Ribosome biogenesis is required in hemogenic endothelial cells to generate hematopoietic stem cells

doi:10.21203/rs.3.rs-3929690/v1

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Ribosome biogenesis is required in hemogenic endothelial cells to generate hematopoietic stem cells

https://doi.org/10.21203/rs.3.rs-3929690/v1

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Undergoing endothelial-to-hematopoietic transition, a small fraction of embryonic aortic endothelial cells specializes into hemogenic endothelial cells (HECs) and eventually gives rise to hematopoietic stem cells (HSCs). Previously we have found that the activity of ribosome biogenesis (RiBi) is highly enriched in the HSC-primed HECs as compared with adjacent arterial endothelial cells, however, whether RiBi is required in HECs for the generation of HSC remain to be determined. Here, we found that robust RiBi was markedly augmented from HEC stage along the HSC ontogeny. Pharmacological inhibition of RiBi completely impeded the generation of HSCs in explant cultures. Moreover, disrupting RiBi selectively interrupted the HSC generation potential of HECs rather than T1 pre-HSCs, which was in line with its influence on cell cycle activity. Further investigation revealed that upon HEC specification the master transcription factor Runx1 dramatically bound to the loci of genes involved in RiBi, thereby facilitating this biological process. Taken together, our study provided functional evidence showing the indispensable role of RiBi in HSC-primed HECs to generate HSCs, providing novel insights that may contribute to improving HSC regeneration strategies.

Biological sciences/Developmental biology/Haematopoiesis

Biological sciences/Stem cells/Haematopoietic stem cells

Hematopoietic stem cells (HSCs) have the capacity to both self-renew and generate all types of blood cells, supporting the life-long hematopoiesis of an organism. The first HSC is widely believed to be generated in the aorta–gonad–mesonephros (AGM) region though the process termed endothelial to hematopoietic transition (EHT)^1–3. Briefly, a small fraction of aortic endothelial cells (AECs) acquires hemogenic potential and specializes into hemogenic endothelial cells (HECs), then orderly differentiate into pre-hematopoietic stem cells (pre-HSCs) and HSCs along the developmental trajectory^{4, 5}. Nevertheless, the underpinning mechanisms orchestrating HSC ontogeny remain poorly understood.

Muti-signaling pathways, such as developmental signals (Notch, Tgf, Fgf et al.), inflammatory signals (tumor necrosis factor, interleukin, interferon, lipopolysaccharide) and fluid biomechanical force signal have been proved to participate in regulating HSC development^6–17. These signals could mediate the expression of key hematopoietic transcription factors, including Runx1, Gata2, Meis1 and Gata3, or interplay with them to elaborately regulate EHT^18–22. Several cellular biology processes, like glycolysis to oxidative phosphorylation metabolic switch, is also revealed to determine the differential potential of HECs²³. Beyond this, a highly active cell cycle state is reported to be necessary for HEC specialization²⁴. Recently, taken advantage of single cell/low input omics technologies, the dynamic developmental path of EHT and molecular signature of HECs have been deeply dissected in multiple dimensions^25–28. Especially in our previous study, the HSC-primed HECs have been precisely identified with the use of single-cell technologies. Specific molecular characteristics and biological processes of HSC-primed HECs were further disclosed, revealing a highly enrichment in cell cycle and ribosome biogenesis (RiBi)²⁵. RiBi is an elaborate process primarily involving initial rRNA biosynthesis and subsequent ribosome assembly. Many studies have indicated the critical role of RiBi in sustaining stem cell homeostasis and fate decisions^29–32. And distinct RiBi levels were required in lineage commitment of both human and mouse hematopoiesis^{33, 34}. Notably, one recent study showing that rRNA biosynthesis, the initial step of ribosome biogenesis, directly regulates the fate transition of mouse embryonic stem cells via maintaining the nucleolar integrity and 3D chromatins configurations³⁵. Otherwise, RiBi is also involved in regulating cell death, metabolism and cell cycle progress relying on different cell contexts^{31, 36}. However, the exact roles and mechanisms underlying RiBi in regulating HSC ontogeny have yet to be elucidated.

