
==== Front
Stem Cell Reports
Stem Cell Reports
Stem Cell Reports
2213-6711
Elsevier

S2213-6711(24)00220-0
10.1016/j.stemcr.2024.07.011
Article
mTORC1 mediates the expansion of hematopoietic stem and progenitor cells through ribosome biogenesis protein Urb2 in zebrafish
Huang Wenming 1234
Yue Yu 1234
Hao Weifeng 1234
Zhang Zhenan 1234
Cai Pengcheng pchengcai@swu.edu.cn
5∗
Yang Deqin yangdeqin@hospital.cqmu.edu.cn
123467∗∗
1 Department of Endodontics, Stomatological Hospital of Chongqing Medical University, Chongqing 404100, China
2 Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Stomatological Hospital of Chongqing Medical University, Chongqing 404100, China
3 Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 404100, China
4 Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, 426 Songshi North Road, Yubei Distrinct, Chongqing 401147, China
5 Institute of Developmental Biology and Regenerative Medicine, Southwest University, Beibei, Chongqing 400715, China
6 Department of Conservative Dentistry and Endodontics, Shanghai Stomatological Hospital & School of Stomatology, Fudan University, Shanghai, China
∗ Corresponding author pchengcai@swu.edu.cn
∗∗ Corresponding author yangdeqin@hospital.cqmu.edu.cn
7 Lead contact

22 8 2024
10 9 2024
22 8 2024
19 9 12771288
26 1 2024
26 7 2024
27 7 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Mammalian target of rapamycin (mTOR) serves as the key sensor to control protein synthesis, cell growth, and survival. Despite mTOR is reported to regulate hematopoietic stem and progenitor cell (HSPC) engraftment and multiple-lineage hematopoiesis in mice, the roles of unique mTOR complexes (mTORCs) in early HSPC development and HSPC pool formation have not been adequately elucidated. Here, we uncover that mTORC1 is essential for early HSPC expansion in zebrafish. mTORC1 signaling was highly activated in definitive HSPCs during the emerging and expanding stages. Pharmacological or genetic inactivation of mTORC1 would cause defective HSPC expansion and migration due to disrupted cell proliferation. Interestingly, mTORC2 is dispensable for early HSPC development. Ribosome biogenesis protein Urb2 was downregulated upon mTORC1 inhibition, and urb2 overexpression partially rescued the hematopoietic defects in mTORC1-deficient embryos. These data demonstrate that mTORC1 signaling regulates early HSPC expansion through Urb2, and this work will deepen our understanding of mTOR in different physiological processes.

Highlights

• mTORC1 signaling is required for HSPC development

• mTORC1 is essential for HSPC proliferation

• mTORC1 regulates HSPC expansion through ribosome biogenesis protein Urb2

In this article, Yang and colleagues show the essential roles of mTORC1 in zebrafish hematopoiesis and demonstrate that Urb2 acts downstream of mTORC1 to regulate HSPC expansion. They prove that mTORC1 mediates early definitive hematopoiesis in a P53-independent way.

Keywords

mTOR
hematopoietic
proliferation
Urb2
zebrafish
Published: August 22, 2024
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pmcIntroduction

In vertebrate organisms, the earliest definitive hematopoietic stem and progenitor cells (HSPCs) can migrate to the fetal liver, which provides an appropriate environment for HSPC proliferation. Ultimately, the bone marrow will be occupied by HSPCs, leading to the formation of adult hematopoietic organs (Cumano and Godin, 2007). The definitive HSPCs are able to generate all blood lineages, which is essential for the physiological homeostasis of blood systems (Zhang et al., 2013). Thus, the research of HSPC development will be beneficial for the understanding of blood systems, which can provide therapeutic ideas for hematological diseases.

