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Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1.

ABSTRACT: Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.

SUBMITTER: Madakashira BP

PROVIDER: S-EPMC8304062 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.

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Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1.

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