In this study, we found that robust RiBi is significantly enriched in HECs along the HSC ontogeny. Utilizing extensive bioinformatic and functional analyses, we have corroborated the essential role of RiBi in HSC-primed HECs to generate HSCs. Consequently, our study provided a new regulatory mechanism underlying HSC generation, and offered theoretical perspective for guiding in vitro regeneration of HSCs.

Dynamic changes of ribosome biosynthesis during HSC development

Our prior studies indicated that ribosome biogenesis (RiBi) process was enriched both in human and mouse HECs^{25, 37}. To substantiate these findings, we utilized our single-cell RNA transcriptome data^{38, 39} and conducted gene set enrichment analysis (GSEA), the results reaffirmed the substantial enrichment of RiBi-related terms in both mouse and human HECs when compared to AECs (Fig S1A). Subsequently, employing a total of 529 genes associated with RiBi, we investigated their expression profiles throughout entire HSC ontogeny in mice. We found that the RiBi score was dramatically elevated during the differentiation of AEC into T1 pre-HSC, then gradually decreased (Fig. 1A). In contrast to the random selection of 529 genes, it is noteworthy that these RiBi genes were able to effectively distinguish consecutive developmental stages. Additionally, several of these genes, including ribosomal subunit genes and assembly factors displayed developmental stage-specific expression patterns, indicating the acquirement of distinct RiBi features during HSC development (Fig. 1B, Fig S1B-D). Further functional annotation of these RiBi genes unveiled that massive rRNA biosynthesis-related terms were prominently enriched in HECs and pre-HSCs, encompassing rRNA transcription, rRNA modification and Ribosome assembly (Fig. 1C, Fig S1E-H). Meanwhile, there was a notable upregulation in the expression levels of RNA polymerase I constituents responsible for rRNA synthesis in both HECs and pre-HSCs, which was in harmony with the amplified transcription of the 47S rRNA precursor (47S pre-rRNA), as evidenced by quantitative PCR assays (Fig. 1D, E).

Moreover, the expression of these RiBi-related genes positively correlated with hematopoietic feature genes, whereas it negatively correlated with genes featuring arterial endothelial identity during AEC to pre-HSCs transition (Fig. 1F, Fig S1I). Collectively, these results indicated that RiBi is notably activated and may play a crucial role during HSC development.

Disruption of RiBi impairs the generation of HSCs

To determine the role of RiBi in HSC generation, we sorted CD31⁺CD45⁻ cells from E10.0 AGM, which encompass the majority of hematopoietic precursors. Subsequently, these cells were treated with CX-5461, a specific inhibitor of RNA polymerase I that has been previously reported to disturb rRNA transcription and ultimately RiBi^{33, 36}. CX-5461 treatment could effectively restrain the expression level of 47S pre-rRNA in CD31⁺CD45⁻ cells (Fig S2C). Of note, following the co-culture assay, the proportion of hematopoietic cells (CD45⁺Kit⁺) exhibited a significant decrease concomitant with increasing concentrations of CX-5461 (Fig S2A, B). This observation was further substantiated by explant culture assay, which demonstrated a notable decline in both the number and proportion of CD45⁺ hematopoietic cells as well as immunophenotypic hematopoietic stem and progenitor cells (HSPCs, CD45⁺Kit⁺Sca1⁺CD201⁺) after treatment of CX5461 (Fig. 2A-F). Importantly, the transplantation experiments utilizing the derivatives of explant cultures showed significantly lower chimerism in the peripheral blood at 16 weeks post-transplantation in CX5461 treatment groups when compared to controls (Fig. 2G), suggesting blocking RiBi dramatically hindered the production of adult repopulating HSCs. Taken together, these results validated that RiBi was pivotal for the generation of HSCs during embryonic development.

