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TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

Gao L, Li D, Ma K, Zhang W, Xu T, Fu C, Jing C, Jia X, Wu S, Sun X, Dong M, Deng M, Chen Y, Zhu W, Peng J, Wan F, Zhou Y, Zon LI, Pan W - PLoS Genet. (2015)

Bottom Line: Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs.Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment.Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

No MeSH data available.


Related in: MedlinePlus

The defect of definitive hematopoiesis is due to the increased apoptosis in the HSPCs of topbp1cas003 mutants.(A-D’) The c-myb in situ hybridization and phospho-histone H3 (pH3) immunostaining at 3.5dpf in sibling and topbp1cas003 mutant embryos, indicating no significant difference in the number of pH3+ HSPCs (pH3 and c-myb double positive cells, white arrows) between wild-type sibling and topbp1cas003 mutant embryos. The images show the stain in the enlarged CHT region. Scale bars represent 25μm. (E-H’) Double immunostaining of c-myb: EGFP and BrdU at 3.5dpf in the sibling and topbp1cas003 mutant embryos. The number of BrdU+ HSPCs (BrdU and EGFP double positive cells, white arrows) in topbp1cas003 mutant embryos is comparable to that in wild-type siblings. Scale bars represent 25μm. (I-P’) Double immunostaining of c-myb: EGFP and TUNEL at 3.5dpf (I-L’) and 4dpf (M-P’) in the sibling and topbp1cas003 mutant embryos. The apoptotic HSPCs (TUNEL and EGFP double positive cells, white arrows) are increased in the topbp1cas003 mutants comparing to siblings, which is more profound at 4dpf. Scale bars represent 25μm. (Q) Quantification of the number of the pH3+ HSPCs in sibling and topbp1cas003 mutant embryos at 3.5dpf. (R) Statistics result of the percentage of BrdU+ HSPCs at 3.5dpf. (Q) and (R) indicate no significant difference in the number of proliferating HSPCs between topbp1cas003 mutant embryos and siblings at 3.5dpf. (S) Quantitative analysis of apoptotic HSPCs in the CHT region in sibling and topbp1cas003 mutant embryos at 3.5dpf and 4dpf, showing the increased apoptotic HSPCs in the topbp1cas003 mutant embryos. For the Quantitative analysis, at least 6 embryos were analysis for each experimental group. Error bars represent SEM. *, p<0.05; ***, p<0.001.
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pgen.1005346.g004: The defect of definitive hematopoiesis is due to the increased apoptosis in the HSPCs of topbp1cas003 mutants.(A-D’) The c-myb in situ hybridization and phospho-histone H3 (pH3) immunostaining at 3.5dpf in sibling and topbp1cas003 mutant embryos, indicating no significant difference in the number of pH3+ HSPCs (pH3 and c-myb double positive cells, white arrows) between wild-type sibling and topbp1cas003 mutant embryos. The images show the stain in the enlarged CHT region. Scale bars represent 25μm. (E-H’) Double immunostaining of c-myb: EGFP and BrdU at 3.5dpf in the sibling and topbp1cas003 mutant embryos. The number of BrdU+ HSPCs (BrdU and EGFP double positive cells, white arrows) in topbp1cas003 mutant embryos is comparable to that in wild-type siblings. Scale bars represent 25μm. (I-P’) Double immunostaining of c-myb: EGFP and TUNEL at 3.5dpf (I-L’) and 4dpf (M-P’) in the sibling and topbp1cas003 mutant embryos. The apoptotic HSPCs (TUNEL and EGFP double positive cells, white arrows) are increased in the topbp1cas003 mutants comparing to siblings, which is more profound at 4dpf. Scale bars represent 25μm. (Q) Quantification of the number of the pH3+ HSPCs in sibling and topbp1cas003 mutant embryos at 3.5dpf. (R) Statistics result of the percentage of BrdU+ HSPCs at 3.5dpf. (Q) and (R) indicate no significant difference in the number of proliferating HSPCs between topbp1cas003 mutant embryos and siblings at 3.5dpf. (S) Quantitative analysis of apoptotic HSPCs in the CHT region in sibling and topbp1cas003 mutant embryos at 3.5dpf and 4dpf, showing the increased apoptotic HSPCs in the topbp1cas003 mutant embryos. For the Quantitative analysis, at least 6 embryos were analysis for each experimental group. Error bars represent SEM. *, p<0.05; ***, p<0.001.

