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

BackgroundAMBRA1 is an intrinsically disordered protein, working as a\r\nscaffold molecule to coordinate, by protein-protein interaction, many\r\ncellular processes, including autophagy, mitophagy, apoptosis and cell\r\ncycle progression. The zebrafish genome contains two ambra1 paralogous\r\ngenes (a and b), both involved in development and expressed at high\r\nlevels in the gonads. Characterization of the zebrafish paralogous genes\r\nmutant lines generated by CRISPR/Cas9 approach showed that ambra1b\r\nknockout leads to an all-male population.ResultsWe demonstrated that the\r\nsilencing of the ambra1b gene determines a reduction of primordial germ\r\ncells (PGCs), a condition that, in the zebrafish, leads to the\r\ndevelopment of all-male progeny. PGC reduction was confirmed by\r\nknockdown experiments and rescued by injection of ambra1b and human\r\nAMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued\r\nby injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding\r\nregion, thus suggesting that interaction with this complex is involved\r\nin PGC protection from loss. Results from zebrafish embryos injected\r\nwith murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could\r\nindirectly regulate this protein through CUL4-DDB1 interaction.\r\nAccording to this, Ambra1(+/-) mice showed a reduced Stat3 expression in\r\nthe ovary together with a low number of antral follicles and an increase\r\nof atretic follicles, indicating a function of Ambra1 in the ovary of\r\nmammals as well. Moreover, in agreement with the high expression of\r\nthese genes in the testis and ovary, we found significant impairment of\r\nthe reproductive process and pathological alterations, including tumors,\r\nmainly limited to the gonads.ConclusionsBy exploiting ambra1a and\r\nambra1b knockout zebrafish lines, we prove the sub-functionalization\r\nbetween the two paralogous zebrafish genes and uncover a novel function\r\nof Ambra1 in the protection from excessive PGC loss, which seems to\r\nrequire binding with the CUL4-DDB1 complex. Both genes seem to play a\r\nrole in the regulation of reproductive physiology.