Zebrafish and mammals share a highly conserved regulatory network of hematopoiesis. In zebrafish, the definitive hematopoiesis initiates in the aorta-gonad-mesonephros(AGM) region at 24 h postfertilization (hpf). Then the earliest definitive HSPCs emerge from the aortic endothelium through endothelial hematopoietic transition between 28 and 32 hpf (Kissa and Herbomel, 2010). Subsequently, those budding HSPCs will migrate to the caudal hematopoietic tissue (CHT). Then the expanded HSPCs will populate the thymus and kidney to sustain lifelong hematopoiesis (De Jong and Zon, 2005). Many key genes and signaling pathways have been identified in the regulation of zebrafish hematopoiesis (Zhang et al., 2013); however, the regulatory mechanisms remain to be fully understood.

Mammalian target of rapamycin (mTOR) is a protein kinase that functions as the catalytic subunit of mTOR complex (mTORC)1 and mTORC2 (Zoncu et al., 2011). mTORC1 and mTORC2 are distinguished by different accessory proteins, in which regulatory-associated protein of mTOR (Raptor) defines mTORC1 and rapamycin-insensitive companion of mTOR (Rictor) defines mTORC2 (Hara et al., 2002). Rapamycin is an effective inhibitor of mTORC1 (Abraham and Gibbons, 2007), making it convenient to explore the physiological roles of mTORC1. mTOR acts as a sensor of growth and starvation and is essential for transcription, protein synthesis, and cell proliferation and survival (Zoncu et al., 2011). Previous studies have validated the central role of mTOR in adult mouse hematopoiesis (Wang et al., 2016). Conditional mTOR deletion in mouse leads to the loss of HSPC quiescence and disrupted HSPC transplantation (Guo et al., 2013). However, mTOR activation by Tsc1 deletion causes impaired HSPC quiescence and reduced hematopoiesis and self-renewal of HSPCs. In addition, mTOR activity is associated with aged HSPCs, and mTOR inhibition by rapamycin can restore the function of HSPCs in aged mice, implying a negative role of mTOR in HSPC aging (Chen et al., 2009). Conditional Raptor deletion in mouse HSPCs ablates mTORC1 activity, leading to HSPC mobilization and differentiation (Kalaitzidis et al., 2012). However, mTORC1 is dispensable for HSPC survival, and it only affects granulocyte and B cell development. Collectively, the research on mouse suggests that mTOR regulates HSPC quiescence and differentiation and thus is important for blood homeostasis. However, mTOR, Raptor, and Rictor mutant mice are lethal in early stages (Shiota et al., 2006), making it infeasible to investigate the roles of mTOR in early HSPC development.

The roles of mTORC1 in zebrafish embryogenesis and tissue regeneration have been identified in recent years. mTORC1 is essential for digestive organ development in zebrafish, and morpholino-mediated knockdown of mtor or raptor results in hypoplastic liver and pancreas (He et al., 2017). Upon extreme hepatocytes injury, mTORC1 plays important roles in the biliary dedifferentiating stage, and loss of mTORC1 leads to the liver regenerative defects (He et al., 2019). Besides, mTORC1 inhibition can rescue the expansive myelopoiesis in Pten-deficient zebrafish embryos (Dong et al., 2014), implying a negative role of mTORC1 in zebrafish definitive myelopoiesis. Despite these identified roles in zebrafish, little is known about the roles of mTORC1 in early HSPC development. Meanwhile, ribosomes are essential machines for protein synthesis, and the biogenesis of ribosome is a highly conserved process in different species. Ribosome biogenesis is crucial to cellular adaptation, growth, and proliferation. In this study, we found that pharmacological inhibition of mTORC1 or genetic inactivation of mtor/raptor blocked the HSPC proliferation after its emergence from dorsal aorta, leading to the reduced HSPCs in the CHT and thymus. Besides, overexpression of ribosome biogenesis protein Urb2 could partially correct the defective HSPC development. Taken together, these findings facilitate our understanding on the roles of mTORC1 in definitive hematopoiesis.

