Distinct roles of core autophagy-related genes in zebrafish definitive hematopoiesis.

Despite the well-described discrepancy between (macroautophagy/autophagy-related) genes in the regulation of hematopoiesis, varying essentiality of core ATG proteins in vertebrate definitive hematopoiesis remains largely unclear. Here, we employed zebrafish () to compare the functions of six core genes, including , (beclin1), , , , and , in vertebrate definitive hematopoiesis via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein and morpholino targeting. Zebrafish with various mutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six mutations led to a declined number of (Spi-1 proto-oncogene b) myeloid progenitor cells. However, only mutation resulted in the expansion of (v-myb avian myeloblastosis viral oncogene homolog) hematopoietic stem and progenitor cells (HSPCs) and transiently increased (coronin, actin binding protein, 1A) leukocytes, whereas mutation decreased the number of HSPCs and leukocytes. Proteomic analysis of caudal hematopoietic tissue identified (SIN3 transcription regulator family member Aa) as a potential modulator of - and -regulated definitive hematopoiesis. Disruption of rescued the expansion of HSPCs and leukocytes in mutants and exacerbated the decrease of HSPCs in mutants. Double mutations were also performed to examine alternative functions of various genes in definitive hematopoiesis. Notably, mutation failed to induce HSPCs expansion with one of the other five mutations. These findings demonstrated the distinct roles of genes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an gene-dependent manner.: AGM: aorta-gonad-mesonephros; AO: acridine orange; : autophagy related; : beclin 1, autophagy related; CHT: caudal hematopoietic tissue; CKO: conditional knockout; : coronin, actin binding protein, 1A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; dpf: days post fertilization; FACS: fluorescence-activated cell sorting; : hemoglobin, alpha embryonic 1.1; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; KD: knockdown; KO: knockout; : microtubule-associated protein 1 light chain 3; MO: morpholino; : macrophage expressed 1, tandem duplicate 1; : myeloid-specific peroxidase; : v-myb avian myeloblastosis viral oncogene homolog; PE: phosphatidylethanolamine; : phospho-H3 histone; PtdIns3K: class 3 phosphatidylinositol 3-kinase; : recombination activating 1; : RB1-inducible coiled-coil 1; RFLP: restriction fragment length polymorphism; RNP: ribonucleoprotein; : SIN3 transcription regulator family member Aa; : Spi-1 proto-oncogene b; : unc-51 like autophagy activating kinase; : vitellogenin 1; WISH: whole-mount in situ hybridization.