1. Volume 8, Issue 2, Pages 153-166 (February 2005)
c-Myb and p300 Regulate Hematopoietic Stem Cell Proliferation and Differentiation 
Mark L. Sandberg, Susan E. Sutton, Mathew T. Pletcher, Tim Wiltshire, Lisa M. Tarantino, John B. Hogenesch, Michael P. Cooke 
Developmental Cell 
Volume 8, Issue 2, Pages (February 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Mapping and Identification of an ENU-Induced Mutation in c-Myb in Family 17
(A) Peripheral blood platelet levels in G3 offspring from ENU-mutagenized mice from the first 29 families screened. Mice with peripheral blood platelet levels > 3 SD from the mean are labeled in red. Family 17, which had greatly elevated platelet levels, is boxed.
(B) Peripheral blood platelet levels in B6/129 F2 animals. Squares and circles represent male and female mice, respectively.
(C) Mapping of the high-platelet phenotype. Shown are the results from fine mapping of the region of chromosome 10 linked to the elevated platelet phenotype. Markers are listed in the first column and indicate chromosome and megabase position. The genotype for each SNP marker is represented as: B, homozygous C57BL/6J genotype; S, homozygous 129SvIm/J; H, heterozygous. Italics indicate inferred genotypes. On the right is a diagram of mouse chromosome 10 showing the interval to which the high-platelet phenotype mapped with the relative locations of 3 of the 125 genes reported within the interval.
(D) Sequence from representative unaffected and affected mice in the region encoding amino acids 300–306 of c-Myb. An A-to-G transition that was found only in family 17 is boxed.
(E) Domain structure of c-Myb: DNA binding, R1, R2, and R3; transactivation/acidic, TA; negative regulatory, NR.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2
Transcriptional Activity and Association with p300 of Gal4-c-Myb Fusion Proteins
(A) Schematic depiction of six different Gal4-c-Myb fusion proteins that were assayed for transcriptional activity: WT, c-Myb amino acids (aa) 230–636; M303V, Met-to-Val substitution at aa 303; 304GP, Gly-Pro inserted after amino acid 304; WT (230–330), M303V (230–330), and 304GP (230–330) contain aa 230–330 with the indicated mutations. Triangles represent the relative location of the mutations in the context of the TA and NR domain of c-Myb.
(B and C) Fold induction of luciferase observed, compared to vector control, when increasing amounts of the Gal4-c-Myb derivatives were transfected into K562 cells. Shown are averages of three independent experiments. Standard deviations were less than 40% of the average in all cases.
(D) Coimmunoprecipitation of c-Myb and Gal4-c-Myb with P300 from nuclear extracts. Shown are immunoblots of lysates (top) or eluates from p300 immunoprecipitates (bottom) from K562 transfected with the indicated Gal4-c-Myb expression vectors. Bands corresponding to endogenous c-Myb or the transfected Gal4-c-Myb are indicated. At the bottom are the ratios of Gal4-c-Myb to endogenous c-Myb obtained after band intensity quantification.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Hematological Analysis of Peripheral Blood from c-Mybwt/wt, c-Mybwt/M303V, and c-MybM303V/M303V Mice
(A) Analysis of peripheral blood from c-Mybwt/wt (n = 22), c-Mybwt/M303V (n = 35), and c-MybM303V/M303V (n = 19) mice by a hematology analyzer. Squares and circles represent male and female mice, respectively.
(B) Peripheral blood levels of B220+ B cells and CD4+ and CD8+ single-positive T cells measured by flow cytometry.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
Hematological Analysis of Lymphoid Organs and Bone Marrow Transplants from c-Mybwt/wt and c-MybM303V/M303V Mice
(A) Cellularity of thymus, spleen, and bone marrow of c-Mybwt/wt and c-MybM303V/M303V mice. Symbols and color coding are as in Figure 1.
(B) Left side: Flow cytometry plots of CD41+ cells in the bone marrow and spleen of a c-Mybwt/wt and a c-MybM303V/M303V mouse. Right side: Wright’s-Giemsa staining of bone marrow and splenocytes from a c-Mybwt/wt and a c-MybM303V/M303V mouse. Arrows point to megakaryocytes.