Results

mTORC1 signaling is required for HSPC development in zebrafish

mTORC1 signaling plays important roles in HSPC quiescence and differentiation (Wang et al., 2016), but whether mTORC1 is involved in early HSPC development remains elusive. To evaluate the mTORC1 activity during definitive hematopoiesis, we first checked the protein level of phosphorylated eukaryotic initiation factor 4E binding protein 1 (p-4E-BP1), which acts as a downstream effector of mTORC1 to regulate protein synthesis (Musa et al., 2016). The expression of p-4E-BP1 kept high levels in HSPCs at 34 , 60 , 72 , and 96 hpf (Figures 1A and 1B), implicating a role of mTORC1 in the development of definitive HSPCs. To test this hypothesis, we used an effective mTORC1 inhibitor rapamycin, which is widely applied in mouse and zebrafish studies (Abraham and Gibbons, 2007; He et al., 2017, 2019). Rapamycin treatment did not affect cmyb expression in the aorta-gonad mesonephros at 34 hpf (Figure 1C). Similarly, rapamycin treatment of Tg(flk1:mCherryRas;runx1:GFP) transgenic line showed no significant changes of GFP-positive HSPCs in the CHT region at 34 hpf (Figures S1A and S1B), indicating that HSPCs could normally emerge from the dorsal aorta. However, rapamycin treatment significantly reduced cmyb expression in the CHT region at 72 and 96 hpf (Figures 1C and S1C). At the same time, the erythrocyte marker ae1-globin and neutrophil marker lyz were also significantly reduced in the rapamycin treatment group (Figure S1D), implicating that HSPC development is defective upon mTORC1 inhibition. We also analyzed HSPC development using the Tg(cmyb:GFP) transgenic line, in which HSPCs are labeled by GFP. Consistently, as compared to DMSO-treated embryos, the rapamycin-treated group exhibited less GFP-positive HSPCs in the CHT region (Figures 1D and 1E), but the HSPCs migration was not affected by rapamycin (Figures S1E and S1F). Moreover, the expression of immature T cells marker rag1 was notably decreased in rapamycin-treated embryos (Figures 1F and 1G), confirming the role of mTORC1 in HSPC development. Collectively, these results show that mTORC1 is required for zebrafish definitive hematopoiesis.Figure 1 mTORC1 signaling is required for HSPC development in zebrafish

(A) Single-optical section images showing GFP and p-4E-BP1 antibody staining under Tg(cmyb:GFP) background at 34 , 60 , 72 , and 96 hpf.

(B) Quantification of the percent of p-4E-BP1+ cells among all GFP+ cells.

(C) WISH images showing the cmyb expression at 34 , 72 , and 96 hpf. Arrows indicate AGM at 34 hpf and CHT at 72 and 96 hpf.

(D) Confocal projection images showing the cmyb:GFP expression in CHT at 72 and 96 hpf.

(E) Quantification of the number of GFP+ HSPCs. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments).

(F) WISH images showing the rag1 expression at 96 hpf.

(G) Quantification of the area of rag1+ cells in the thymus. Circles indicate the thymus. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments). Error bars represent SEM. Scale bars: 100 μm. Rapa, rapamycin.

mTORC1 inhibition leads to compromised proliferation in the CHT region

To determine how HSPC development was disrupted upon mTORC1 inhibition, we first performed proliferating cell nuclear antigen (PCNA) antibody staining and EdU (5-ethynyl-2'-deoxyuridine) to check the proliferating signals. In DMSO-treated embryos, we observed strong PCNA and EdU signals in the CHT region at 60 and 72 hpf (Figures 2A, 2B, S2A, and S2B). However, PCNA and EdU signals were notably repressed in rapamycin-treated embryos at 60 and 72 hpf (Figures 2A, 2B, S2A, and S2B), suggesting that mTORC1 signaling is essential for HSPC proliferation in the CHT region. Next, we checked cell apoptosis in mTORC1-deficient embryos by assessing the expression of TUNEL and γH2AX, which is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks (Bonner et al., 2008). Antibody staining result showed that γH2AX+ and TUNEL+ cells in the CHT region of rapamycin-treated embryos were comparable with DMSO-treated group at 60 and 72 hpf (Figures 2C, 2D, S2C, and S2D), implying that rapamycin treatment would not cause abnormal apoptosis. These data suggest that mTORC1 signaling is essential for HSPC proliferation rather than maintenance in definitive hematopoiesis.Figure 2 mTORC1 inhibition leads to compromised proliferation but normal apoptosis in the CHT region

(A) Confocal projection images showing PCNA antibody and DAPI stainings at 60 and 72 hpf.

