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. 2014 Mar;46(3):245-52.
doi: 10.1038/ng.2889. Epub 2014 Feb 2.

Lis1 Regulates Asymmetric Division in Hematopoietic Stem Cells and in Leukemia

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Free PMC article

Lis1 Regulates Asymmetric Division in Hematopoietic Stem Cells and in Leukemia

Bryan Zimdahl et al. Nat Genet. .
Free PMC article

Abstract

Cell fate can be controlled through asymmetric division and segregation of protein determinants, but the regulation of this process in the hematopoietic system is poorly understood. Here we show that the dynein-binding protein Lis1 is critically required for hematopoietic stem cell function and leukemogenesis. Conditional deletion of Lis1 (also known as Pafah1b1) in the hematopoietic system led to a severe bloodless phenotype, depletion of the stem cell pool and embryonic lethality. Further, real-time imaging revealed that loss of Lis1 caused defects in spindle positioning and inheritance of cell fate determinants, triggering accelerated differentiation. Finally, deletion of Lis1 blocked the propagation of myeloid leukemia and led to a marked improvement in survival, suggesting that Lis1 is also required for oncogenic growth. These data identify a key role for Lis1 in hematopoietic stem cells and mark its directed control of asymmetric division as a critical regulator of normal and malignant hematopoietic development.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Genetic deletion of Lis1 impairs establishment of the hematopoietic system during embryonic development
(a) Representative image of Control (Lis1f/+, upper left) and Lis1−/− (Lis1f/f; Vav-Cre, upper right) littermates at E14.5. Hematoxylin and eosin (H&E) stain of fetal liver from Control (bottom left) and Lis1−/− (bottom right) littermates at E14.5. 40X. Scale bars, 55μm. (b) Fetal liver cells from Control (Lis1f/+) and Lis1−/− mice were analyzed for frequency of HSCs (cKit+ Lin AA4.1+; KL AA4.1+). Dot plots are shown for representative Control (left) and Lis1−/− (right) E14.5 embryos. (c) Absolute number of HSCs (KL AA4.1+) from Control (Lis1f/+ or Lis1f/f; solid squares) or Lis1−/− (open squares) mice at different gestational ages; n=3–5 mice for each genotype for each gestational age; **P=0.0014 for E14.5, **P=0.0018 for E15.5. (d) Number of colonies generated from Control (Lis1f/+ or Lis1f/f) and Lis1−/− fetal liver. Cells are isolated from 3–6 embryos of each genotype; *P=0.0110 (n=3, technical replicates). (e) Representative FACS profile of donor chimerism (4 months) in CD45.1+ recipients transplanted with HSC-enriched cells (Lin AA4.1+) from either Control (Lis1f/+) or Lis1−/− E14.5 embryos. (f) Average donor chimerism at different times post-transplantation (2–4 donor mice were used for each genotype and 4–6 recipient mice in each cohort). Control: solid squares; Lis1−/−: open squares; **P=0.0088 for 60 days, **P=0.0021 for 90 days and ***P=0.0006 for 120 days. Error bars show standard error of the mean (SEM).
Figure 2
Figure 2. Lis1 is cell-autonomously required for adult hematopoietic stem cell self-renewal
(a) Average frequency of HSCs (KLSFlt3, KLSF) in whole bone marrow from tamoxifen-treated control (Lis1+/+; Rosa-creER, indicated as WT) and tamoxifen-treated (Lis1f/f; Rosa-creER (indicated as Lis1−/−) mice; n=4 for control (WT), n=3 for Lis1−/−; *P=0.0140. (b) Representative FACS plots of HSCs (KLSF) from WT and Lis1−/− mice. (c) Repopulation efficiency of Lis1−/− HSCs. Representative FACS plots shows donor chimerism (CD45.2+ cells) in recipients transplanted with HSCs (KLS CD150+ CD48) from WT or Lis1−/− mice. FACS analysis was performed 28 weeks post-transplantation. (d) Average donor chimerism at different times after transplantation (4–5 mice per cohort). WT is shown with solid squares and Lis1−/− is shown with open squares. (e) Genome wide expression analysis of Lis1-deficient HSC-enriched cells. Heat map of known regulators of stem and progenitor cell activity significantly affected by the loss of Lis1. (f–i) Lis1 chimeras with hematopoietic-specific Lis1 deletion, (f) Donor chimerism prior to tamoxifen (tam) treatment was assessed two months post-transplantation. (WT) indicates control Lis1+/+; Rosa-creER and (f/f) indicates Lis1f/f; Rosa-creER