(C) Analysis of peripheral blood populations in lethally irradiated WT Ly5.1/5.1 mice that were transplanted with 5 × 106 bone marrow cells from Ly5.2/5.2 c-Mybwt/wt or c-MybM303V/M303V mice together with 5 × 106 bone marrow cells from Ly5.1/5/1 WT mice. Shown is the percentage of Ly5.2/5.2 cells for each cell population. Individual mice are indicated by symbols, with the horizontal line representing the average of all the mice. Asterisks denote statistically significant differences between WT and M303V bone marrow (Student’s t test, **p < 0.01, *p < 0.05).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5 Altered Lymphocyte Development in c-MybM303V/M303V Mice
(A and B) B and T cell development in the (A) bone marrow and (B) thymus of representative c-Mybwt/wt and c-MybM303V/M303V mice. In parentheses is the name of the respective population as defined in the text.
(C) T cell development in the thymus 12 weeks after transplant with 5 × 106 bone marrow cells from Ly5.2/5.2 c-Mybwt/wt or c-MybM303V/M303V mice together with an equal number of WT Ly5.1/5.1 bone marrow cells into lethally irradiated WT Ly5.1/5.1 mice. The example shown is representative of four animals of each genotype analyzed.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
Number and Function of Hematopoietic Stem and Progenitor Cell Populations in the Bone Marrow of c-Mybwt/wt and c-MybM303V/M303V Mice
(A) Flow cytometric analysis of Lin−c-Kit+ myeloid progenitor cells.
(B) Hematopoietic stem cell (HSC) or common lymphoid progenitor (CLP) in bone marrow of c-Mybwt/wt and c-MybM303V/M303V mice. Numbers represent the percentage of each population as a percentage of total bone marrow cells.
(C) Cell cycle analysis of stem and progenitor cells from c-Mybwt/wt and c-MybM303V/M303V mice. The indicated populations of cells were electronically gated, and DNA content was determined by using DAPI. The experiment shown is representative of two different experiments.
(D) Amount and type of colonies formed in methylcellulose following culture of sorted MEPs and HSCs from c-Mybwt/wt and c-MybM303V/M303V mice.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7
Competitive Repopulation Assay Shows an Increased Number of Functional HSCs in Bone Marrow of c-MybM303V/M303V Mice
(A) Mixed bone marrow transplants were conducted by using the indicated ratio of bone marrow from c-MybM303V/M303V or c-Mybwt/wt donor (Ly5.2/5.2) mice along with 5 × 105 competitor bone marrow cells from Ly5.1/5.2 mice into lethally irradiated Ly5.1/5.1 recipient mice.
(B) Representative flow cytometric analysis of primary recipient animals receiving Ly5.2/5.2 c-Mybwt/wt (left side) or Ly5.2/5.2 c-MybM303V/M303V (right side) bone marrow.
(C) Peripheral blood analysis of primary recipients. Shown is the percentage of donor-derived (Ly5.2/5.2) cells for each of the recipients 8 weeks after transplant (top left panel). Ratio of donor to competitor for each of the indicated cell populations in the peripheral blood 8 weeks after transplant. Each point represents the average ± SEM of 3–4 animals for each group.
(D) HSC composition in bone marrow of Ly5.1/5/.1 recipients. Bone marrow of mice that received a transplant containing the indicated ratio of donor:competitor cells 12 weeks earlier was analyzed for HSC (Lin−Kit+Sca+) content by flow cytometry. Shown is the final ratio of donor:competitor cells within the HSC compartment (upper panel) and the absolute percentage of HSCs in each recipient for the wt:wt chimeras (lower panel, left half) or M303V:wt chimeras (lower panel, right half).
(E) Contribution of Ly5.2/5.2 bone marrow in the peripheral blood of secondary recipients 8 weeks following transplant. Mice showing donor contribution above 10% were used for tertiary transplants.
(F) Limit dilution analysis of HSC numbers in tertiary transplants. The indicated number of bone marrow cells from secondary recipients was transplanted into lethally irradiated Ly5.1/5.1 recipients, and blood was analyzed 8 weeks later for Ly5.2/5.2 donor cells (left panel). Animals containing greater than 1% Ly5.2/5.2 cells in the peripheral blood were scored as positive and were used to calculate the frequency of HSCs in the secondary recipients (right panel).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

9. Figure 8 Summary of Hematopoiesis in c-MybM303V/M303V Mice
(A) Total number of cells measured at different points of hematopoiesis of multiple c-Mybwt/wt and c-MybM303V/M303V mice. Cell populations were identified as described in Figures 4, 5, and 6.
(B) Model of abnormal hematopoiesis in c-MybM303V/M303V mice. Shown is a schematic overview of hematopoiesis; green circles with (+) and red circles with (−) represent populations or differentiation pathways that are enhanced or repressed, respectively, in c-MybM303V/M303V mice. See text for details.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions