In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs)
via
...endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an
Hlf-tdTomato
reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45
–
and CD45
+
pre-HSCs in the embryonic day (E) 10.5 AGM region. By
in vitro
co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45
–
counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45
+
E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel
Hlf-CreER
mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.
Hematopoietic stem cells (HSCs) are situated at the top of the adult hematopoietic hierarchy in mammals and give rise to the majority of blood cells throughout life. Recently, with the advance of ...multiple single-cell technologies, researchers have unprecedentedly deciphered the cellular and molecular evolution, the lineage relationships, and the regulatory mechanisms underlying HSC emergence in mammals. In this review, we describe the precise vascular origin of HSCs in mouse and human embryos, emphasizing the conservation in the unambiguous arterial characteristics of the HSC-primed hemogenic endothelial cells (HECs). Serving as the immediate progeny of some HECs, functional pre-HSCs of mouse embryos can now be isolated at single-cell level using defined surface marker combinations. Heterogeneity regrading cell cycle status or lineage differentiation bias within HECs, pre-HSCs, or emerging HSCs in mouse embryos has been figured out. Several epigenetic regulatory mechanisms of HSC generation, including long noncoding RNA, DNA methylation modification, RNA splicing, and layered epigenetic modifications, have also been recently uncovered. In addition to that of HSCs, the cellular and molecular events underlying the development of multiple hematopoietic progenitors in human embryos/fetus have been unraveled with the use of series of single-cell technologies. Specifically, yolk sac-derived myeloid-biased progenitors have been identified as the earliest multipotent hematopoietic progenitors in human embryo, serving as an important origin of fetal liver monocyte-derived macrophages. Moreover, the development of multiple hematopoietic lineages in human embryos such as T and B lymphocytes, innate lymphoid cells, as well as myeloid cells like monocytes, macrophages, erythrocytes, and megakaryocytes has also been depicted and reviewed here.
ABSTRACTHematopoietic stem cells (HSCs) have the capacity for self‐renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production ...of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self‐renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low‐density lipoprotein receptor‐related protein 5 (Lrp5) and low‐density lipoprotein receptor‐related protein 6 (Lrp6) mice in adult hematopoiesis via Vav‐Cre Loxp system. Inactivation of Lrp5 and ‐6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and ‐6 double deficiency HSCs showed intrinsic defects in self‐renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up‐regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down‐regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and ‐6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and ‐6 are required for the self‐renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.—Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.‐H., Yu, S. Lrp5 and Lrp6 are required for maintaining self‐renewal and differentiation of hematopoietic stem cells. FASEB J. 33, 5615–5625 (2019). www.fasebj.org
Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors ...including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.
Long noncoding RNAs (lncRNAs) are emerging as critical mediators of various biological processes in the immune system. The current data showed that the lncRNA Malat1 is highly expressed in T cell ...subsets, but the function of Malat1 in T cell remains unclear. In this study, we detected the T cell development and both CD8
+
and CD4
+
T cell response to LCMV infection using Malat1
−/-
mice model. To our surprise, there were no significant defects in thymocytes at different developmental stages and the peripheral T cell pool with ablation of Malat1. During LCMV infection, Malat1
−/-
mice exhibited normal effector and memory CD8
+
T cells as well as T
FH
cells differentiation. Our results indicated that Malat1 is not essential for T cell development and T cell-mediated antiviral response though it expresses at very high level in different T cell populations.
