiple mRNAs and proteins with high sensitivity, and enables detection at single-cell resolution. The protocol can be completed within 6 days, overcoming the shortage of reliable antibodies available for zebrafish and exploiting the advantages of zebrafish for studying organ development and regeneration.


Assuntos
Embrião não Mamífero/diagnóstico por imagem , Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Animais , Anticorpos/metabolismo , Larva/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética
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