A Single Copy of Large Tumor Suppressor 1 or Large Tumor Suppressor 2 Is Sufficient for Normal Hematopoiesis
Hematopoietic stem cells (HSCs) possess the remarkable ability to self-renew and differentiate into all subsets of blood cells, playing a crucial role in maintaining hematopoietic homeostasis. The quiescent state of HSCs is essential for their long-term function and preservation of self-renewal capacity. Understanding the molecular mechanisms regulating HSC quiescence is vital for insights into tissue regeneration and stress responses. The Hippo signaling pathway, a key regulator of organ size and stem cell function, has been extensively studied in various tissues. However, its role in the hematopoietic system remains unclear. Large tumor suppressor 1 (LATS1) and large tumor suppressor 2 (LATS2) are critical components of the Hippo pathway, functioning as tumor suppressors and stem cell regulators. This study investigates the role of LATS1 and LATS2 in normal hematopoiesis using knockout mouse models.
Expression of LATS1 and LATS2 in Hematopoietic Cells
The expression levels of LATS1 and LATS2 were examined in subsets of primitive and mature murine hematopoietic cells. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that both LATS1 and LATS2 are widely expressed in hematopoietic cells, with higher expression levels in primitive hematopoietic cells compared to mature cells. Specifically, LATS1 and LATS2 expression was higher in hematopoietic progenitor cells (HPCs) than in long-term HSCs (LT-HSCs), lymphoid-primed multipotent progenitors (LMPPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs). Notably, LATS1 exhibited higher expression levels than LATS2 in hematopoietic cells, suggesting a more significant role for LATS1 in hematopoiesis.
Generation of LATS1 and LATS2 Knockout Mice
To investigate the function of LATS1 and LATS2 in normal hematopoiesis, knockout mice were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Two small guide RNAs (sgRNAs) were designed to target exon 4 of LATS1 and exon 2 of LATS2. Two independent knockout lines for each gene were obtained, with frameshift mutations confirmed by DNA sequencing. Homozygous mutants were generated by inter-crossing heterozygous mice, but no homozygotes were born, indicating embryonic lethality. Heterozygous mice were viable and fertile, with no visible differences compared to wild-type littermates. The expression levels of LATS1 and LATS2 in bone marrow (BM) were halved in heterozygous mice, confirming the successful generation of knockout models.
Normal Hematopoiesis in LATS1 and LATS2 Heterozygotes
The impact of LATS1 and LATS2 heterozygous mutations on hematopoiesis was assessed in 8 to 12-week-old mice. Complete blood cell counts revealed no differences in the numbers of circulating white blood cells, neutrophils, lymphocytes, monocytes, platelets, and red blood cells, as well as hemoglobin concentration between heterozygous mice and their littermate controls. The total number of BM cells was also similar between the two groups. Flow cytometry analyses showed no significant differences in the frequency or total number of early HPCs (HPC, CMP, GMP, MEP, LMPP, and Lin-Sca-1+c-Kit+ [LSK] cells) or HSCs (LT-HSCs and CD34 negative LSK [CD34-LSK]) between heterozygous mice and controls. These findings suggest that a single allele of LATS1 or LATS2 is sufficient for normal hematopoiesis.
Effect of LATS1 and LATS2 Depletion on Overall Survival
To evaluate the effects of LATS1 and LATS2 depletion on hematopoietic reconstitution, heterozygous and control mice were treated with 5-fluorouracil (5-FU), a chemotherapeutic agent that kills rapidly proliferating cells and replenishes the hematopoietic system. Mice were monitored daily for three weeks after weekly injections of 5-FU. Both heterozygous and wild-type control mice died gradually after the second injection, with the remaining mice dying after the third injection. There were no significant differences in overall survival between heterozygous and wild-type control mice, indicating that the depletion of LATS1 or LATS2 did not affect the long-term self-renewal of HSCs.
Discussion
The Hippo signaling pathway is a critical regulator of organ size and stem cell function in various tissues. LATS1 and LATS2, key mediators of Hippo signaling, function to negatively regulate the transcriptional co-activators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). This study demonstrates that the inhibition of Hippo signaling through the depletion of LATS1 and LATS2 does not affect hematopoiesis or HSC function. The knockout of LATS1 or LATS2 resulted in early embryonic lethality, consistent with previous studies. However, heterozygous mice exhibited normal hematopoiesis and HSC function, indicating that a single allele of LATS1 or LATS2 is sufficient for these processes.
Haploinsufficiency, where a single functional copy of a gene is insufficient to preserve normal function, is a common cause of dominant diseases. Several genes, such as Apc, Npm1, and macroH2A1.1, are haploinsufficient in normal hematopoiesis. However, LATS1 and LATS2 are not haploinsufficient genes, as their functions are not dosage-dependent. The remaining LATS1 or LATS2 in heterozygous mice were sufficient for normal hematopoiesis and HSC function.
The Hippo signaling pathway is associated with various upstream regulatory molecules, many of which are still poorly characterized in mammalian cells. Hippo signaling plays a critical role in cell-cell interactions that regulate cell proliferation through contact-mediated inhibition. However, hematopoietic cells reside in a liquid environment (blood and BM), distinct from solid organs. In solid organs, contact-mediated inhibition of proliferation is crucial for tissue homeostasis, but this mechanism may not be effective in liquid tissue. This could explain why the depletion of LATS1 or LATS2 leads to overgrowth and aberrant cell proliferation in solid tissue but not in hematopoietic cells.
Neurofibromatosis 2 (NF2) is a well-studied upstream regulator of Hippo signaling in mammalian cells. NF2 is essential for contact-mediated inhibition and inhibits YAP activity in the liver. Loss of NF2 in the liver leads to an overgrowth phenotype, similar to that observed in the livers of Mst1 and Mst2 depletion mice and mice with enforced expression of YAP. However, the loss of NF2 in hematopoietic cells does not affect the functions of progenitor cells and HSCs, consistent with the observations in LATS1 or LATS2 knockout mice. In contrast, the depletion of NF2 has significant effects on non-hematopoietic cells of the BM microenvironment, resulting in secondary effects on HSC function and localization. This suggests that NF2 may function through the Hippo signaling pathway in this specific context, warranting further investigation into the role of LATS1 and LATS2 within BM microenvironments.
In conclusion, this study demonstrates that the inhibition of the Hippo signaling pathway through the depletion of LATS1 and LATS2 does not affect hematopoiesis or HSC function. The findings indicate that a single allele of LATS1 or LATS2 is sufficient for normal hematopoiesis. However, further research is needed to explore the protein levels and kinase activity of LATS1 and LATS2, as well as their roles under conditions of drastic hematopoietic stress, such as serial transplantations. Additionally, the function of LATS1 and LATS2 in a hematopoietic system-specific knockout mouse model should be investigated to gain a comprehensive understanding of their roles in hematopoiesis.
doi.org/10.1097/CM9.0000000000000934
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