The heterogeneity of erythroid cells: insight at the single-cell transcriptome level.

Erythroid cells, the most prevalent cell type in blood, are one of the earliest products and permeate through the entire process of hematopoietic development in the human body, the oxygen-transporting function of which is crucial for maintaining overall health and life support. Previous investigations into erythrocyte differentiation and development have primarily focused on population-level analyses, lacking the single-cell perspective essential for comprehending the intricate pathways of erythroid maturation, differentiation, and the encompassing cellular heterogeneity. The continuous optimization of single-cell transcriptome sequencing technology, or single-cell RNA sequencing (scRNA-seq), provides a powerful tool for life sciences research, which has a particular superiority in the identification of unprecedented cell subgroups, the analyzing of cellular heterogeneity, and the transcriptomic characteristics of individual cells. Over the past decade, remarkable strides have been taken in the realm of single-cell RNA sequencing technology, profoundly enhancing our understanding of erythroid cells. In this review, we systematically summarize the recent developments in single-cell transcriptome sequencing technology and emphasize their substantial impact on the study of erythroid cells, highlighting their contributions, including the exploration of functional heterogeneity within erythroid populations, the identification of novel erythrocyte subgroups, the tracking of different erythroid lineages, and the unveiling of mechanisms governing erythroid fate decisions. These findings not only invigorate erythroid cell research but also offer new perspectives on the management of diseases related to erythroid cells.

Metabolite and protein shifts in mature erythrocyte under hypoxia.

As the only cell type responsible for oxygen delivery, erythrocytes play a crucial role in supplying oxygen to hypoxic tissues, ensuring their normal functions. Hypoxia commonly occurs under physiological or pathological conditions, and understanding how erythrocytes adapt to hypoxia is fundamental for exploring the mechanisms of hypoxic diseases. Additionally, investigating acute and chronic mountain sickness caused by plateaus, which are naturally hypoxic environments, will aid in the study of hypoxic diseases. In recent years, increasingly developed proteomics and metabolomics technologies have become powerful tools for studying mature enucleated erythrocytes, which has significantly contributed to clarifying how hypoxia affects erythrocytes. The aim of this article is to summarize the composition of the cytoskeleton and cytoplasmic proteins of hypoxia-altered erythrocytes and explore the impact of hypoxia on their essential functions. Furthermore, we discuss the role of microRNAs in the adaptation of erythrocytes to hypoxia, providing new perspectives on hypoxia-related diseases.

SETD7 promotes lateral plate mesoderm formation by modulating the Wnt/ß-catenin signaling pathway.

The role of SET domain containing 7 (SETD7) during human hematopoietic development remains elusive. Here, we found that deletion of SETD7 attenuated the generation of hematopoietic progenitor cells (HPCs) during the induction of hematopoietic differentiation from human embryonic stem cells (hESCs). Further analysis specified that SETD7 was required for lateral plate mesoderm (LPM) specification but dispensable for the generation of endothelial progenitor cells (EPCs) and HPCs. Mechanistically, rather than depending on its histone methyltransferase activity, SETD7 interacted with ß-catenin at lysine residue 180 facilitated its degradation. Diminished SETD7 expression led to the accumulation of ß-catenin and the consequent activation of the Wnt signaling pathway, which altered LPM patterning and facilitated the production of paraxial mesoderm (PM). Taken together, the findings indicate that SETD7 is related to LPM and PM patterning via posttranslational regulation of the Wnt/ß-catenin signaling pathway, providing novel insights into mesoderm specification during hematopoietic differentiation from hESCs.

Molecular analysis of phenotypic heterogeneity in JAK2V617F-positive myeloproliferative neoplasms reveals a potential target for therapy.

