TY - JOUR
T1 - Distinct roles of core autophagy-related genes in zebrafish definitive hematopoiesis
AU - Chen, Xiang-Ke
AU - Yi, Zhen-Ni
AU - Lau, Jack Jark Yin
AU - Ma, Alvin Chun Hang
N1 - Publisher Copyright:
© 2023 Informa UK Limited, trading as Taylor & Francis Group.
PY - 2023/11/3
Y1 - 2023/11/3
N2 - Despite 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 atg genes, 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 atg mutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six atg mutations led to a declined number of spi1b + (Spi-1 proto-oncogene b) myeloid progenitor cells. However, only becn1 mutation 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 atg3 mutation 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 sin3aa rescued the expansion of HSPCs and leukocytes in becn1 mutants and exacerbated the decrease of HSPCs in atg13 mutants. Double mutations were also performed to examine alternative functions of various atg genes in definitive hematopoiesis. Notably, becn1 mutation failed to induce HSPCs expansion with one of the other five atg mutations. These findings demonstrated the distinct roles of atg genes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an atg gene-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.
AB - Despite 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 atg genes, 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 atg mutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six atg mutations led to a declined number of spi1b + (Spi-1 proto-oncogene b) myeloid progenitor cells. However, only becn1 mutation 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 atg3 mutation 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 sin3aa rescued the expansion of HSPCs and leukocytes in becn1 mutants and exacerbated the decrease of HSPCs in atg13 mutants. Double mutations were also performed to examine alternative functions of various atg genes in definitive hematopoiesis. Notably, becn1 mutation failed to induce HSPCs expansion with one of the other five atg mutations. These findings demonstrated the distinct roles of atg genes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an atg gene-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.
KW - Autophagy-related genes
KW - CRISPR-Cas9 ribonucleoprotein
KW - definitive hematopoiesis
KW - hematopoietic stem and progenitor cells
KW - zebrafish
UR - http://www.scopus.com/inward/record.url?scp=85176111520&partnerID=8YFLogxK
U2 - 10.1080/15548627.2023.2274251
DO - 10.1080/15548627.2023.2274251
M3 - Journal article
C2 - 37921505
AN - SCOPUS:85176111520
SN - 1554-8627
VL - 20
SP - 830
EP - 846
JO - Autophagy
JF - Autophagy
IS - 4
ER -