Distinct roles of core autophagy-related genes in zebrafish definitive hematopoiesis
Creators: name: Chen, Xiang-Ke
name: Yi, Zhen-Ni
name: Lau, Jack Jark-Yin
name: Chun-Hang, Alvin
Publisher: name: Taylor & Francis
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Evolutionary Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Hematology
subject: Computational Biology

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