JAK2V617F is the most frequent mutation in BCR-ABL-negative myeloproliferative neoplasms (MPNs). It is an important but not the only determinant of MPN phenotype. We performed high-throughput sequencing on JAK2V617F+ essential thrombocythaemia (ET) and polycythaemia vera (PV) patient samples to unveil factors involved in phenotypic heterogeneity and to identify novel therapeutic targets for MPN. Two concurrent mutations that may affect phenotype were identified, including mutations in SH2B3, which is primarily prevalent in PV, and SF3B1, which is more commonly mutated in ET. Next, we conducted transcriptomic analysis at the haematopoietic stem cell (HSC) and megakaryocyte (MK)-erythroid progenitor (MEP) levels. Inflammatory signalling pathways were elevated in both ET HSCs and MEPs, unlike in PV HSCs and MEPs. Notably, Wnt/ß-catenin signalling was uniquely upregulated during ET haematopoietic differentiation from HSC to MEP, and inhibiting Wnt/ß-catenin signalling blocked MK differentiation in vitro. Consistently, Wnt/ß-catenin inhibitor administration decreased platelet counts in JAK2V617F+ MPN mice by blocking MEPs and MK progenitors and by inhibiting maturation of MKs, while in wild-type mice, Wnt/ß-catenin inhibitor did not significantly reduce platelet counts. In conclusion, our findings provide new insights into the mechanisms underlying phenotypic differentiation of JAK2V617F+ PV and ET and indicate Wnt/ß-catenin signalling as a potential therapeutic target for MPN.

Expression profiles analysis identifies specific interferon-stimulated signatures as potential diagnostic and predictive indicators of JAK2V617F + myelofibrosis.

Objective: This study aimed to identify specific dysregulated genes with potential diagnostic and predictive values for JAK2V617F + myelofibrosis. Methods: Two gene expression datasets of CD34+ hematopoietic stem and progenitor cells (HSPCs) from patients with JAK2V617F + myeloproliferative neoplasm (MPN) [n = 66, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)] and healthy controls (HC) (n = 30) were acquired from the GEO (Gene Expression Omnibus) database. The differentially expressed genes (DEGs) were screened between each JAK2V617F + MPN entity and HC. Subsequently, functional enrichment analyses, including Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and Gene Set Enrichment Analysis (GSEA), were conducted to decipher the important biological effects of DEGs. Protein-protein interaction (PPI) networks of the DEGs were constructed to identify hub genes and significant modules. Another two gene expression profiles of patients with JAK2V617F + MPN [n = 23, including PV, ET, secondary myelofibrosis (SMF), and PMF] and HC (n = 6) from GEO were used as external validation datasets to prove the reliability of the identified signatures. Results: KEGG analysis revealed the upregulated genes in three JAK2V617F + MPN entities compared with HC were essentially enriched in inflammatory pathways and immune response signaling pathways, and the number of these pathways enriched in PMF was obviously more than that in PV and ET. Following the PPI analysis, 10 genes primarily related to inflammation and immune response were found upregulated in different JAK2V617F + MPN entities. In addition, Reactome enrichment analysis indicated that interferon signaling pathways were enriched specifically in PMF but not in PV or ET. Furthermore, several interferon (IFN)-stimulated genes were identified to be uniquely upregulated in JAK2V617F + PMF. The external datasets validated the upregulation of four interferon-related genes (OAS1, IFITM3, GBP1, and GBP2) in JAK2V617F + myelofibrosis. The receiver operating characteristic (ROC) curves indicate that the four genes have high area under the ROC curve (AUC) values when distinguishing JAK2V617F + myelofibrosis from PV or ET. Conclusion: Four interferon-stimulated genes (OAS1, IFITM3, GBP1, and GBP2) exclusively upregulated in JAK2V617F + myelofibrosis might have the potential to be the auxiliary molecular diagnostic and predictive indicators of myelofibrosis.

Decoding the pathogenesis of Diamond-Blackfan anemia using single-cell RNA-seq.

Ribosomal protein dysfunction causes diverse human diseases, including Diamond-Blackfan anemia (DBA). Despite the universal need for ribosomes in all cell types, the mechanisms underlying ribosomopathies, which are characterized by tissue-specific defects, are still poorly understood. In the present study, we analyzed the transcriptomes of single purified erythroid progenitors isolated from the bone marrow of DBA patients. These patients were categorized into untreated, glucocorticoid (GC)-responsive and GC-non-responsive groups. We found that erythroid progenitors from untreated DBA patients entered S-phase of the cell cycle under considerable duress, resulting in replication stress and the activation of P53 signaling. In contrast, cell cycle progression was inhibited through induction of the type 1 interferon pathway in treated, GC-responsive patients, but not in GC-non-responsive patients. Notably, a low dose of interferon alpha treatment stimulated the production of erythrocytes derived from DBA patients. By linking the innately shorter cell cycle of erythroid progenitors to DBA pathogenesis, we demonstrated that interferon-mediated cell cycle control underlies the clinical efficacy of glucocorticoids. Our study suggests that interferon administration may constitute a new alternative therapeutic strategy for the treatment of DBA. The trial was registered at www.chictr.org.cn as ChiCTR2000038510.