(B) Quantification of the number of PCNA+ cells in the CHT. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = 6 embryos from three independent experiments).

(C) Confocal projection images showing γH2AX antibody and DAPI stainings at 60 and 72 hpf.

(D) Quantification of the number of γH2AX+ cells in the CHT. ns, no significant difference (n = 6 embryos from three independent experiments). Error bars represent SEM. White dashed lines mark the CHT region. Scale bars: 100 μm. Rapa, rapamycin.

The mtor and raptor mutants exhibit defective HSPC expansion

mTOR is the key catalytic subunit of mTORC1, thus important for the regulation of downstream effectors (Zoncu et al., 2011). To investigate the role of mTORC1 in hematopoiesis in a genetic way, we applied the mtorcq69 mutant. First, we assessed primitive hematopoiesis by the expression of etsrp, scl, and gata1, which exhibited comparable levels between siblings and mtor mutant group at 22 hpf (Figure S3A). Besides, mtor mutation had no effect on the vascular development and integrity, which was assessed by the transgenic line Tg(flk1:mCherryRas) and the expression of dorsal aorta marker efnb2a, posterior cardinal vein marker dab2, and endothelial marker cdh5 (Figures S3B and S3C). Moreover, mtor mutants showed normal runx1 expression at 24 and 29 hpf, and normal cmyb expression at 34 hpf (Figures 3A and 3B), indicating that mTOR is dispensable for the early phase of definitive hematopoiesis. However, loss of mtor resulted in reduced HSPCs in the CHT region and thymus (Figures 3B–3D), suggesting the defective HSPC development in the mtor mutant. Similar to the rapamycin treatment, mtor mutation caused reduced PCNA+ cells in the CHT region but did not induce excessive apoptosis (Figures 3E–3H).Figure 3 The mtor mutant exhibits defective HSPC expansion

(A) WISH images showing the runx1 expression in siblings and mtor mutants at 24 and 29 hpf.

(B) WISH images showing the cmyb expression in siblings and mtor mutants at 34, 72, and 96 hpf. Arrows indicate AGM at 34 hpf and CHT at 72 and 96 hpf.

(C) Confocal projection images showing the runx1:GFP expression in CHT at 72 and 96 hpf. Quantification of the number of GFP+ HSPCs. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments).

(D) WISH images showing the rag1 expression at 96 hpf. Circles indicate the thymus. Quantification of the area of rag1+ cells in the thymus. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments).

(E) Confocal projection images showing PCNA antibody and DAPI stainings in siblings and mtor mutants at 60 and 72 hpf.

(F) Quantification of the number of PCNA+ cells in the CHT. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = 10 embryos from three independent experiments).

(G) Confocal projection images showing γH2AX antibody and DAPI stainings at 60 and 72 hpf.

(H) Quantification of the number of γH2AX+ cells in the CHT. ns, no significant difference (n = 7 embryos from three independent experiments). Error bars represent SEM. White dashed lines mark the CHT region. Scale bars: 100 μm.