Interfering RiBi prevents the generation of HSCs from HECs rather than pre-HSCs

The generation of HSCs is a complex and tightly regulated process that primarily involves initial HEC specification, followed by a fate transition to pre-HSCs, which ultimately acquire definitive hematopoietic function. We then separately sorted HSC-primed HECs (CD41⁻CD43⁻CD45⁻CD31⁺CD201⁺Kit⁺CD44⁺, PK44) at E10.0 and the functional T1 pre-HSCs at E11.0 as previously identified^{25, 39}, and combined flow cytometric analysis and transplantation assays to determine the stages at which the defect occurred due to the inhibition of RiBi (Fig. 3A). Following the co-culture of HSC-primed HECs, the number of CD45⁺ hematopoietic cells was significantly reduced in groups treated with CX-5461 compared to the controls (Fig. 3B). Particularly, both the quantity and proportion of the immunophenotypic HSPCs (CD45⁺Kit⁺Sca1⁺CD201⁺) also substantially diminished upon treatment with CX-5461 (Fig. 3C-E). This finding was further validated by transplantation experiments, which demonstrated a failure in reconstitution in CX-5461 treatment groups at 16 weeks post-transplantation, in contrast to the controls (Fig. 3F). PK44-represented HECs exhibit a continuum of cellular states of EHT, encompassing the endothelial, dual endothelial and hematopoietic, and hematopoietic potential^{25, 40}. We further found CX-5461 treatment only led to diminishing the hemogenic capacity of PK44 cells, while leaving endothelial potential unaffected (Fig S3A-C). Nevertheless, upon conducting co-culture experiments with T1 pre-HSCs (CD31⁺CD41^lowCD45⁻), we observed that although the progenies of T1 pre-HSCs presented a slight reduction in CX-5461 treatment groups, inhibition of RiBi in T1 pre-HSCs did not compromise their ability to generate functional HSCs (Fig. 3G-K). Overall, these results suggest that RiBi acts at HEC stage but is not required for subsequent pre-HSC maturation to form functional HSCs.

Perturbation of RiBi in HECs specifically induces cell cycle arrest

Previous study indicated the activation of cell cycle is essential for EHT and the ensuing subsequent HSPCs generation²⁴. When compared to AECs and T1 pre-HSCs, HECs showed a more active cycling status, characterized by a greater proportion of cells in the G1/S phase (Fig. 4A, B). Meanwhile, cells with higher RiBi level were more likely to enter the G1/S phase, and the expression of genes related to RiBi exhibited a positive correlation with those associated with the S/G2/M phases (Fig. 4C-E). As several studies have clarified that RiBi might be involved in regulating cell cycle progress ^{32, 36}, we therefore hypothesized that inhibition of RiBi in HECs could induce cell cycle arrest, subsequently disrupting their hemogenic capacity. Of note, when treated HECs with CX-5461 for a 48-hour short duration, there was a marked increase in the proportion of cells in the G0 phase and a corresponding decrease in the S/G2/M phase, indicating the occurrence of cell cycle arrest in HECs upon RiBi inhibition (Fig. 4F, G). To exclude the effect of CX-5461 on cell apoptosis, additional experiments were conducted to assess the cell death status, which revealing that there was no significant difference in HECs for two groups (Fig S3D, E). Intriguingly, we also found that the distribution of each cell cycle phase in the progenies of T1 pre-HSCs remained unchanged after treatment with CX-5461 (Fig. 4H, I). In summary, these findings implied that RiBi might preserve the ability of HECs to generate HSCs though modulating progression of the cell cycle.

