Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Role of histone deacetylase activity in the developing lateral line neuromast of zebrafish larvae

Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.