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First published online 11 June 2008
doi: 10.1242/dev.022707

Development 135, 2455-2465 (2008)
Published by The Company of Biologists 2008
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Placental rescue reveals a sole requirement for c-Myc in embryonic erythroblast survival and hematopoietic stem cell function

Nicole C. Dubois1,*, Christelle Adolphe1, Armin Ehninger1, Rong A. Wang2, Elisabeth J. Robertson3 and Andreas Trumpp1,{dagger}

1 Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC-Swiss Institute for Experimental Cancer Research, School of Life Science, 1066 Epalinges, Switzerland.
2 Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgery, University of California, San Francisco, CA 94143, USA.
3 Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.


Figure 1
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  		Fig. 1. Sox2-Cre mediated epiblast-specific deletion of c-myc reveals viable embryos up to E11.5. (A) Schematic diagram showing the conditional c-mycflox allele (Trumpp et al., 2001Go). Exons are indicated as boxes 1-3. The open reading frame (ATG to Stop; yellow segments) is flanked by loxP sites (black triangles). Restriction sites and probe (blue ovals) used for Southern blot analysis are indicated. (B) Southern blot showing recombination at the c-myc locus in c-mycflox/{Delta}ORFrec (cont) and Sox2Cre;c-mycflox/flox (exp) embryo and c-mycflox/flox (cont) and Sox2Cre;c-mycflox/flox (exp) placenta at E10.5. (C) Real-time RT-PCR analysis of c-myc, N-myc and L-myc expression in Sox2Cre;c-mycflox/flox embryos at E10.5. Expression levels in mutants are normalized to that in controls, which was set to 1. (D) Phenotypes of control (left) and Sox2Cre;c-mycflox/flox embryos (right) at (i) E9.5, (ii) E10.5 and YS, and (iii) E11.5.

 

Figure 2
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  		Fig. 2. Severe placental defects in c-myc{Delta}ORF{Delta}/ORF embryos. (A) Immunohistochemical analysis of c-Myc expression in the E10.5 wild-type placenta. c-Myc expression is detected in the embryonic parts of the placenta, including the chorionic plate (CP), labyrinth layer (La), spongiotrophoblast (Sp), trophoblast giant cells (TGC) and in hematopoietic cells derived from the embryo, which are located in the fetal blood spaces (inset A'). (B-D) Comparison of control (left) and c-Myc null (c-myc{Delta}ORF/{Delta}ORF) embryos (right) at E10.5. (B) Hematoxylin and eosin (H&E) staining of control and c-Myc-deficient placenta at E10.5. The bottom panels show an enlargement of the boxed areas in the top panels. (C) Analysis of placental vascularization. Laminin (pink) is expressed on embryonic vessels and defines fetal blood spaces. Cytokeratin (brown) marks trophoblast cells and defines maternal blood spaces. The bottom panels show an enlargement of the boxed areas in the top panels. The white line demarcates the failure of vessels (pink) to invade into the trophoblast (brown) layer (D) Bromodeoxyuridine (BrdU) incorporation analysis of E9.5 control and c-Myc-deficient placenta. Highly proliferating (BrdU+; arrows) tissues are present in control (left) but not in c-myc{Delta}ORF/{Delta}ORF (right) placentas. (E) Hematoxylin and eosin (H&E) staining of control and Sox2Cre;c-mycflox/flox placenta at E10.5. Placental developmental defects are rescued with respect to c-myc{Delta}ORF/{Delta}ORF in B.

 

Figure 3
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  		Fig. 3. c-Myc deficiency causes apoptotic loss of primitive erythroblasts. (A) H&E staining of control and Sox2Cre;c-mycflox/flox YS at E10.5. Control YS vessels contain numerous hematopoietic cells (arrows), whereas mutant vessels are mostly devoid of hematopoietic cells (arrows), although individual cells are sometimes observed (arrowheads). (B) FACS analysis of the primitive embryonic system at E11.0. CD71 and Ter119 are used to define primitive proerythroblasts (PPE; CD71-Ter119+) and primitive erythroblasts (PE; CD71+Ter119+). (C) Quantitative analysis of total PPE and PE number in control and c-Myc-deficient embryos at E11.0 (**P≤0.001). (D) Quantitative analysis of TUNEL-positive CD71+ cells in E9.5 and E10.5 embryos (**P≤0.001).