transplanted mice (5 mice in each cohort). (g) Frequency of donor-derived KLS cells in chimeric mice post-deletion. ((WT) +tam) indicates mice that received donor cells from Lis1+/+; Rosa-creER and ((f/f) +tam) indicates mice that received donor cells from Lis1f/f; Rosa-creER mice; n=3 for each cohort, *P=0.0277. (h–i) Repopulation ability of whole bone marrow (WBM) cells isolated from Lis1 chimera mice. (h) Representative FACS plots show donor chimerism (CD45.2+ cells) in recipients that received cells from either control (f/f +vehicle) or (f/f +tam) Lis1 chimeras. (i) Average donor chimerism at 16 weeks post-transplantation (n=3–4 recipients per cohort; *P=0.0369).
Figure 3
Figure 3. Lis1 deficiency leads to accelerated differentiation of hematopoietic stem cells
(a–c) Cell cycle status of hematopoietic cells following Lis1 deletion. Control (Lis1+/+; Rosa-creER; WT) and Lis1f/f; Rosa-creER (Lis1−/−) mice were treated with tamoxifen and cell cycle analyzed after BrdU incorporation. Average frequency of KLS (a), KLS CD48+ CD150 (b), and KLS CD150+ CD48 (c) in G0/G1, S, and G2/M cell cycle phases in control (WT) and Lis1−/− mice. Data shown are from two independent experiments (n=2–3 per cohort). (d) Percentage of HSCs (KLS CD150+ CD48) undergoing apoptosis (AnnexinV+ 7AAD) in control (WT) and Lis1−/− mice. Data shown are from three independent experiments (n=2–3 per cohort). (e) Analysis of rate of differentiation of Lis1−/− cells. KLS cells from control (Lis1+/+; Rosa-creER; WT) and Lis1f/f; Rosa-creER (Lis1−/−) mice were treated with tamoxifen in vitro. Representative FACS plot shows frequency of cells expressing lineage markers in WT (shown in gray) and Lis1−/− (shown in black) populations 24 hours post-deletion. (f) Average frequency of cells expressing lineage markers (Lin+) in WT and Lis1−/− cells. Data shown are from three independent experiments; ***P=0.0002. (g) Analysis of apoptosis in Lin and Lin+ fraction of Lis1−/− cells. Percentage of Annexin V+ cells is shown 24 hours post-deletion. Data shown are from two independent experiments. Error bars show the standard error of mean (SEM).
Figure 4
Figure 4. Loss of Lis1 impairs inheritance of fate determinants in hematopoietic development
(a) Expression of Numb in HSCs and progenitor cells. Representative image with magnified inlay (dotted white box) shows HSCs (KLS CD48 CD150+) and progenitor cells (KLS CD48+) stained with anti-Numb antibody (green) and DAPI (blue), 63X. Scale bars, 10μm. (b) Average fluorescence intensity of Numb in individual HSCs and progenitor cells. Data shown are from two independent experiments (n=34 cells for each genotype); ****P<0.0001. (c) Realtime RT-PCR analysis of Numb expression in Lin and Lin+ cells (n=2 independent experiments). (d) Representative images of individual HSC-enriched cells with polarized or non-polarized Numb (Numb in red, DAPI in blue, zoomed 63x images, Scale bars, 2.5μm). (e) Frequency of control (WT) or Lis1−/− cells with polarized Numb. Frequencies were determined out of 100 tracked cells for each genotype. (f) Model illustrates how two dividing cells may equivalently polarize Numb (shown in red) to one side of the cell, yet direct the cleavage plane in such a way to ensure either equal or unequal inheritance of Numb into the incipient daughter cells. (g) Representative image of a tracked cell inheriting Numb symmetrically (top) or asymmetrically (bottom) into incipient daughter cells (Numb in red, DAPI in blue, zoomed 63x images, Scale bars, 2.5μm). (h) Relative ratios of symmetric:asymmetric division in vitro. Data shown are from two independent experiments; n=25–27 dividing cells were assessed for each experiment per cohort; **P=0.0032. (i) Representative image of symmetric (top) and asymmetric (bottom) inheritance of Numb by incipient daughter cells in vivo (Numb in red, DAPI in blue, zoomed 63x images, Scale bars, 2.5μm). (j) Relative ratio of symmetric:asymmetric division in vivo (nine dividing cells were assessed for the control (WT) group; eight dividing cells were assessed for the Lis1−/− group. Data analyzed using three independent chimeric mice for each genotype).
Figure 5
Figure 5. Loss of Lis1 impairs spindle orientation in hematopoietic development
