Fig 1.
In vivo reconstitution potential of booreana fetal liver HSCs.
Chimeras were generated to investigate the in vivo reconstitution ability of booreana (boo) versus WT fetal liver. Analyses involved a quantitative and kinetic study of the lineage potential of 1 x 106 (1M) or 2 x105 (200K) fetal liver cells from boo/boo and WT mice of CD45.2 origin transplanted into lethally irradiated CD45.1 mice. Donor cell reconstitution was assessed via flow cytometric analysis of subsets in peripheral blood of chimeras at 8 and 16 weeks post reconstitution. The proportional representation of T cells (CD3+), B cells (B220+), myeloid cells (CD11b+) and granulocytes (Gr1+) was measured within the donor-derived (CD45.2) leukocyte compartment of peripheral blood. Individual data points are shown, with mean value indicated by a horizontal bar. All comparisons between equivalent boo/boo and wild type chimeras were significantly different (p ≤ 0.05) except for T cells at 8 weeks, and macrophages and granulocytes at 16 weeks. More complete data at 4, 8, 12 and 16 weeks are summarised in S3 Fig. Significantly different data sets are shown by *.
Fig 2.
Peripheral blood analysis of chimeras reconstituted with booreana fetal liver.
Chimeras were prepared as described in Fig 1. At 16-weeks post-transplant, blood composition was analyzed. Individual data points are shown for boo/boo and WT chimeras, with the mean value indicated by a horizontal bar. All comparisons between equivalent boo/boo and wild type chimeras were significantly different (p ≤ 0.05), except for reticulocyte counts in chimeras reconstituted with 1 x 106 cells. HGB = hemoglobin, HCT = hematocrit, RBC = red blood cells, WBC = white blood cells. Significantly different data sets are shown by *.
Fig 3.
Analysis of bone marrow progenitors in booreana (boo/boo) and WT mice.
Bone marrow from adult mice was prepared and stained with antibodies to distinguish hematopoietic progenitors flow cytometrically. A lineage cocktail of antibodies was used to gate Lin- subsets, and Sca-1 and c-kit staining used to identify the Lin-c-kit+Sca-1+ (LSK) subset. Staining for Flt3 and CD150 was used to distinguish LT-HSCs and MPPs, and staining for IL-7R and Flt3 was used to distinguish CLPs. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells as PI-. Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin-PI- bone marrow cells. (A) Staining profiles for representative individual mice are shown. (B) Percent cells amongst viable Lin- bone marrow is shown for 3 individual mice, with mean shown by a bar and statistically significant results (p ≤ 0.05) boxed in red.
Fig 4.
Booreana (boo/boo) mice have increased numbers of dendritic cell progenitors in bone marrow.
Cells from boo/boo and WT mice were prepared and stained with antibodies to distinguish macrophage dendritic cell progenitors (MDP) and common dendritic cell progenitors (CDP) flow cytometrically. A lineage cocktail of antibodies was used to gate the Lin- cells and so exclude mature cells. Sca-1 and c-kit staining was used to identify the Lin-c-kithiSca-1- and Lin-c-kitloSca-1- subsets, and staining for Flt3 and CD115 used to distinguish MDP and CDP amongst these subsets. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI-). Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin-PI- bone marrow cells. (A) Profiles for representative individual mice are shown. (B) Percent cells amongst viable Lin- bone marrow is shown for 3 individual mice, with mean value shown as a bar, and statistically significant results (p ≤ 0.05) boxed in red.
Fig 5.
Booreana (boo/boo) mice show a reduction in myeloid progenitors in bone marrow.
Cells from boo/boo and WT mice were prepared and stained with antibodies to distinguish hematopoietic progenitors flow cytometrically. A lineage cocktail of antibodies was used to gate the Lin- subsets, so excluding mature cells. Sca-1 and c-kit staining was used to identify the Lin-c-kit+Sca-1+ subset. Staining for CD34 and CD16/32 was used to distinguish CMPs, GMPs, and MEPs. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI-). Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin-PI- bone marrow cells. (A) Profiles for representative individual mice are shown. (B) Percent cells amongst viable Lin- bone marrow is shown for 3 individual mice, with mean value shown as a bar, and statistically significant results (p ≤ 0.05) boxed in red.
