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ABSTRACTDespite the well-described discrepancy between ATG(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 (Danio rerio) to compare the functions of six core atggenes, including atg13, becn1(beclin1), atg9a, atg2a, atg5, and atg3, in vertebrate definitive hematopoiesis via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein and morpholino targeting. Zebrafish with various atgmutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six atgmutations led to a declined number of spi1b+(Spi-1 proto-oncogene b) myeloid progenitor cells. However, only becn1mutation resulted in the expansion of myb+(v-myb avian myeloblastosis viral oncogene homolog) hematopoietic stem and progenitor cells (HSPCs) and transiently increased coro1a+(coronin, actin binding protein, 1A) leukocytes, whereas atg3mutation decreased the number of HSPCs and leukocytes. Proteomic analysis of caudal hematopoietic tissue identified sin3aa(SIN3 transcription regulator family member Aa) as a potential modulator of atg13- and becn1-regulated definitive hematopoiesis. Disruption of sin3aarescued the expansion of HSPCs and leukocytes in becn1mutants and exacerbated the decrease of HSPCs in atg13mutants. Double mutations were also performed to examine alternative functions of various atggenes in definitive hematopoiesis. Notably, becn1mutation failed to induce HSPCs expansion with one of the other five atgmutations. These findings demonstrated the distinct roles of atggenes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an atggene-dependent manner.Abbreviations: AGM: aorta-gonad-mesonephros; AO: acridine orange; atg: autophagy related; becn1: beclin 1, autophagy related; CHT: caudal hematopoietic tissue; CKO: conditional knockout; coro1a: coronin, actin binding protein, 1A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; dpf: days post fertilization; FACS: fluorescence-activated cell sorting; hbae1.1: hemoglobin, alpha embryonic 1.1; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; KD: knockdown; KO: knockout; map1lc3/lc3: microtubule-associated protein 1 light chain 3; MO: morpholino; mpeg1.1: macrophage expressed 1, tandem duplicate 1; mpx: myeloid-specific peroxidase; myb: v-myb avian myeloblastosis viral oncogene homolog; PE: phosphatidylethanolamine; p-H3: phospho-H3 histone; PtdIns3K: class 3 phosphatidylinositol 3-kinase; rag1: recombination activating 1; rb1cc1/fip200: RB1-inducible coiled-coil 1; RFLP: restriction fragment length polymorphism; RNP: ribonucleoprotein; sin3aa: SIN3 transcription regulator family member Aa; spi1b: Spi-1 proto-oncogene b; ulk: unc-51 like autophagy activating kinase; vtg1: vitellogenin 1; WISH: whole-mount in situ hybridization.

After two hours' travel through driving rain, Lucio arrived in the village with his two wives and children aboard a raft, all soaked to the skin. Four weeks earlier, they [...]