Runx1 pre-configurates RiBi signature in AECs prior to HECs

A previous study has elucidated that Runx1 appears to directly regulate RiBi in mouse adult bone marrow HSPCs³³. To explore whether RiBi is also regulated by Runx1 during EHT, we employed and re-analyzed Runx1 ChIP-seq data from AECs to HECs in our previous report⁴¹. First, the number of Runx1 binding peaks in the genome are similar between AECs and HECs (Fig. 5A, C; Fig S4A). Consistent with the observation that the amount of open genomic regions tending to be occupied by Runx1 was also similar between AECs and HECs as evidenced by ATAC-seq data (Fig. 5B, C). Second, we observed that over half of the Runx1-binding peaks were annotated to promoter regions and exhibited three major enrichment patterns, comprising AEC-specific (C1), HEC-specific (C3) and common shared in AECs and HECs (C2) (Fig. 5C, D). Furthermore, we conducted Gene Ontology (GO) analysis on the genes that exhibit Runx1 binding within their promoter regions accompanying with upregulated expression, which indicated enrichment of several RiBi-related terms as early as in C1 including ribonucleoprotein complex export from nucleus and ribonucleoprotein complex localization, while more specific terms, such as ribonucleoprotein complex biogenesis, regulation of transcription by RNA polymerase I, and ribosome biogenesis were significantly enriched in C2 (Fig. 5E, Fig S4B-D). Accurately, we found that Runx1 exhibited a preference for binding to promoters rather than introns of RiBi-related genes as well as the genes encoding ribosomal proteins in both AECs and HECs, and the expression level of these genes was up-regulated in HECs compared with AECs (Fig. 5F-H). Collectively, these results suggested that Runx1 already binds to RiBi-related genes in AECs to facilitate the subsequent up-regulation of these genes in HECs.

Next, we utilized and deep-analyzed the published scRNA-seq data²⁶ of EHT-related cells from Runx1^+/+ and Runx1^+/− littermates, in which the identified pre-HEC transcriptomically corresponds to the AEC we annotated^{25, 38}. We indeed observed a continuous increase in RiBi level along the development path from conflux AECs to IACs in Runx1^+/+ (wild type, WT) embryos (Fig. 5I). Notably, Runx1 haploinsufficiency resulted in the increase of the proportion of pre-HEC/AEC compared to control²⁶. Strikingly, the expression levels of several ribosomal protein genes in pre-HEC/AEC were indeed found to be reduced in Runx1^+/− compared with in Runx1^+/+ (Fig. 5J), further confirming the notion that Runx1 was involved in modulating RiBi gene expression as early as in AECs. In summary, these results together demonstrated that the binding of Runx1 onto the genome participating in regulating RiBi gene expression might contribute to HSC development.

Excavating molecular regulatory mechanisms of HSC ontogeny can provide new clues for HSC regeneration in vitro. Many studies have shown that EHT is precisely regulated by several master transcription factors and signaling pathways^6–22. Some fundamental biological processes, such as metabolism and cell cycle, have also been reported to be involved in the regulation of HSC development^{23, 24, 42}. Recent studies indicated the pivotal role of RiBi in orchestrating the homeostasis and fate decisions of stem cell^{30, 34, 35, 43}. However, the precise significance of RiBi in HSC development remains inadequately elucidated.

Preliminary, we found that robust RiBi was markedly augmented from HEC stage along the HSC ontogeny. Further screening reveled that the synthesis of rRNA, the initial stage of RiBi, as well as muti-steps of RiBi were significantly enhanced in HECs. Currently, selectively inhibiting RiBi within a specific cell population in vivo is exceedingly challenging. Therefore, a selective molecule CX-5461 identified as an effective RiBi inhibitor was used to study the function of RiBi in HSC generation^{31, 36, 44}. Functionally, through organ culture and transplantation experiments, we observed that reduced RiBi blocked the generation of HSCs in AGM region. Subsequently, in vitro and ex vivo experiments clarified decreased RiBi hampers the generation of HSCs from HECs rather than T1 pre-HSCs. Mechanistically, we discovered that reduced RiBi in HECs leads to more cells arresting in G0 phase. It has been documented that blocking cell cycle in HECs would interrupt the generation of HSPCs²⁴.