The complexes mTORC1 and mTORC2 are distinguished by different accessory proteins Raptor and Rictor, respectively (Hara et al., 2002). To validate the regulation of mTORC1 in zebrafish hematopoiesis, we examined HSPC development in the raptorcq70 mutant. In accord with the phenotype in the mtor mutant, primitive hematopoiesis, vascular development, and early definitive HSPC emergence were unaffected in the raptor−/− mutant (Figures 4A and S4A–S4C). Nevertheless, the decreased expressions of cmyb and rag1 and reduced GFP+ HSPCs in raptor mutants (Figures 4B–4D) suggested that raptor is required for zebrafish definitive HSPC development. Similarly, raptor mutants exhibited compromised proliferation while apoptosis remained unaffected (Figures 4E–4H). Then we investigated if mTORC2 also functions in definitive hematopoiesis by detecting cmyb and rag1 expressions in the rictorcq72 mutant. Surprisingly, no significant changes occurred in rictor mutants at 72 and 96 hpf (Figures S5A and S5B),but the rictor and raptor expressions were significantly increased at 60 and 72 hpf(Figures S5C and S5D), implying that mTORC2 is dispensable for zebrafish early HSPC development. Taken together, these data suggest the essential roles of mTORC1 signaling in zebrafish definitive HSPC expansion.Figure 4 The raptor mutant exhibits defective HSPC expansion

(A) WISH images showing the runx1 expression in siblings and raptor mutants at 24 and 29 hpf.

(B) WISH images showing the cmyb expression in siblings and raptor mutants at 34, 72, and 96 hpf. Arrows indicate AGM at 34 hpf and CHT at 72 and 96 hpf.

(C) Confocal projection images showing the runx1:GFP expression in CHT at 72 and 96 hpf. Quantification of the number of GFP+ HSPCs. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments).

(D) WISH images showing the rag1 expression at 96 hpf. Circles indicate the thymus. Quantification of the area of rag1+ cells in the thymus. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments).

(E) Confocal projection images showing PCNA antibody and DAPI stainings in siblings and raptor mutants at 60 and 72 hpf.

(F) Quantification of the number of PCNA+ cells in the CHT. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = 7 embryos from three independent experiments).

(G) Confocal projection images showing γH2AX antibody and DAPI stainings at 60 and 72 hpf.

(H) Quantification of the number of γH2AX+ cells in the CHT. ns, no significant difference (n = 7 embryos from three independent experiments). Error bars represent SEM. White dashed lines mark the CHT region. Scale bars: 100 μm.

Urb2 acts as a downstream effector of mTORC1 to regulate HSPC expansion

To explore the regulatory mechanism of mTORC1 signaling in hematopoiesis, the factors that are relevant to mTORC1 signaling were analyzed. Ribosome biogenesis protein Urb1 is a downstream effector of mTORC1 that is essential for protein synthesis (He et al., 2017). In contrast to the roles of Urb1 in digestive organ development, Urb2 exerts its roles in definitive hematopoiesis (Cai et al., 2018). To examine if mTORC1 regulates definitive hematopoiesis through Urb1, we performed the Urb1 overexpression assay using the Tg(hsp70l:urb1-flag) transgenic line (He et al., 2017). However, Urb1 could not correct the reduced HSPC number in rapamycin-treated embryos, implying that Urb1 is not involved in mTORC1-regulated hematopoiesis (Figures S6A and S6B). Then we explored if Urb2 participates in the regulation of mTORC1 in definitive hematopoiesis. By performing the fluorescent in situ hybridization (FISH) and qPCR, we found that the expression of urb2 was dramatically decreased in the CHT region of rapamycin-treated embryos at 60 and 72 hpf (Figures 5A and 5B). After urb2 overexpression, cmyb expression in the CHT region and rag1 expression in thymus were notably increased in rapamycin-treated embryos (Figures 5C–5F); also the PCNA signals in the CHT region were significantly increased in rapamycin-treated embryos (Figures S6C and S6D), indicating that the defective hematopoiesis was partially rescued. Similarly, urb2 overexpression could also correct the defective HSPC expansion in the mtor and raptor mutants (Figures 5G and 5H). Collectively, mTORC1 signaling controls early HSPC expansion partially through Urb2.Figure 5 Urb2 acts as a downstream effector of mTORC1 to regulate HSPC expansion

(A) FISH images showing the urb2 expression in DMSO- or rapamycin-treated embryos at 60 and 72 hpf. White dashed lines mark the CHT region.