Hematopoietic Stem Cell Heterogeneity Is Linked to the Initiation and Therapeutic Response of Myeloproliferative Neoplasms.

The implications of stem cell heterogeneity for disease pathogenesis and therapy are poorly defined. JAK2V617F+ myeloproliferative neoplasms (MPNs), harboring the same mutation in hematopoietic stem cells (HSCs), display diverse phenotypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These chronic malignant disorders are ideal models to analyze the pathological consequences of stem cell heterogeneity. Single-cell gene expression profiling with parallel mutation detection demonstrated that the megakaryocyte (Mk)-primed HSC subpopulation expanded significantly with enhanced potential in untreated individuals with JAK2V617F+ ET, driven primarily by the JAK2 mutation and elevated interferon signaling. During treatment, mutant HSCs were targeted preferentially in the Mk-primed HSC subpopulation. Interestingly, homozygous mutant HSCs were forced to re-enter quiescence, whereas their heterozygous counterparts underwent apoptosis. This study provides important evidence for the association of stem cell heterogeneity with the pathogenesis and therapeutic response of a malignant disease.

A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells.

Alternative splicing (AS) leads to transcriptome diversity in eukaryotic cells and is one of the key regulators driving cellular differentiation. Although AS is of crucial importance for normal hematopoiesis and hematopoietic malignancies, its role in early hematopoietic development is still largely unknown. Here, by using high-throughput transcriptomic analyses, we show that pervasive and dynamic AS takes place during hematopoietic development of human pluripotent stem cells (hPSCs). We identify a splicing factor switch that occurs during the differentiation of mesodermal cells to endothelial progenitor cells (EPCs). Perturbation of this switch selectively impairs the emergence of EPCs and hemogenic endothelial progenitor cells (HEPs). Mechanistically, an EPC-induced alternative spliced isoform of NUMB dictates EPC specification by controlling NOTCH signaling. Furthermore, we demonstrate that the splicing factor SRSF2 regulates splicing of the EPC-induced NUMB isoform, and the SRSF2-NUMB-NOTCH splicing axis regulates EPC generation. The identification of this splicing factor switch provides a new molecular mechanism to control cell fate and lineage specification.

Heat shock transcription factor 1 regulates the fetal γ-globin expression in a stress-dependent and independent manner during erythroid differentiation.

Heat shock transcription factor 1 (HSF1) is a highly versatile transcription factor that, in addition to protecting cells against proteotoxic stress, is also critical during diverse developmental processes. Although the functions of HSF1 have received considerable attention, its potential role in ß-globin gene regulation during erythropoiesis has not been fully elucidated. Here, after comparing the transcriptomes of erythrocytes differentiated from cord blood or adult peripheral blood hematopoietic progenitor CD34+ cells in vitro, we constructed the molecular regulatory network associated with ß-globin genes and identified novel and putative globin gene regulators by combining the weighted gene coexpression network analysis (WGCNA) and context likelihood of relatedness (CLR) algorithms. Further investigation revealed that one of the identified regulators, HSF1, acts as a key activator of the γ-globin gene in human primary erythroid cells in both erythroid developmental stages. While during stress, HSF1 is required for heat-induced globin gene activation, and HSF1 downregulation markedly decreases globin gene induction in K562 cells. Mechanistically, HSF1 occupies DNase I hypersensitive site 3 of the locus control region upstream of ß-globin genes via its canonical binding motif. Hence, HSF1 executes stress-dependent and -independent roles in fetal γ-globin regulation during erythroid differentiation.

Long non-coding RNA-dependent mechanism to regulate heme biosynthesis and erythrocyte development.