Fig 6.
Analysis of splenic myeloid and DC subsets in booreana versus WT mice.
(A) Chimeras were prepared as described in Fig 1 and sacrificed at 52 weeks. The total splenic dendritic and myeloid subset was purified via red blood cell lysis and T/B cell depletion. Cells were stained with antibody to detect CD45.2+ cells, and the population gated as CD11b+ and/or CD11c+ to estimate % dendritic/myeloid cells, respectively. Data represent mean ± SE (n = 4), and p values for statistically significant pairs of data shown. (B) Spleens of adult booreana and WT mice were enriched for DC and myeloid cells via red blood cell lysis and T/B cell depletion. Cells were then stained with antibodies to delineate subsets of CD8+ cDCs (CD11chiCD11b-MHCII+CD8+), CD8- cDCs (CD11chiCD11bloMHCII+CD8-), L-DCs (CD11cloCD11bhiLy6C-Ly6G-), resident monocytes (resi mono: CD11bhiCD11clo/-Ly6C+Ly6G-), inflammatory monocytes (infl mono: CD11bhiCD11c-Ly6ChiLy6G-), and neutrophils (neu: CD11bhiCD11c-Ly6C+Ly6G+). The proportional representation of each subset was calculated relative to the total dendritic and myeloid population in spleen (i.e. CD11b+ and/or CD11c+ cells, respectively). Data (mean) are shown for boo/boo (n = 3) and WT (n = 4) mice. Statistically significant findings (p ≤ 0.01) are boxed and p values are shown.
Fig 7.
Hematopoiesis due to booreana (boo/boo) and WT bone marrow in co-cultures.
Bone marrow was prepared from one-year old chimeras described in Fig 1. Donor-derived CD45.2+ bone marrow cells were sorted as a Lin- population of boo/boo or WT origin, and equal numbers co-cultured over 5G3 splenic stroma. Individual co-cultures were established from 4 boo/boo chimeras and 2 WT chimeras. Cell production was monitored over time by staining non-adherent cells using antibodies to CD11c, CD11b, MHC-II, and CD8α, or with isotype controls. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI-). Gates were set on bivariate plots using isotype control antibodies, and numbers shown in gates reflect % positive cells. (A) Data shows staining of 21 day co-cultures established from one of each chimera type. L-DCs were gated as CD11cloCD11bhiMHC II-CD8α-B220- cells, and cDC-like cells as CD11chiCD11bloMHC II+CD8α-B220-. (B) Cell production was calculated and shown as mean ± SE for co-cultures established from boo/boo (n = 4) and wild-type (WT) (n = 2) chimeras. Significantly different data pairs are indicated.
Fig 8.
Changes in hematopoiesis attributable to the c-Myb308G mutation.
The summary diagram combines analysis of stem/progenitor subsets in mutant mouse bone marrow, with subset analysis of mature cell subsets in blood and spleen of chimeras reconstituted with mutant fetal liver cells and analysed at 16 weeks after reconstitution. Significant changes in cell production in relation to wild type mice or wild type control chimeras are shown in red indicating increase, or blue indicating a decrease, in cell production. Subsets unchanged in mutant and wild type are shown in grey. The diagram is not a complete lineage map since only subsets analysed are shown. These include: LT-HSC, longterm hematopoietic stem cell; MPP, multipotential progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MDP, myeloid dendritic progenitor; CDP common dendritic progenitor; MEP, myeloid/erythroid progenitor; GMP, granulocyte/macrophage progenitor. The lineage relationship between the MDP subset and CMP and CDP is shown as a dashed line since it is still in doubt [45]]. Similarly, the derivation of L-DC directly from bone marrow HSC and MPP is shown as a dashed line, since this has only been confirmed in vitro [28].