Browsing by Subject "Hematopoietic stem cells"

Background

Methods

Results

Conclusions

Stem cells are defined by their ability to make more stem cells, a property known as self-renewal and their ability to generate cells that enter differentiation. One mechanism by which fate decisions can be effectively controlled in stem cells is through asymmetric division and the correct partitioning and inheritance of cell fate determinants. While hematopoietic stem cells have the capacity to divide through asymmetric division, the molecular machinery that regulates this process is unknown and whether its activity is required in vivo remains unclear. Here we show that Lis1, a dynein-binding protein and regulator of asymmetric division, is critically required for blood development and for hematopoietic stem cell renewal in fetal and adult life. In particular, conditional deletion of Lis1 led to a severe bloodless phenotype and embryonic lethality in vivo. In both fetal and adult mice, loss of Lis1 led to a failure of normal self-renewal, which included impaired colony-forming ability in vitro and defects in long-term reconstitution ability following transplantation. As a possible mechanism, we find that the absence of Lis1 in hematopoietic cells, in part, accelerates differentiation linked to the incorrect inheritance of cell fate determinants. Furthermore, using a live cell imaging strategy, we find that the incorrect inheritance of cell fate determinants observed following the loss of Lis1 is due defects in spindle positioning and orientation. Finally, using two animal models of undifferentiated myeloid leukemia, we show that Lis1 is critical for the aberrant cell growth that occurs in cancer. Deletion of Lis1 both at the early and late stages of myeloid leukemia blocked its propagation in vivo and led to a marked improvement in survival. Together, these data identify Lis1 and the directed control of asymmetric division as key regulators of normal and malignant hematopoietic development.

The tristetraprolin (TTP) protein family of RNA-binding proteins contains three widely expressed mammalian protein members: TTP (ZFP36), ZFP36L1, and ZFP36L2, all of which can regulate gene expression by binding to specific AU-rich sequences located in the 3'-untranslated regions (3’-UTR) of mRNAs and accelerating their decay. Unique among the three, ZFP36L2 plays a pivotal role in maintaining hematopoietic stem cells (HSC) during development. ZFP36L2-deficient mice exhibit severely impaired definitive hematopoiesis and die approximately two weeks after birth due to severe anemia, thrombocytopenia, and internal hemorrhage. Recent single-cell RNA sequencing (scRNA-seq) studies have demonstrated widespread Zfp36l2 expression in HSC and the hematopoietic system during both development and adulthood.

Despite the recognized importance of ZFP36L2 in maintaining HSC, there are still numerous aspects of its role that are not fully understood, hindering our understanding of hematopoietic regulation. While prior studies have provided valuable insights into the physiological function of ZFP36L2, its molecular mechanisms in HSC development and hematopoietic system maintenance remain poorly defined. To decipher its involvement in hematopoiesis, it is crucial to identify the mRNA targets and pathways regulated by ZFP36L2 and determine whether this regulation is intrinsic to HSC. Moreover, the premature death of ZFP36L2-deficient mice makes it unclear to what extent this protein governs adult HSC function. Lastly, considering the pivotal position of HSC in the hematopoietic hierarchy, it is important to investigate how ZFP36L2's activity in HSC affects the subsequent differentiation of hematopoietic lineages. Clarifying this relationship could yield valuable insights into the post-transcriptional mechanisms that govern HSC biology and potentially lead to the identification of new therapeutic targets for hematological disorders.

To address the knowledge gap, we employed a detailed analysis combining flow cytometry and scRNA-seq to examine HSC and hematopoietic progenitor cells (HSPC) at several critical developmental stages. Our studies revealed that the absence of ZFP36L2 resulted in significant reductions in both HSC and immature progenitors during mouse development, primarily due to HSC-autonomous dysregulation. In addition, scRNA-seq analysis of HSC and progenitors revealed that ZFP36L2 deficiency caused abnormal upregulation of transcripts related to cell cycle regulation and lymphoid specification, leading to aberrant cell cycle progression and premature lymphoid lineage commitment. This ultimately resulted in cellular damage and HSC exhaustion at birth. These findings demonstrate that ZFP36L2 is essential for maintaining the homeostasis of HSC, and emphasizes the significance of restraining lineage commitment and excessive self-renewal during HSC development.

In a related study, we investigated possible functional overlap of ZFP36L2 and TTP. We developed mice (L2KO/TTP∆ARE) that lacked Zfp36l2 but modestly overexpressed TTP throughout the body. L2KO/TTP∆ARE mice not only survived but also exhibited normal peripheral blood counts, except for residual moderate thrombocytopenia. We took advantage of this rescued ZFP36L2-deficient model and investigated the role of ZFP36L2 in adult hematopoiesis. We discovered that megakaryocyte (MK) progenitors and MK-biased HSC were decreased in bone marrow from L2KO/TTP∆ARE mice and exhibited enriched erythroid and decreased MK gene signatures. In addition, L2KO/TTP∆ARE HSC failed to reconstitute hematopoiesis upon non-competitive transplantation, and showed molecular features of stress and reduced cycling. Thus, TTP can assume some functions of ZFP36L2 in a genetic dose-dependent manner, but ZFP36L2 may be specifically required for the maintenance of megakaryopoiesis and HSC function.In summary, our studies provide novel insights into the essential role of ZFP36L2 in the maintenance of HSC throughout both developmental stages and adulthood. We demonstrate that ZFP36L2 is essential for restraining abnormal cell cycling and lymphoid commitment during HSC development, thereby ensuring proper HSC maintenance. Interestingly, our results also suggest that TTP may partially compensate for ZFP36L2 deficiency during the development of the hematopoietic system, but that ZFP36L2 may have specific functions in maintaining megakaryopoiesis and HSC function in adulthood. Overall, our research provides a strong foundation for future studies aimed at elucidating the underlying mechanisms that govern ZFP36L2's function in hematopoiesis, further advancing our understanding of the intricate regulatory paradigm of HSC biology.