(B) qPCR data showing the relative expression level of urb2 in the CHT at 60 and 72 hpf. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 on unpaired two-tailed t test (three independent experiments, with >20 embryos pooled per condition, per experiment).

(C) Experimental scheme illustrating the heat-shocked condition.

(D) WISH images showing the cmyb expression after urb2 overexpression in DMSO- and rapamycin-treated groups at 72 hpf.

(E) WISH images showing the rag1 expression after urb2 overexpression in DMSO- and rapamycin-treated groups at 96 hpf. Circles indicate the thymus.

(F) Quantification of the area of rag1+ cells in the thymus. ns, no significant difference. ∗∗∗p < 0.001 on unpaired two-tailed t test (n = number of total embryos from three independent experiments).

(G and H) WISH images showing the cmyb expression after urb2 overexpression in the mtor mutant (G) and raptor mutant (H) at 72 hpf. Error bars represent SEM. Scale bars: 100 μm. Rapa, rapamycin; HS, heat shock.

Discussion

In this study, we report that mTORC1 inhibition by rapamycin resulted in defective HSPC expansion, which was attributed to compromised cell proliferation. Genetic inactivation of mtor or raptor well recapitulated the defective phenotypes in rapamycin-treated embryos. Mechanistically, ribosome biogenesis protein Urb2 was significantly downregulated in the CHT region of mTORC1-deficient embryos, and HSPC developmental defects could be corrected by urb2 overexpression.

mTOR signaling is essential for embryonic development in mice; loss of mTOR, Raptor, or Rictor is lethal in early stages (Shiota et al., 2006). Thus, the lethality of mTORC loss limits the previous studies to later HSPC differentiation or maintenance. However, zebrafish mtor, raptor, and rictor mutants could survive to later developmental stages and showed quite normal body morphologies (He et al., 2019), making it possible to investigate the roles of mTORC in early HSPC development. Surprisingly, primitive hematopoiesis was unaffected in the mtor and raptor mutants (Figures S3A and S4A), indicating that the requirements of nutrients may be low for primitive HSPCs. However, our work discovers the roles of mTORC1 in early HSPC proliferation, which is identical to its roles in mouse embryonic stem cell proliferation. Although mTORC2 is reported to be essential for cell proliferation and fetal growth in mice (Shiota et al., 2006), we found that mTORC2 is dispensable for early HSPC expansion in zebrafish (Figure S5A), indicating a specific regulation of mTORCs in HSPC development. The distinct functions of mTORC1 and mTORC2 also existed in other biological processes, such as in mouse HSPC engraftment (Kalaitzidis et al., 2012; Magee et al., 2012) and zebrafish liver regeneration (He et al., 2019).

mTOR signaling regulates ribosomal RNA transcription, ribosomal protein synthesis, and ribosome assembly and thus is essential for ribosome biogenesis (Iadevaia et al., 2014). Loss of ribosomal protein, such as Rpl11, Rps19, and Rps29, resulted in hematopoietic defects in zebrafish (Bibikova et al., 2014; Danilova et al., 2011; Taylor et al., 2012), implying the significant roles of ribosome biogenesis in hematopoiesis. Our study shows that the expression of ribosome biogenesis protein Urb2 was disrupted in the CHT region upon mTORC1 inhibition (Figures 5A and 5B), which is accorded with the research in liver regeneration (He et al., 2019). Moreover, urb2 overexpression could partially rescue the defective hematopoiesis in mTORC1- deficient embryos (Figures 5D–5H). Thus, this work validates the control of mTORC1 in ribosome biogenesis during HSPC development. Although Urb1 is proved to be a downstream effector of mTORC1 to regulate zebrafish digestive organ development (He et al., 2017), we found that Urb1 could not correct the defects in mTORC1-deficient embryos (Figure S6B), proving that Urb1 and Urb2 play distinct roles in mTORC1-mediated developmental processes. Urb2 is reported to regulate HSPC development partially through the P53 pathway (Cai et al., 2018). However, our work proves that mTORC1 mediates early definitive hematopoiesis in a P53-independent way (Figures 6A–6D). This difference indicates that there must be other effectors that act downstream of mTORC1-Urb2 axis to regulate early HSPC expansion.Figure 6 mTORC1 regulates HSPC expansion in a P53-independent way

(A) WISH images showing the p53 expression in siblings and mtor mutants at 72 hpf.