In addition to serving as a prosthetic group for enzymes and a hemoglobin structural component, heme is a crucial homeostatic regulator of erythroid cell development and function. While lncRNAs modulate diverse physiological and pathological cellular processes, their involvement in heme-dependent mechanisms is largely unexplored. In this study, we elucidated a lncRNA (UCA1)-mediated mechanism that regulates heme metabolism in human erythroid cells. We discovered that UCA1 expression is dynamically regulated during human erythroid maturation, with a maximal expression in proerythroblasts. UCA1 depletion predominantly impairs heme biosynthesis and arrests erythroid differentiation at the proerythroblast stage. Mechanistic analysis revealed that UCA1 physically interacts with the RNA-binding protein PTBP1, and UCA1 functions as an RNA scaffold to recruit PTBP1 to ALAS2 mRNA, which stabilizes ALAS2 mRNA. These results define a lncRNA-mediated posttranscriptional mechanism that provides a new dimension into how the fundamental heme biosynthetic process is regulated as a determinant of erythrocyte development.

[Knockout of LSD1 Gene by CRISPR/Cas9 System Significantly Inhibited Proliferation and Expression of CD235a in K562 Cells].

OBJECTIVE: To study the effect of LSD1 knock-out on human chronic myeloid leukemia cells(K562 cells). METHODS: The LSD1 gene in K562 cells was knocked-out specifically by using CRISPR/Cas9 system, the single cells were gained by flow cytometric sorting technique, the LSD1+/- and LSD1-/- cell lines were gained after amplificantion and culture, identification of Western blot and sequencing. The MTS assay was used to detect the effect of LSD1 knockout on the proliferation of K562 cells, the flow cytometry was used to examine the expression of K562 cell surface marker after LSD1 knockout. RESULTS: The LSD1 stable knockout cell line of K562 (LSD1+/- and LSD1-/-)were successfully costructed. It was found that knockout of LSD1 significantly inhibited the proliferation of K562 and the expression of CD235a. CONCLUSION: LSD1 plays a key role in the regulation of K562 cell proliferation and CD235a expression.

[Rbb4l enhances TGF-ß/Nodal signaling and promotes zebrafish embryonic dorsolization].

The TGF-ß/Nodal signaling pathway plays an important role in the zebrafish dorsoventral patterning process. To further explore the function and mechanism of this signaling pathway, we identified a set of Smad2/3a interacting proteins by the yeast two-hybrid screen. Rbb4l (Retinoblastoma binding protein 4, like) is one of the identified proteins. Human RBBP4 (Retinoblastoma binding protein 4), the homolog of zebrafish Rbb4l, has been shown to form complexes with other chromatin modifiers, but its roles in embryonic development remain unknown. In this study, we showed that Rbb4l directly interacted with Smad3a and enhances TGF-ß/Nodal signaling. In zebrafish embryos, rbb4l overexpression resulted in an expanded expression of dorsal markers with a reduction of ventral markers expression, suggesting a dorsalizing function. On the contrary, rbb4l knockdown caused ventralized phenotype of the embryos at 24 hours post-fertilization (hpf). Furthermore, a series of rescue experiments showed that rbb4l failed to cause embryonic dorsalization in the absence of Nodal signal. Together, our data suggested that Rbb4l acts as an enhancer of Nodal/Smad2/3 signaling during embryogene-sis, and depends on the existence of Nodal signaling.

Loss of zygotic NUP107 protein causes missing of pharyngeal skeleton and other tissue defects with impaired nuclear pore function in zebrafish embryos.

The Nup107-160 multiprotein subcomplex is essential for the assembly of nuclear pore complexes. The developmental functions of individual constituents of this subcomplex in vertebrates remain elusive. In particular, it is unknown whether Nup107 plays an important role in development of vertebrate embryos. Zebrafish nup107 is maternally expressed and its zygotic expression becomes prominent in the head region and the intestine from 24 h postfertilization (hpf) onward. In this study, we generate a zebrafish mutant line, nup107(tsu068Gt), in which the nup107 locus is disrupted by an insertion of Tol2 transposon element in the first intron and as a result it fails to produce normal transcripts. Homozygous nup107(tsu068Gt) mutant embryos exhibit tissue-specific defects after 3 days postfertilization (dpf), including loss of the pharyngeal skeletons, degeneration of the intestine, absence of the swim bladder, and smaller eyes. These mutants die at 5-6 days. Extensive apoptosis occurs in the affected tissues, which is partially dependent on p53 apoptotic pathways. In cells of the defective tissues, FG-repeat nucleoporins are disturbed and nuclear pore number is reduced, leading to impaired translocation of mRNAs from the nucleus to the cytoplasm. Our findings shed new light on developmental function of Nup107 in vertebrates.