The underlying mechanisms of thymocyte maturation remain largely unknown. Here, we report that serine/arginine-rich splicing factor 1 (SRSF1) intrinsically regulates the late stage of thymocyte ...development. Conditional deletion of SRSF1 resulted in severe defects in maintenance of late thymocyte survival and a blockade of the transition of TCRβ
CD24
CD69
immature to TCRβ
CD24
CD69
mature thymocytes, corresponding to a notable reduction of recent thymic emigrants and diminished periphery T cell pool. Mechanistically, SRSF1 regulates the gene networks involved in thymocyte differentiation, proliferation, apoptosis, and type I interferon signaling pathway to safeguard T cell intrathymic maturation. In particular, SRSF1 directly binds and regulates
and
expression via alternative splicing in response to type I interferon signaling. Moreover, forced expression of interferon regulatory factor 7 rectifies the defects in SRSF1-deficient thymocyte maturation via restoring expression of type I interferon-related genes. Thus, our work provides new insight on SRSF1-mediated posttranscriptional regulatory mechanism of thymocyte development.
Efficient immune responses rely on the proper differentiation of CD8
T cells into effector and memory cells. Here, we show a critical requirement of N
-Methyladenosine (m
A) methyltransferase Mettl3 ...during CD8
T cell responses upon acute viral infection. Conditional deletion of Mettl3 in CD8
T cells impairs effector expansion and terminal differentiation in an m
A-dependent manner, subsequently affecting memory formation and the secondary response of CD8
T cells. Our combined RNA-seq and m
A-miCLIP-seq analyses reveal that Mettl3 deficiency broadly impacts the expression of cell cycle and transcriptional regulators. Remarkably, Mettl3 binds to the Tbx21 transcript and stabilizes it, promoting effector differentiation of CD8
T cells. Moreover, ectopic expression of T-bet partially restores the defects in CD8
T cell differentiation in the absence of Mettl3. Thus, our study highlights the role of Mettl3 in regulating multiple target genes in an m
A-dependent manner and underscores the importance of m
A modification during CD8
T cell response.
N
-methyladenosine (m
A) methyltransferase Mettl3 is involved in conventional T cell immunity; however, its role in innate immune cells remains largely unknown. Here, we show that Mettl3 ...intrinsically regulates invariant natural killer T (iNKT) cell development and function in an m
A-dependent manner. Conditional ablation of Mettl3 in CD4
CD8
double-positive (DP) thymocytes impairs iNKT cell proliferation, differentiation, and cytokine secretion, which synergistically causes defects in B16F10 melanoma resistance. Transcriptomic and epi-transcriptomic analyses reveal that Mettl3 deficiency disturbs the expression of iNKT cell-related genes with altered m
A modification. Strikingly, Mettl3 modulates the stability of the Creb1 transcript, which in turn controls the protein and phosphorylation levels of Creb1. Furthermore, conditional targeting of Creb1 in DP thymocytes results in similar phenotypes of iNKT cells lacking Mettl3. Importantly, ectopic expression of Creb1 largely rectifies such developmental defects in Mettl3-deficient iNKT cells. These findings reveal that the Mettl3-m
A-Creb1 axis plays critical roles in regulating iNKT cells at the post-transcriptional layer.
T follicular helper (T
) cells are specialized effector CD4
T cells critical to humoral immunity. Whether post-transcriptional regulation has a function in T
cells is unknown. Here, we show ...conditional deletion of METTL3 (a methyltransferase catalyzing mRNA N
-methyladenosine (m
A) modification) in CD4
T cells impairs T
differentiation and germinal center responses in a cell-intrinsic manner in mice. METTL3 is necessary for expression of important T
signature genes, including Tcf7, Bcl6, Icos and Cxcr5 and these effects depend on intact methyltransferase activity. m
A-miCLIP-seq shows the 3' UTR of Tcf7 mRNA is subjected to METTL3-dependent m
A modification. Loss of METTL3 or mutation of the Tcf7 3' UTR m
A site results in accelerated decay of Tcf7 transcripts. Importantly, ectopic expression of TCF-1 (encoded by Tcf7) rectifies T
defects owing to METTL3 deficiency. Our findings indicate that METTL3 stabilizes Tcf7 transcripts via m
A modification to ensure activation of a T
transcriptional program, indicating a pivotal function of post-transcriptional regulation in promoting T
cell differentiation.
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