(a) Schematic showing how spindle angle is measured relative to the retronectin base. (b) Representative side view images of a control (WT) and Lis1−/− cell undergoing cell division and their spindle angles. Scale bars, 1μm. (c) Average metaphase and telophase spindle angles of control (WT) and Lis1−/− cells relative to substrate; data shown are from three independent experiments; n=5–7 cells per genotype; **P=0.0054. (d) Numb distribution in dividing HSC-enriched cells relative to mitotic spindle orientation. Representative images of control (WT) cells (I and II) or Lis1−/− cells (III and IV) with examples of symmetric (I) or asymmetric (II, III, IV) inheritance of Numb by incipient daughter cells. Numb (green), α-tubulin (magenta); representative videos of a cell undergoing symmetric or asymmetric cell division are shown in Supplementary Videos 2–4. Scale bars, 1μm. On far right panel, each cell is displayed in spectrum color format to facilitate accurate identification of spindle position (dotted black line connecting the two centrosomes highlighted in red) and the cleavage furrow (solid lines; white and black) which partitions the dividing cell into incipient daughter 1 (D1) and daughter 2 (D2). (e) Quantification of fluorescence intensity of Numb in D1 and D2 for each representative control (WT; I and II) or Lis1−/− cell (III, IV) shown in (d). (f) Frequency of cells undergoing asymmetric inheritance of Numb in control (WT) or Lis1−/− cells; data are shown for four independent experiments; n=23 cells for WT and n=9 cells for Lis1−/−. All error bars show the standard error of mean (SEM).
Figure 6
Figure 6. Loss of Lis1 impairs the development and propagation of myeloid leukemia in mouse models and human leukemia cells
(a) Impact of loss of Lis1 on bcCML initiation. KLS cells from control (Lis1+/+; Rosa-creER) and Lis1f/f; Rosa-creER mice transduced with BCR-ABL-IRES-YFP and NUP98-HOXA9-IRES-GFP were transplanted into recipients, treated with tamoxifen and survival monitored. Data shown are from three independent experiments (n=9 for Lis1f/f +tamoxifen (black), n=9 for Lis1f/f +vehicle (blue) and n=7 for Lis1+/+ +tamoxifen (green)) (b) Impact of loss of Lis1 on propagation of established bcCML. Lin-negative cells from established control Lis1+/+creER or Lis1f/fcreER bcCML were transplanted into secondary recipients. Recipients were administered tamoxifen and survival monitored (n=4 for Lis1+/+, green and n=3 for Lis1f/f, black). (c) Impact of loss of Lis1 on de novo AML. KLS cells were isolated from control (Lis1+/+; Rosa-creER) and Lis1f/f; Rosa-creER mice and co-transduced with MLL-AF9-IRES-GFP and NRASG12V-IRES-YFP, treated with tamoxifen or corn oil and survival was monitored. Data shown are from two experiments (n=5 for Lis1f/f +tamoxifen (black), n=4 for Lis1f/f +vehicle (blue) and n=3 for Lis1+/+ +tamoxifen (green)). (d–f). Growth and cellular behavior of bcCML in vivo. Lineage-negative (Lin) cells from control Lis1+/+; Rosa-creER or Lis1f/f; Rosa-creER established bcCML were transplanted into secondary recipients and (d) average tumor burden was tracked before (% of peripheral blood) and after (% of spleen cells) tamoxifen delivery; n=4 for control (WT) and n=3 for Lis1−/− mice; **P=0.0065. (e) Representative FACS plots show frequency of bcCML cells expressing lineage markers in control (WT) or Lis1−/− populations 1 day post-tamoxifen treatment. (f) Average frequency of bcCML cells expressing lineage markers (Lin+). Data shown are from two independent experiments (n=4 for WT and n=6 for Lis1−/−, *P=0.0282). (g) Frequency of leukemia cells undergoing asymmetric inheritance of Numb in control (WT) or Lis1−/− cells; n=22 cells for WT and n=7 cells for Lis1−/−. (h–k) Influence of Lis1 loss on human leukemia growth. Human leukemia cells were infected with either control (shLuc) or lentiviral shRNA targeting human LIS1 (shLIS1). Subsequently, infected cells were sorted and plated in methylcellulose. Colony formation was assessed in K562 blast crisis CML cells (h), MV4-11 AML cells (i), Imatinib, Nilotinib and Dastinib-resistant primary human CD34+ bcCML (j) and primary human CD34+ AML (k); One patient sample was tested for each leukemia with n=3 technical replicates, *P<0.05, **P<0.01, ***P<0.001. Error bars represent standard error of the mean (SEM).

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