Mentions: To explore how topbp1 affected maintenance of HSPCs in CHT region, we first investigated the expression pattern of topbp1 during embryonic development. WISH analysis data indicated that topbp1 was a maternal mRNA, and was ubiquitously expressed during embryogenesis (S5A–S5J Fig). Previous reports had showed that topbp1 knock-out or knock-down could result in either cell proliferation blockage or cell apoptosis activation [44,45]. To investigate the cause of HSPCs abrogation, we conducted cell biology assessment of HSPCs in topbp1cas003 mutants in Tg(c-myb: EGFP) transgenic background. Double staining of c-myb and phospho-histone 3 (pH3) showed no significant difference in topbp1cas003 mutants, compared with siblings at 3.5dpf (Fig 4A–D’, quantified in Q), suggesting that the cell cycle of HSPCs was not affected in topbp1cas003 mutants. Furthermore, we performed 5-bromo-2-deoxyuridine (BrdU) incorporation assay on HSPCs, BrdU and EGFP double immunostaining results indicated that there was no significant difference in the percentage of BrdU+ HSPCs between siblings and topbp1cas003 mutants at 3.5dpf (Fig 4E–H’, quantified in R). However, TUNEL assay showed a significant increase of apoptotic EGFP+ HSPCs in CHT region of topbp1cas003 mutants, compared with that in wild-type siblings at 3.5dpf (Fig 4I–L’, quantified in S). At 4dpf, the percentage of apoptotic EGFP+ HSPCs was even more significantly increased in topbp1cas003 mutants in comparison with siblings (Fig 4M–P’, quantified in S), while the number of EGFP+ HSPCs were dramatically decreased (Fig 2N and 2Q). Notably, we could also detect the increased apoptosis in the cranial region and the neural tube in the topbp1cas003 mutants at 3.5dpf and 4dpf. Collectively, we concluded that the increased apoptosis in HSPCs was linked to the defective hematopoiesis in topbp1cas003 mutants.


TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

Gao L, Li D, Ma K, Zhang W, Xu T, Fu C, Jing C, Jia X, Wu S, Sun X, Dong M, Deng M, Chen Y, Zhu W, Peng J, Wan F, Zhou Y, Zon LI, Pan W - PLoS Genet. (2015)

