Identification of Annexin A4 as a hepatopancreas factor involved in liver cell survival

Abstract

To gain insight into liver and pancreas development, we investigated the target of 2F11, a monoclonal antibody of unknown antigen, widely used in zebrafish studies for labeling hepatopancreatic ducts. Utilizing mass spectrometry and in vivo assays, we determined the molecular target of 2F11 to be Annexin A4 (Anxa4), a calcium binding protein. We further found that in both zebrafish and mouse endoderm, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including the insulin producing β-cells. Although Anxa4 is a known target of several monogenic diabetes genes and its elevated expression is associated with chemoresistance in malignancy, its in vivo role is largely unexplored. Knockdown of Anxa4 in zebrafish leads to elevated expression of caspase 8 and Δ113p53, and liver bud specific activation of Caspase 3 and apoptosis. Mosaic knockdown reveal that Anxa4 is required cell-autonomously in the liver bud for cell survival. This finding is further corroborated with mosaic anxa4 knockout studies using the CRISPR/Cas9 system. Collectively, we identify Anxa4 as a new, evolutionarily conserved hepatopancreatic factor that is required in zebrafish for liver progenitor viability, through inhibition of the extrinsic apoptotic pathway. A role for Anxa4 in cell survival may have implications for the mechanism of diabetic β-cell apoptosis and cancer cell chemoresistance.

Sox4 regulates choroid fissure closure by limiting Hedgehog signaling during ocular morphogenesis

“…High resolution melt curve analysis (HRMA) and sequencing of the sox4a and sox4b target regions in individual injected embryos confirmed that short indels were generated in the sox4 gRNA/Cas9 injected individuals with high efficiency (Figure S1P-Q and data not shown), with the mutation frequency ranging from 52.78% to 94.44%. These data suggest that the CRISPRs induced bi-allelic gene inactivation in mosaic injected embryos, as has been reported previously in zebrafish and mice (Jao et al, 2013; Yen et al, 2014; Zhang et al, 2014), resulting in ocular phenotypes similar to those seen in the sox4 morphants. Taken together, these results strongly support the specificity of the sox4 morphant coloboma phenotype.…”

Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish

“…The limitation of this method is that rare Tg(Tp1-MmHbb:EGFP) um14 -negative terminal cells will be excluded from this computational assay. The Anxa4 antibody (mAb 2F11) recognizes most of the biliary epithelial cells (Sakaguchi et al, 2008;Zhang et al, 2014), and this algorithm can also analyze the Anxa4 expression pattern (Fig. S6).…”

The lure of zebrafish in liver research: regulation of hepatic growth in development and regeneration

“…For example, sorting nexon 7 (snx7) and annexin A4 (anxa4) are both required for hepatoblast survival[40,41]. Similarly, the small nuclear RNA (snRNA) transcription complex component snap4c is required for BEC survival[42].…”