Angiomotin-like2 gene (amotl2) is required for migration and proliferation of endothelial cells during angiogenesis.

Angiogenesis involves sprouting, migration, and proliferation of endothelial cells. The angiomotin-like2 gene (amotl2) has been found in blood vessels in zebrafish embryos, but its function in angiogenesis and underlying mechanisms remain unknown. In this study, we demonstrate that knockdown of amotl2 in zebrafish Tg(fli1:EGFP)(y1) and Tg(fli1:nEGFP)(y7) transgenic embryos impairs the intersegmental vessel growth and suppresses proliferation of endothelial cells. Transplantation experiments indicate that function of amotl2 in intersegmental vessel growth is cell-autonomous. AMOTL2 knockdown in cultured human umbilical vein endothelial cells also inhibits cell proliferation and migration and disrupts cell polarity, ultimately interrupting the formation of vascular tube-like structures. Amotl2 promotes MAPK/ERK activation via c-Src, which is dependent on phosphorylation of tyrosine residue at position 103 but independent of the C-terminal PDZ-binding domain. Taking together, our data indicate that Amotl2 plays a pivotal role in polarity, migration and proliferation of angiogenic endothelial cells.

Grhl2 deficiency impairs otic development and hearing ability in a zebrafish model of the progressive dominant hearing loss DFNA28.

Congenital and progressive hearing impairment is a common distressing disease. The progressive dominant hearing loss DFNA28 in human is associated with a frameshift mutation of Grainyhead-like 2 (GRHL2) but its etiology and mechanism remain unknown. Here we report a zebrafish grhl2b(T086) mutant line in which grhl2b expression is interrupted by an insertion of a Tol2 transposon element. The mutants exhibit enlarged otocysts, smaller or eliminated otoliths, malformed semicircular canals, insensitiveness to sound stimulation and imbalanced swimming motion. Since grainyhead-like family members can regulate epithelial adhesion, we examined the expression of some genes encoding junction proteins in mutants. We show that the expression of claudin b (cldnb) and epcam is abolished or dramatically reduced and apical junctional complexes are abnormal in otic epithelial cells of mutant embryos. Co-injection of cldnb and epcam mRNA could largely rescue the mutant phenotype. Injection of human wild-type GRHL2 mRNA but not the mutant GRHL2 mRNA derived from DFNA28 patients into grhl2b(T086) mutant embryos could rescue the inner-ear defects. Furthermore, we demonstrate that Grhl2b directly binds to the enhancers and promotes the expression of cldnb and epcam. Thus, this work reveals an evolutionarily conserved function of Grhl2 in otic development and provides a fish model for further studying mechanisms of Grhl2-related hearing loss.

Interruption of cenph causes mitotic failure and embryonic death, and its haploinsufficiency suppresses cancer in zebrafish.

Kinetochore proteins associate with centromeric DNA and spindle microtubules and play essential roles in chromosome segregation during mitosis. In this study, we uncovered a zebrafish mutant, stagnant and curly (stac), that carries the Tol2 transposon element inserted at the kinetochore protein H (cenph) locus. Mutant embryos exhibit discernible cell death as early as 20 hours postfertilization, extensive apoptosis, and upward curly tail during the pharyngula period and deform around 5 days postfertilization. The stac mutant phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph deficiency for stac mutants. We demonstrate that the intrinsic apoptosis pathway is hyperactivated in stac mutants and that p53 knockdown partially blocks excess apoptosis in stac mutants. Mitotic cells in stac mutants show chromosome missegregation and are usually arrested in G(2)/M phase. Furthermore, compared with wild type siblings, heterozygous stac fish develop invasive tumors at a dramatically reduced rate, suggesting a reduced cancer risk. Taken together, our findings uncover an essential role of cenph in mitosis and embryonic development and its association with tumor development.