Fig 2: Myb function is required for both Hoxb8 and non-Hoxb8 hematopoietic progenitors during fetal liver hematopoiesis. (A-C) Representative flow cytometry plots of Ter119− Kit+ cells gated for tdTomato from the fetal liver (FL) of (A) E11.5, (B) E12.5 and (C) E14.5 Myb+/+ and Myb−/− embryos. (D,E) Representative flow cytometry plots of CD16/32+ EMPs (CD41+ Kit+) gated for tdTomato from fetal livers of E12.5 (D) Myb+/+ and (E) Myb−/− embryos. (F-H) Number of Hoxb8 lineage (B8L) and non-Hoxb8 lineage (nB8L) cells in the Kit+ population in fetal livers of (F) E11.5, (G) E12.5 and (H) E14.5 Myb+/+ and Myb−/− embryos. E11.5: n=2 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb−/− in B8L (P=0.0351) and nB8L (P<0.0001). E12.5: n=2 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+with Myb−/− in B8L (P=0.0047) and nB8L (P<0.0001). E14.5: n=5 biological replicates (Myb+/+) and n=3 biological replicates (Myb−/−); two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb−/− in B8L (P<0.0001) and nB8L (P=0.9406). (I) Number of B8L and nB8L cells in the CD16/32+ EMP population of the E12.5 fetal liver. n=2 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb−/− in B8L (P=0.0040) and nB8L (P=0.0150). Gray circles are individual data points. Data are mean±s.e.m. n.s., non-significant, *P<0.05, **P<0.01, ****P<0.0001. Data are from two or three independent experiments per time point.

Fig 3: Hoxb8 hematopoietic progenitors in the AGM are selectively affected in the absence of Myb function. (A) Representative flow cytometry plots of total live Ter119- Kit+ cells gated for tdTomato from yolk sac of E10.5 Myb+/+ and Myb-/- embryos. (B) Number and (C) percentage of Hoxb8 lineage (B8L) and non-Hoxb8 lineage (nB8L) cells in the Kit+ population in the yolk sac of E10.5 Myb+/+ and Myb-/- embryos. n=5 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb-/- in B8L [P=0.9909 (number), P=0.8153 (percentage)] and nB8L [P=0.2052 (number) and P=0.8594 (percentage)]. (D) Representative flow cytometry plots of total live Ter119- Kit+ cells gated for tdTomato from AGM of E10.5 Myb+/+ and Myb-/- embryos. (E) Number of B8L and nB8L cells in the Kit+ population in AGM of E10.5 Myb+/+ and Myb-/- embryos. n=4 (Myb+/+) and n=5 (Myb-/-) biological replicates; two-way ANOVA with post-hoc analysis comparing Myb+/+with Myb-/- in B8L (P=0.0040) and nB8L (P=0.3300). Gray circles are individual data points. Data are mean±s.e.m. n.s. non-significant, **P<0.01. Data in all graphs are from three or four independent experiments.

Fig 4: Osteo-angiogenic development of long bones is disrupted in VEGF cTg mice(A) Total VEGF mRNA (qRT-PCR) and VEGF164 protein levels (ELISA) in bones of control and VEGF cTg embryos (n = 4–8).(B) VEGF serum levels (ELISA) (n = 6).(C) Skeletal preparations at E18.5. Middle panels, dissected hindlimbs of control (top) and VEGF cTg (bottom).(D) Longitudinal (top) and transversal (bottom) 3D micro-computed tomography (micro-CT) reconstructions of E18.5 tibias.(E) Histology of E18.5 tibias; (left) Safranin O and (right) H&E. Brackets, BM cavity.(F) BM cavity volume at E18.5 (n = 7).(G) CD31 IHC on tibia sections. Arrows, aberrant perichondrial vascularization.(H) Maximum intensity projection (MIP) of 20-µm depth confocal imaging of Osx-Cre:GFP (nuclear green, OPCs), Emcn (red, blood vessels), and Hoechst (blue, nuclei) in E18.5 tibias. Left, overview; middle, metaphysis; right, diaphysis.(I) Flow cytometry of CD45-Ter119-CD31-Osx/GFP+ cells in E18.5 bones (n = 4–5).All graphs represent mean ± SEM and depict individual data points derived from different mice (also in the next figures); *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t test between the genotypes for each time point; no symbol implies p > 0.05. See also Figure S1.

Fig 5: The Myb -68 enhancer is active in Ly6C- GMPs in Myb -68 GFP transgenic mice. GFP activity in whole bone marrow (a), LS-K cells (b), and GMPs (c) of Myb -68 GFP mice. FACS gating strategies are shown in Supplementary Fig. 12. Representative FACS plots of indicated cells from control (grey) and Myb -74 GFP mice (green) are shown. Numbers shown are mean percentages±SD of GFP+ cells in Myb -68 GFP mice. The percentages of GFP+ cells are also shown in the right graphs. a n = 10 mice for LSK cells and LS-K cells, and n = 4 mice for others. p = 4.1 × 10-4 by one-way Welch’s ANOVA. ***p < 0.001 by the Games-Howell post hoc test. p = 4.1 × 10-4 between LSK and LS-K, p = 3.7 x 10-4 between CD11b+Gr1+ and LS-K, p = 3.5 x 10-4 between B220+ and LS-K, p = 3.5 x 10-4 between CD4+ and LS-K, p = 3.5 x 10-4 between CD8+ and LS-K, and p = 3.9 × 10-4 between Ter119+- and LS-K. (b) n = 6 mice. p = 5.8 × 10-7 by one-way Welch’s ANOVA. **p < 0.01 by the Games-Howell post hoc test. p = 0.0014 between PreGM and GMP, p = 0.0008 between PreMegE and GMP, p = 0.0007 between PreCFU-E and GMP, p = 0.0006 between CFU-E/ProEry and GMP, and p = 0.0006 between MkP and GMP. c n = 10 mice for Ly6C- GMPs and n = 7 mice for others. p = 8.5 × 10-6 by one-way Welch’s ANOVA. ***p < 0.001 by the Games-Howell post hoc test. p = 1.1 × 10-5 between Ly6C- GMPs and GP, and p = 1.1 × 10-5 between Ly6C- GMPs and MP. LSK Lin-Sca1+cKit+ cells, LS-K Lin-Sca1-cKit+ cells, MkP megakaryocyte progenitor, GMP granulocyte/monocyte progenitor, PreGM pre-granulocyte/macrophage, CFU-E colony-forming unit-erythroid, ProEry pro-erythrocytes, PreCFU-E pre-colony-forming unit-erythroid, PreMegE pre-megakaryocyte/erythrocyte, GP granulocyte progenitor, MP monocyte progenitor.