However, how RiBi regulated the cell cycle progression in HECs still needs to be explored. Shi et al. discovered that core ribosomal proteins exhibit heterogeneity in constituting translating ribosomes, which seems to facilitate the preferential translation of certain mRNAs⁴⁵. In line with this observation, our findings indicated that numerous ribosomal proteins and associated factors display distinct stage-specific expression patterns (Fig S1B). This implies the existence of specialized translation events that could be substantiated by single-cell ribosome profiling (scRibo-seq) as the technology becomes more widespread in future study. Moreover, through the integration analysis of omics data, we uncovered that Runx1 bound to RiBi-related genes locus and upregulated their expression in HECs compared with AECs.

Summarizing, our work shed light on the essential role of RiBi in HSC development and preliminarily expounded its possible upstream and downstream regulatory mechanisms. These findings enhance the comprehension of the precise regulatory mechanisms underlying EHT and offer a novel perspective for the in vitro regeneration of HSCs.

Mice

All mice were maintained on C57BL/6 genetic background and bred in Specific Pathogen Free (SPF) condition at the Laboratory Animal Center of Academy of Military Medical Sciences. E9.5-E11.5 embryos were confirmed by counting the somite pairs (sp). The caudal half or AGM region was dissected as previously reported⁴. The experimental manipulations of mice were approved by the Animal Care and Use Committee of the Institute.

Explant culture

Caudal half regions were isolated from E9.5 embryos and cultured using an ex vivo explant culture system as previously described⁴⁶. The medium used for the caudal half region culture contained IMDM (Hyclone) and 20% fetal bovine serum (Hyclone) in the presence or absence of 100 nM CX-5461 (MCE, HY-13323). After being cultured for 48 hours, caudal half regions were dissociated in collagenase for flow cytometry analysis and transplantation.

Transplantation assay

For transplantation of freshly isolated cells from explant culture or co-cultured cells from HECs, ten to twelve-week-old female recipients (CD45.2/2) were subjected to a split dose of 9 Gy γ-irradiation (⁶⁰Co) at an interval of two hours. Donor cells (CD45.1/2), together with 2 × 10⁴ nucleated bone marrow cells (CD45.2/2), were injected into irradiated adult recipients (CD45.2/2) via the tail vein. For transplantation of co-cultured cells from T1 pre-HSCs, ten to twelve-week-old female recipients (CD45.1/1) were subjected to a split dose of 9 Gy γ-irradiation (⁶⁰Co) at an interval of two hours. Donor cells (CD45.2/2), together with 2 × 10⁴ nucleated bone marrow cells (CD45.1/1), were injected into irradiated adult recipients (CD45.1/1) via the tail vein. The recipients demonstrating ≥ 5% donor-derived chimaerism in peripheral blood were counted as successfully reconstituted.

Curated gene sets

Several gene sets were curated from the published data. The set of genes associated with RiBi was curated from the Gene Ontology (GO) database (http://geneontology.org/)⁴⁷. To accomplish this, genes involved in biological processes were meticulously searched using the keywords “mouse,” “ribosome,” and “rRNA”. Ultimately, 529 genes were obtained as RiBi-related genes for subsequent analysis. The genes including Gja4, Unc5b, Mecom, Hey1, Efnb2, Dll4, Vegfc, Epas1, Cxcr4, Igfbp3 were characterized as arterial endothelia features²⁵. While a comprehensive set of 98 genes has been designated to depict the features of hematopoietic cells³⁹.

Processing and integration of scRNA-seq data

Cellranger was employed for the trimming, alignment, and quantitation of 10x scRNA-seq data. The following outlines the quantification process for STRT scRNA-seq analysis. Herein, the UMI-based scRNA-seq approach was utilized to quantify the gene expression profile within individual cells. Initially, Cutadapt (v4.4) was applied to trim Read 1 to remove the TSO sequence and polyA tail segment. Reads contaminated with adapter sequences or plagued by low-quality bases (N > 10%) are excluded. Subsequently, TopHat v2.0.12⁴⁸ performs the alignment against the mm10 mouse transcriptome database (UCSC). Unique molecular identifiers (UMIs) coupled with gene pairs were quantified using HTSeq⁴⁹, with subsequent categorization according to cell-specific barcodes. Culminating the analysis, the abundance of unique UMIs attributed to each gene within solitary cells was equated to the corresponding transcript copy number.