The defect of definitive hematopoiesis is due to the increased apoptosis in the HSPCs of topbp1cas003 mutants.(A-D’) The c-myb in situ hybridization and phospho-histone H3 (pH3) immunostaining at 3.5dpf in sibling and topbp1cas003 mutant embryos, indicating no significant difference in the number of pH3+ HSPCs (pH3 and c-myb double positive cells, white arrows) between wild-type sibling and topbp1cas003 mutant embryos. The images show the stain in the enlarged CHT region. Scale bars represent 25μm. (E-H’) Double immunostaining of c-myb: EGFP and BrdU at 3.5dpf in the sibling and topbp1cas003 mutant embryos. The number of BrdU+ HSPCs (BrdU and EGFP double positive cells, white arrows) in topbp1cas003 mutant embryos is comparable to that in wild-type siblings. Scale bars represent 25μm. (I-P’) Double immunostaining of c-myb: EGFP and TUNEL at 3.5dpf (I-L’) and 4dpf (M-P’) in the sibling and topbp1cas003 mutant embryos. The apoptotic HSPCs (TUNEL and EGFP double positive cells, white arrows) are increased in the topbp1cas003 mutants comparing to siblings, which is more profound at 4dpf. Scale bars represent 25μm. (Q) Quantification of the number of the pH3+ HSPCs in sibling and topbp1cas003 mutant embryos at 3.5dpf. (R) Statistics result of the percentage of BrdU+ HSPCs at 3.5dpf. (Q) and (R) indicate no significant difference in the number of proliferating HSPCs between topbp1cas003 mutant embryos and siblings at 3.5dpf. (S) Quantitative analysis of apoptotic HSPCs in the CHT region in sibling and topbp1cas003 mutant embryos at 3.5dpf and 4dpf, showing the increased apoptotic HSPCs in the topbp1cas003 mutant embryos. For the Quantitative analysis, at least 6 embryos were analysis for each experimental group. Error bars represent SEM. *, p<0.05; ***, p<0.001.
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pgen.1005346.g004: The defect of definitive hematopoiesis is due to the increased apoptosis in the HSPCs of topbp1cas003 mutants.(A-D’) The c-myb in situ hybridization and phospho-histone H3 (pH3) immunostaining at 3.5dpf in sibling and topbp1cas003 mutant embryos, indicating no significant difference in the number of pH3+ HSPCs (pH3 and c-myb double positive cells, white arrows) between wild-type sibling and topbp1cas003 mutant embryos. The images show the stain in the enlarged CHT region. Scale bars represent 25μm. (E-H’) Double immunostaining of c-myb: EGFP and BrdU at 3.5dpf in the sibling and topbp1cas003 mutant embryos. The number of BrdU+ HSPCs (BrdU and EGFP double positive cells, white arrows) in topbp1cas003 mutant embryos is comparable to that in wild-type siblings. Scale bars represent 25μm. (I-P’) Double immunostaining of c-myb: EGFP and TUNEL at 3.5dpf (I-L’) and 4dpf (M-P’) in the sibling and topbp1cas003 mutant embryos. The apoptotic HSPCs (TUNEL and EGFP double positive cells, white arrows) are increased in the topbp1cas003 mutants comparing to siblings, which is more profound at 4dpf. Scale bars represent 25μm. (Q) Quantification of the number of the pH3+ HSPCs in sibling and topbp1cas003 mutant embryos at 3.5dpf. (R) Statistics result of the percentage of BrdU+ HSPCs at 3.5dpf. (Q) and (R) indicate no significant difference in the number of proliferating HSPCs between topbp1cas003 mutant embryos and siblings at 3.5dpf. (S) Quantitative analysis of apoptotic HSPCs in the CHT region in sibling and topbp1cas003 mutant embryos at 3.5dpf and 4dpf, showing the increased apoptotic HSPCs in the topbp1cas003 mutant embryos. For the Quantitative analysis, at least 6 embryos were analysis for each experimental group. Error bars represent SEM. *, p<0.05; ***, p<0.001.
Mentions: To explore how topbp1 affected maintenance of HSPCs in CHT region, we first investigated the expression pattern of topbp1 during embryonic development. WISH analysis data indicated that topbp1 was a maternal mRNA, and was ubiquitously expressed during embryogenesis (S5A–S5J Fig). Previous reports had showed that topbp1 knock-out or knock-down could result in either cell proliferation blockage or cell apoptosis activation [44,45]. To investigate the cause of HSPCs abrogation, we conducted cell biology assessment of HSPCs in topbp1cas003 mutants in Tg(c-myb: EGFP) transgenic background. Double staining of c-myb and phospho-histone 3 (pH3) showed no significant difference in topbp1cas003 mutants, compared with siblings at 3.5dpf (Fig 4A–D’, quantified in Q), suggesting that the cell cycle of HSPCs was not affected in topbp1cas003 mutants. Furthermore, we performed 5-bromo-2-deoxyuridine (BrdU) incorporation assay on HSPCs, BrdU and EGFP double immunostaining results indicated that there was no significant difference in the percentage of BrdU+ HSPCs between siblings and topbp1cas003 mutants at 3.5dpf (Fig 4E–H’, quantified in R). However, TUNEL assay showed a significant increase of apoptotic EGFP+ HSPCs in CHT region of topbp1cas003 mutants, compared with that in wild-type siblings at 3.5dpf (Fig 4I–L’, quantified in S). At 4dpf, the percentage of apoptotic EGFP+ HSPCs was even more significantly increased in topbp1cas003 mutants in comparison with siblings (Fig 4M–P’, quantified in S), while the number of EGFP+ HSPCs were dramatically decreased (Fig 2N and 2Q). Notably, we could also detect the increased apoptosis in the cranial region and the neural tube in the topbp1cas003 mutants at 3.5dpf and 4dpf. Collectively, we concluded that the increased apoptosis in HSPCs was linked to the defective hematopoiesis in topbp1cas003 mutants.

Bottom Line: Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs.Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment.Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

No MeSH data available.


Related in: MedlinePlus