(B) WISH images showing the p53 expression at 72 hpf in siblings and raptor mutants.

(C) WISH images showing the cmyb expression in siblings and mtor mutants after p53 morpholino injection at 72 hpf.

(D) WISH images showing the cmyb expression in siblings and raptor mutants after p53 morpholino injection at 72 hpf. Scale bars: 100 μm. Arrows indicate the CHT region. MO, morpholino.

Oncogenic mTOR signaling activation promotes leukemic cell growth and survival (Wang et al., 2016), so mTOR signaling could be a therapeutic target for leukemia. However, our study shows that mTORC1 inhibition by rapamycin leads to the defects in early HSPC expansion. Thus, when leukemia patients are treated with mTOR inhibitors, the age of each patient should be considered. If suffering patients are infants, the mTOR inhibitor maybe not suitable for the therapy, because it may affect HSPC development.

In summary, we show the essential roles of mTORC1 in zebrafish hematopoiesis and demonstrate that Urb2 acts downstream of mTORC1 to regulate HSPC expansion. To the best of our knowledge, the roles of mTOR in early HSPC expansion have not been reported before. Considering the difficulty for the therapy of blood disease, the understanding of HSPC development can provide useful insights into the pathogenetic mechanisms.

Experimental procedures

Resource availability

Lead contact

Requests for resources, reagents, and further information should be directed to and will be fulfilled by the lead contacts Deqin Yang (yangdeqin@hospital.cqmu.edu.cn). pchengcai@swu.edu.cn

Materials availability

The materials included in the current story are available from the corresponding author on reasonable request.

Data and code availability

This study did not generate new unique datasets or code.

Zebrafish strains

All animal research and care procedures were approved by the Ethics Committee of the Affiliated Stomatological Hospital of Chongqing Medical University (approval number: 2022-163). Zebrafish of AB genetic background, mtorcq69 mutant line (He et al., 2019), raptorcq70 mutant line (He et al., 2019), rictorcq72 mutant line (He et al., 2019), Tg(hsp70l:urb2-flag) (He et al., 2019), Tg(hsp70l:urb1-flag) (He et al., 2017), Tg(flk1:mCherryRas) (Chi et al., 2008), Tg(cmyb:GFP), and Tg(runx1:GFP) (Zhang et al., 2015) transgenic lines were raised and maintained under standard laboratory conditions. To inhibit pigmentation, embryos were treated with 0.003% PTU (1-phenyl-2-thiourea) (Sigma-Aldrich, Darmstadt, Germany) from 24 hpf.

Genotyping of mtorcq69, raptorcq70, and rictorcq72 mutant

The genotype of mtorcq69 was identified by PCR using the following primers: forward: 5′-GTTGGTGAGTAGACACAATC-3′; reverse: 5′-GACAGTCTTATGAACACTCAC-3′. The genotype of raptorcq70 was identified using the following primers: forward: 5′-CAGCAACAGTAGCAACAGTAAC-3'; reverse: 5′-ATAGTTCACACCTCATCACACAG-3′. The genotype of rictorcq72 was identified using the following primers: forward: 5′-GTTCTAAGACAAGAATGGGT-3′; reverse: 5′-CCATATACACCTAAGATGCC-3′.

