Actb−/− embryos are pale and growth retarded at E10.25.

<p>(A) Pictures of freshly dissected embryos at E10.25. Pale and growth retarded <i>Actb^−/−</i> embryos show no obvious vascular pattern in the embryo proper (middle) or yolk sac (right) compared to the <i>Actb^+/+</i> littermates. 15× magnification. (B) LacZ stainings of <i>Actb^+/+</i> and <i>Actb^−/−</i> yolk sac at E9.5 indicate reduced vascular branching complexity of <i>Actb^−/−</i> yolk sacs. 20× magnification. (C) Whole mount PECAM-1 immunohistochemistry of <i>Actb^+/+</i> and <i>Actb^−/−</i> embryos, processed in parallel, shows less coloring, indicating fewer endothelial cells and red blood cells. 15× magnification. Embryos were imaged on a Leica MS5 (Leica Microsystems) stereomicroscope. Digital images were acquired using a Leica camera.</p

Morphologically normal <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ embryos show improved Gata2 mRNA levels compared to <i>Actb−/−</i> embryos.

<p>(A) Relative E10.25 mRNA levels measured by qRT-PCR of <i>Gata1</i>, <i>Gata2</i>, Hbb-y, and Hbb-bh1 in yolk sacs of <i>Actb+/+</i>, <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ embryos with aberrant morphology and <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ embryos with normal morphology. (B) Relative E10.25 mRNA levels measured by qRT-PCR of Actg1 and <i>Gata2</i> targets: <i>c-kit</i>, and <i>EpoR</i> in yolk sacs of <i>Actb+/+</i>, <i>Actb−/−</i> and the two groups of <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ embryos. (C) Relative E11.5 mRNA levels measured by qRT-PCR of <i>Gata1</i>, <i>Gata2</i>, Hbb-y, and Hbb-bh1 in yolk sacs of <i>Actb+/+</i>, <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ embryos with aberrant morphology (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067855#pone-0067855-g006" target="_blank">Figure 6C</a>) and <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ with normal morphology. White bars  = <i>Actb+/+</i>, dark-grey bars  = <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ with aberrant morphology, light-grey bars  = <i>Actb−/−</i> R26+hGata2EpoR-iCre/+ with normal morphology.</p

Beta-actin binds to specific regions of the <i>Gata2</i> gene.

<p>(A) Immunoprecipitation with the anti beta-actin and anti gamma-actin antibodies followed by qRT-PCR with 9 specific primers for the <i>Gata2</i> promotor region yielded 2 loci of interest: amplicon 3 and 8. Error bars represent mean ±SEM; *P<.05, **P<.01. (B) Genomic alignment with multiple species showing that amplicon 3 is partly overlapping with a highly conserved region in the <i>Gata2</i> promotor, especially in mammals. Also the localization of the specific amplicons relative to the mouse <i>Gata2</i> gene is shown. Amplicon8 is located between exons 1 and 2. Figure was made using UCSC genome bioinformatics software (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>).</p

Absence of beta-actin during primitive erythropoiesis correlates with reduced Gata2 expression levels in the yolk sac.

<p>(A) Relative E8.5 yolk sac mRNA levels measured by qRT-PCR of <i>Gata1</i>, <i>Gata2</i>, Hbb-y, Hbb-bh1, Hba and Hbb. We found a 90% decrease of <i>Gata2</i> expression level in <i>Actb−/−</i> embryos versus <i>Actb+/+</i> embryos. (B) Relative E8.5 yolk sac mRNA levels measured by qRT-PCR of <i>Gata2</i> target genes: <i>Runx1</i>, Tal1/Scl, <i>c-kit</i> and <i>EpoR</i>. <i>c-kit</i> and <i>EpoR</i> show 50% reduced expression levels in <i>Actb−/−</i> embryos versus <i>Actb+/+</i> embryos. (C) <i>Gata2</i> immunohistochemistry on E9.5 yolk sac sections. <i>Gata2</i> expression was detected only in the endoderm of the yolk sac (white arrows). No difference in <i>Gata2</i> expression could be seen between <i>Actb−/−</i> embryos and <i>Actb+/+</i> embryos. 60× magnification. (D) Relative E8.5 and E10.25 yolk sac mRNA levels measured by qRT-PCR of Vegf. At E10.25, we could demonstrate a 50% increase of Vegf mRNA expression. Error bars represent mean ±SEM; *P<.05. Immunofluorescence sections were imaged using an Olympus IX81 confocal microscope with Fluoview FV10 software.</p

Yolk sac erythropoiesis is impaired in Actb−/− embryos.

<p>(A) Primitive erythroid colonies (EryP) from E8.5 yolk sacs measured by methylcellulose assays show a dramatic decrease in colony forming potential in <i>Actb^−/−</i> embryos compared to <i>Actb^+/+</i> embryos. Results are given as percentage of <i>Actb^+/+</i> embryos absolute number of colonies (100%). (B) Definitive erythroid colonies (BFU-E, CFU-GM and CFU-GEMM) from E9.75 yolk sacs measured by methylcellulose assays show an 85 to 90% decrease in colony forming potential of <i>Actb^−/−</i> embryos versus <i>Actb^+/+</i> embryos. Results are given as percentage of <i>Actb^+/+</i> embryos absolute number of colonies (100%). Bars represent mean ±SEM; *P<.05, **P<.01, ***P<.001.</p

Actb−/− embryos show reduced number of red blood cells and abnormal blood island morphology in the yolk sac.

<p>(A) PECAM-1 immunostained sagittal cardiac and somitic section, showing normal development of the left atrium (a), atrioventricular canal (av), left ventricle (v) and regular development of intersomitic vessels. (B) H&E stained yolk sac sections show disrupted blood island morphology with empty, enlarged cavities in E9.5 <i>Actb^−/−</i> embryos. A and B at 20× magnification. (C) H&E stained yolk sac sections show an almost complete absence of blood islands in E10.25 <i>Actb^−/−</i> embryos. 40× magnification. (D) PECAM-1 immunostained yolk sac section showing disorganized endothelial patterning and almost no red blood cells populating the remaining blood islands at E10.25. C and D at 40× magnification. Sections were imaged using a SNAP-COOL camera (Roper Scientific) mounted on an Olympus Bx51 microscope (Olympus), with Plan Olympus 20×/0.40 or 40×/0.65 lens and RSImage Version 1.9.2 software (Roper Scientific).</p