Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

<div><p><i>Meis1</i> is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of <i>Meis1</i>’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of <i>Meis1</i>’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for <i>Meis1</i> have been established. These models highlight a critical role for <i>Meis1</i> in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of <i>Meis1</i> function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of <i>Meis1</i> which we crossed onto the inducible Cre localization/expression strains, B6;129-<i>Gt(ROSA)26Sor</i>^{<i>tm1(Cre/ERT)Nat</i>}/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of <i>Meis1</i> in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of <i>Meis1</i> in ROS regulation.</p></div

Loss of <i>Meis1</i> results in an intrinsic defect in LTRC.

<p><b>a)</b> Experimental plan to test the cell-intrinsic requirement for <i>Meis1</i> in HSCs. <b>b)</b> PB engraftment of primary recipients 4 weeks after transplantation and prior to induction shows no difference in engraftment (n = 11). <b>c)</b> PB engraftment of primary recipients 2 days following PolyI:C administration. <i>MxCre</i>^<i>+</i><i>/Meis1</i>^<i>-/-</i> (n = 7) engraftment is reduced 17% compared to <i>MxCre/Meis1</i>^<i>fl/fl</i> (n = 4; <i>p</i> = 0.0005). <b>d)</b> BM engraftment in primary recipients 3 days following PolyI:C administration. <i>MxCre</i>^<i>+</i><i>/Meis1</i>^<i>-/-</i> (n = 7) engraftment is reduced 38% compared to <i>MxCre/Meis1</i>^<i>fl/fl</i> (n = 4; <i>p</i> = 6x10^-6).</p

NAC treatment does not rescue committed progenitor functional differences between <i>MxCre/Meis1</i>^<i>-/-</i> and <i>MxCre/Meis1</i>^<i>-/+</i> mice.

<p>a) CFC numbers assessed in myeloid-supportive conditions remained reduced in <i>MxCre/Meis1</i>^<i>-/-</i> mice following <i>in vivo</i> NAC administration. b) NAC did not rescue the capacity for <i>MxCre/Meis1</i>^<i>-/-</i> cells to form large BFU-E (>16 clusters/colony) in erythroid supportive media (12.8-fold reduction,). Both FACS data and colony numbers are expressed as absolute numbers isolated from the trunk of mice (2 femurs, 2 tibias, 2 iliac crests).</p

Loss of <i>Meis1</i> results in an intrinsic defect in HSC self-renewal.

<p><b>a)</b> Engraftment of secondary recipients transplanted with bulk CD45.2 cells from primary mice with transplant dose based on the number of CD45.2 cells 24 weeks after transplantation. <b>b)</b> Engraftment of secondary recipients transplanted with sorted CD45.2 cells from primary mice 24 weeks after transplantation.</p

Gating strategy and representative plots for myeloid progenitors, HSCs and MkP from <i>MxCre/Meis1</i>^<i>-/-</i> and <i>MxCre/Meis1</i>^<i>-/+</i> BM

<p>Gating strategy and representative plots for myeloid progenitors, HSCs and MkP from <i>MxCre/Meis1</i>^<i>-/-</i> and <i>MxCre/Meis1</i>^<i>-/+</i> BM</p

Loss of <i>Meis1</i> results in changes to the PB and BM composition of <i>ERTCre/Meis1</i> and <i>MxCre/Meis1</i> mice.

<p><b>a)</b> Loss of <i>Meis1 in vivo</i> results in a significant decrease in red blood cells in the PB in <i>Meis1</i> ^<i>-/-</i> mice. <i>ERTCre/Meis1</i> ^<i>-/-</i> (n = 13) compared to <i>ERTCre/Meis1</i>^{-/+ & +/+} (<i>p =</i> 0.01; <i>ERTCre/Meis1</i>^<i>+/+</i> n = 3; <i>ERTCre/Meis1</i>^<i>-/fl</i> n = 10). The three <i>ERTCre/Meis1</i>^<i>-/-</i> mice with the lowest RBC counts were euthanized due to pallor and lethargy and had profound reductions in BM cellularity, that is, less than 1.3x10⁶ nucleated cells per trunk (2x femur, 2x iliac crest, 2x tibia). Moribund mice are represented as hollow squares on the graph. <i>MxCre/Meis1</i>^<i>-/-</i> mice (n = 5) show no such reduction when compared to <i>MxCre/Meis1</i>^<i>+/+</i>(n = 2) and <i>MxCre/Meis1</i>^<i>-/+</i> (n = 3) mice. <b>b)</b> Loss of <i>Meis1</i> results in a reduction of white blood cells (WBC) in <i>MxCre/Meis1</i> ^<i>-/-</i> mice compared to <i>MxCre/Meis1</i> ^{-/+ & +/+} (<i>p =</i> 0.004, <i>n</i> = 5) 2 days after the final PolyI:C injection. <b>c)</b> Loss of <i>Meis1</i> results in a reduction of peripheral platelets (PLT) in <i>MxCre/Meis1</i> ^<i>-/-</i> <i>ERTCre</i>^<i>+</i><i>/Meis1</i> ^<i>-/-</i> and mice compared to <i>MxCre</i>^<i>+</i> and <i>ERTCre</i>^<i>+</i> control mice 2 days after the final PolyI:C/4-OHT injection (<i>p =</i> 0.02, and <i>p</i> = 0.02, respectively). <b>d)</b> Lineage distribution in the PB of treated mice, 2 days following the last injection. No significant differences were found.</p

Genes with > 2-fold expression change associated with deletion of <i>Meis1</i> as determined by Affymetrix analysis.

<p>Genes with > 2-fold expression change associated with deletion of <i>Meis1</i> as determined by Affymetrix analysis.</p

Targeted <i>Meis1</i>^<i>fl/fl</i> locus.

<p>The sequence is numbered according to the wild-type sequence on the antisense strand of chromosome 11 according to the Dec. 2011 (GRCm38/mm10) sequence from the Mouse Genome Reference Consortium. Non-wild-type sequence introduced by the targeting vector is underlined while the loxP sites are highlighted in grey. Exon 8 is denoted in italicized-bold type.</p

HSC frequency following <i>in vivo</i> deletion of <i>Meis1</i> in primary recipients.

<p>HSC frequency following <i>in vivo</i> deletion of <i>Meis1</i> in primary recipients.</p

LTC-IC frequency in <i>MxCre/Meis1</i>^<i>-/-</i> BM.

<p>LTC-IC frequency in <i>MxCre/Meis1</i>^<i>-/-</i> BM.</p