Whole-mount in situ hybridizations and immunostaining

Whole-mount in situ hybridization (WISH) and FISH were performed as previously described (Cao et al., 2019; He et al., 2014) using etsrp, scl, gata1, efnb2a, dab2, cdh5, runx1, cmyb, rag1, ae1-globin, lyz, and urb2 anti-sense probes. Whole-mount immunostaining was performed as previously described (Yang et al., 2021), and the primary antibodies are listed as follows: p-4E-BP1 (Thr37/46) (1:500; Cell Signaling, USA), γH2AX (1:500; GeneTex, USA), GFP (1:1,000; ab6658, Abcam, Cambridge, MA), and PCNA (1:500; SAB2701819, Sigma, St. Louis, MO). The images of WISH were captured by SteREO Discovery V20 microscope (Carl Zeiss, Jena, Germany). The images of immunostaining were captured by LSM780 and LSM880 confocal microscope (Carl Zeiss).

Chemical treatment

To inhibit mTORC1 signaling, embryos were treated with 1 μM rapamycin in egg water from 8 hpf, and a 0.2% DMSO solution in egg water was used as a control. To maintain the pharmacological effects, the chemical solutions were renewed every 24 h.

Morpholino injection

The p53 morpholino (5′-GCGCCATTGCTTTGCAAGAATTG-3′) was obtained from Gene Tools (USA). Embryos were injected with p53 morpholino at 1–4 cell stage with 4 ng per embryo.

Heat shocked condition

Embryos of Tg(hsp70l:urb2-flag) or Tg(hsp70l:urb1-flag) were heat-shocked at 38.5°C for 40 min and then incubated at 28.5°C.

Quantitative real-time PCR

The CHT tissues were manually dissected from the larvae at 60 and 72 hpf, and then total mRNA was extracted using the TriPure isolation reagent (Roche, Indianapolis, IN). cDNAs of different groups were synthesized using the Omniscript RT kit (QIAGEN, Valencia, CA). Using the FastStart universal SYBR Green Master (Roche), qPCR was performed to quantify the fold changes of urb2 expression as previously reported (Cai et al., 2021). The expression levels of urb2 were normalized by the expression of eef1a1l1. The primers used for qPCR are listed in the following. Primers for urb2, forward: 5′-TCCACTGCTACCCTCAGGTAA-3’; reverse: 5′-TCACTCCGCTGTTTCCCTTC-3’. Primers for eef1a1l1, forward: 5′-CTGGAGGCCAGCTCAAACAT-3’; reverse: 5′-ATCAAGAAGAGTAGTACCGCTAGCATTAC-3’.

Quantification of HSPC number and rag1+ area

Confocal projection images of Tg(cmyb:GFP) or Tg(runx1:GFP) transgenic background were used to count HSPCs manually. The rag1+ areas were assessed using ZEN 2.1 blue edition (Carl Zeiss).

Image acquisition, processing, and statistical analysis

Zeiss LSM780, LSM880 and SteREO DiscoveryV20 microscopes were used to obtain image data as previously reported (He et al., 2020). All figures, labels, arrows, scale bars, and outlines were assembled and drawn using the Adobe Photoshop software. For statistical analysis, the unpaired two-tailed t test was performed using the GraphPad Prism software. p < 0.05 was considered statistically significant. Quantitative data were shown as means ± SEM.

Supplemental information

Document S1. Figures S1–S6, Table S1, and supplemental experimental procedures

Document S2. Article plus supplemental information

Acknowledgments

We thank prof. Lingfei Luo for suggestions and Dr. Xiaoyu Mao for technical assistance. This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China (31970783 , 32270888 , 32200687 ) and the 10.13039/501100012226 Fundamental Research Funds for the Central Universities (SWU-KQ22043 ).

Author contributions

D.Y., W. Huang, and P.C. contributed to the conceptualization, investigation, data analysis, and manuscript preparation. Y.Y. performed the WISH and FISH experiments. W. Hao and Z.Z. raised the zebrafish embryos. W. Huang performed other experiments. D.Y. and P.C. contributed to the project administration, funding acquisition, and figure design.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.07.011.
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