 

Figure 4
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  		Fig. 4. Impaired definitive hematopoiesis in c-Myc-deficient embryos. (A) Expression of the pan-hematopoietic marker CD45 in control and c-Myc-deficient dorsal aorta (top panels) and fetal liver (bottom panels) at E11.0. (B) FACS analysis showing CD45 expression in E11.0 embryos. Numbers in the CD45+ gates represent percentage of cells per embryo. (C) Quantitative analysis of total CD45+ cells per E11.0 embryos (as defined in B) (**P≤0.001). (D) FACS analysis showing the expression of the stem cell markers Kit (CD117) and AA4.1 (CD93) in CD45+ hematopoietic cells of control (left) and c-Myc-deficient (right) embryos at E11.0. Numbers represent percentages in the CD45+ population. (E) Quantitative analysis showing the percentage of Kit+AA4.1+ (stem/progenitor) cells within the CD45+ population in E11.0 control and Sox2Cre;c-mycflox/flox embryos (*P≤0.01). (F) Quantitative analysis showing the number of CD45+Kit+AA4.1+ cells per E11.0 control and Sox2Cre;c-mycflox/flox embryos. (G) CFU-assay at E10.5. Examples of blast-forming unit-erythroid (BFU-E) and colony-forming unit-macrophage/granulocyte (CFU-M/G) are shown for control embryos. Cells derived from Sox2Cre;c-mycflox/flox embryos failed to give rise to typical colonies, but only produced very rare colonies composed of very small cells (bottom right). (H) Quantitative analysis of total colony number per embryo after 8 days in culture.

 

Figure 5
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  		Fig. 5. Proliferation and development of non-hematopoietic tissues, including the vascular system, are normal in c-Myc-deficient embryos. (A) Immunohistochemical analysis of BrdU incorporation in control (left) and c-Myc-deficient (right) embryos at E11.0. (B) PECAM (CD31) staining in wholemounts (top) and histological sections (bottom) show normal development of the vascular system in the embryo, the placenta and the YS of Sox2Cre;c-mycflox/flox embryos at E11.0.

 

Figure 6
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  		Fig. 6. Hematopoietic- and endothelial-specific elimination of c-Myc via Tie2Cre. (A) Phenotypes of control and Tie2Cre;c-mycflox/flox embryos and YS at E11.0. (B) Quantitative analysis of total CD45+ cells per E11.0 embryos (**P≤0.001). (C) FACS analysis showing the expression of the stem cell markers Kit (CD117) and AA4.1 (CD93) in CD45+ hematopoietic cells of control (left) and Tie2Cre;c-mycflox/flox (right) embryos at E11.0. Numbers represent percentages in the CD45+ population.

 

Figure 7
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  		Fig. 7. Fetal liver development requires hematopoietic cells but not c-Myc. (A) c-Myc expression in the fetal liver of E10.5 control (upper panel; higher magnification is shown in the right panel) and Sox2Cre;c-mycflox/flox (bottom left) embryos. The broken line outlines the fetal liver. c-Myc is expressed in hepatoblasts (arrows) and blood cells (arrowheads). (B) Hematoxylin and Eosin (H&E) staining of control and Sox2Cre;c-mycflox/flox fetal livers at E10.5. The top panels show low magnification, whereas the bottom panels show a high magnification. (C) HNF4{alpha} expression in control and Sox2Cre;c-mycflox/flox embryos at E11.0. (D) H&E staining (top panels) and HNF4{alpha} expression (bottom panels) in E11.0 control and Tie2Cre;c-mycflox/flox embryos. (E) Model illustrating the different direct roles for c-Myc in the midgestation embryo and the placenta. First, c-Myc is required to generate a functional embryonic placenta. Second, only in the presence of a normal placenta are the direct roles of c-Myc in the embryo proper revealed. Primitive YS erythropoiesis is drastically decreased. Although definitive AGM derived HSCs are generated in normal numbers, these mutant HSCs are non-functional and only very few hematopoietic cells (of primitive and definitive origin) reach the fetal liver, which as a consequence remains hypoplastic and fails to expand. All other organs and tissues proliferate and develop normally.

 

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