Processing of ChIP-seq/ATAC-seq data

The processing steps of ChIP-seq/ATAC-seq data are as follows. First, FastQC (v.0.11.8) was utilized to assess the quality of the sequencing data. Subsequently, fastp software⁵⁰ was employed to trim low-quality reads and adapter sequences. Then, bowtie2⁵¹ was used to align the processed read segments to the mm10 reference genome, setting the maximum number of allowed mismatches to 1. Only alignments with a mapping quality score exceeding 30 were considered uniquely mapped reads. Next, the aligned reads were sorted using Samtools⁵², and the sorted files were utilized as input for Picard software (v.2.2.4), where the MarkDuplicates function was applied to remove duplicate reads for downstream analysis. To account for potential sequencing depth biases across different batches, Samtools⁵² was again used to randomly down sample the reads and standardize them to a uniform level. Lastly, the bamCoverage function in deepTools (v1.5.12)⁵³ was used to generate bigWig files, which facilitate subsequent visualization in the Integrative Genomics Viewer (IGV) or on the UCSC Genome Browser (https://genome.ucsc.edu/). MACS2 software^{54, 55} (v2.2.6) was employed to identify peaks within ChIP-seq/ATAC-seq data. Here, the enrichment mode selected is "--narrow". Enrichment peak annotations across the entire genome were carried out using the ChIPseeker (v1.20.0)⁵⁶ and clusterProfiler⁵⁷. Specifically, the promoter region has been defined as the area spanning 3 kb both upstream and downstream of the transcription start site (TSS).

Identification of signature genes and GO enrichment

The ‘FindMarkers’ or ‘FindAllMarkers’ function, utilizing the default Wilcoxon rank sum test, was deployed to identify signature genes. For GO enrichment analysis, Metascape (http://metascape.org)⁵⁸ and clusterProfiler⁵⁷ were utilized. Within these analyses, biological processes with P-values corrected by the Benjamini-Hochberg method below 0.05 are defined as significantly enriched categories within the respective population.

Statistical analysis

No statistical methods were used to predetermine sample size. For statistical analysis between groups, unpaired two-tailed Student’s t-test was used to calculate P values, unless otherwise specified. Statistical analysis was carried out using GraphPad Prism 8.

Data availability

The data that support this study are available from the corresponding authors upon reasonable request. The data employed in this study was existing data available in GEO under accession numbers GSE185555, GSE139389, GSE67120, GSE137117, GSE135202 and GSE161328.

Acknowledgments

This work was supported by the National Key R&D Program of China (2020YFA0112400, 2022YFA1103501, 2021YFA1100901) and the National Natural Science Foundation of China (82122004, 82070104, 81900115, 31930054, 82000107).

Author Contribution

YL, BL and JZ conceived and designed the study; HW and HC performed cell sorting with the help from YN; HW performed co-culture and transplantation assay with the help from CW, SH and XT; XT performed qPCR with the help of YJ and YL; DL performed bioinformatics analysis with the help from ZL; YL, JZ and DL wrote the manuscript. All authors reviewed the manuscript.

Disclosures

No conflicts of interest to disclose.

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Ribosome biogenesis is required in hemogenic endothelial cells to generate hematopoietic stem cells

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Abstract

Figures

Introduction

Results

Dynamic changes of ribosome biosynthesis during HSC development

Disruption of RiBi impairs the generation of HSCs

Interfering RiBi prevents the generation of HSCs from HECs rather than pre-HSCs

Perturbation of RiBi in HECs specifically induces cell cycle arrest

Runx1 pre-configurates RiBi signature in AECs prior to HECs

Discussion

Materials and methods

Mice

Explant culture

Transplantation assay

Curated gene sets

Processing and integration of scRNA-seq data

Processing of ChIP-seq/ATAC-seq data

Identification of signature genes and GO enrichment

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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