PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis

Abstract

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A^-/-) is embryonic lethal. Notably, livers of PDE2A^-/- embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A^-/- liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A^-/- livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A^-/- embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A^-/- embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis.

Keywords: ICER; apoptosis; cAMP; fetal liver; hematopoiesis; phosphodiesterase 2A.

Figure 1

Morphological defects in liver of PDE2A^−/− embryos. (a) Volume rendering visualization of coronal sections obtained from E14.5 PDE2A^+/+ and PDE2A^−/− embryos. Sections were chosen referring to the stomach lumen (LS) volume. H heart, L liver, PC peritoneal cavity, IS interlobular space, MV mesencephalic vesicle, SG submandibular gland, RM roof of midbrain, NP nasopharynx. (b) Representative stereomicroscope picture of livers isolated from E14.5 PDE2A^+/+, PDE2A^+/− and PDE2A^−/− embryos. The liver of PDE2A^−/− is hypoplastic. (c) Liver weight compared at E14.5 in PDE2A^+/+, PDE2A^+/− and PDE2A^−/− embryos. (d) Histological analysis of E14.5 embryos stained with H&E. Scale bar 500 µm. (e) Histological analysis of E14.5 liver sections stained with H&E. Scale bars: 200 µm (a,b), 100 µm (c,d) and 50 µm (e,f). (f) cAMP level in E 14.5 PDE2A^+/+ and PDE2A^−/− livers. (g) Expression of ICER in livers of E14.5 PDE2A^+/+ and PDE2A^−/− mice evaluated by qRT-PCR. At least N = 3 embryos/genotype were analyzed in each experiment. * P < 0.05; ** P < 0.01.

Figure 2

Transcript modulation in liver maturation of PDE2A^−/− embryos. qRT-PCR in E14.5 liver embryos shows reduction of expression of liver differentiation markers and increase of stromal and endothelial markers relative to GAPDH expression. (a) Relative mRNA level of albumin, α-fetoprotein and transcription factors cMET, cEBPα, HNF1, HNF4 and HNF6 in liver of PDE2A^−/− embryos. (b) Relative mRNA level of CD31, vimentin and α-SMA in liver of PDE2A^−/− embryos. At least N = 3 embryos/genotype were analyzed in each experiment. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 3

Apoptosis is increased in liver of PDE2A^−/− embryos. (a) Cells dissociated from liver of E14.5 PDE2A^+/+, PDE2A^+/− and PDE2A^−/− embryos display cell cycle similarities by flow cytometry analysis. (b) TUNEL staining shows increased apoptosis in livers sections derived from E14.5 PDE2A^−/− embryos respect to PDE2A^+/+ embryos. Scale bar: 50 µm. N = 3 embryos/genotype. (c) Representative western blot analysis of cleaved caspase-3 expression in liver extracts of E14.5 PDE2A^+/+, PDE2A^+/− and PDE2A^−/− embryos. Densitometry is shown relative to tubulin expression. N = 3 embryos/genotype. (d) qRT-PCR in E14.5 liver embryos showing Bcl2 expression. N = 3 embryos/genotype. (e) E14.5 liver cells isolated from C57BL/6 embryos enter apoptosis after TNFα (5 ng/mL) and CHX (25 µg/mL) treatments if pretreated with the PDE2A inhibitor EHNA (10 µM). Apoptosis was evaluated by cleaved caspase-3 in western blots. Densitometry analysis relative to tubulin is shown. N = 2 embryos. * P < 0.05.

Figure 4

The niche is compromised in liver of PDE2A^−/− embryos. (a–c) Flow cytometry analysis of liver cells isolated from E14.5 PDE2A^+/+ and PDE2A^−/− embryos, stained with Annexin-V and antibodies against CD71 (a), CD45 (b), CD31 (c) markers. Histogram showing the percentage of double stained cells reveals an increase of endothelial cell apoptosis in PDE2A^−/− embryos. N = 3 embryos/genotype. * P < 0.05. (d,e) Immunofluorescence of E14.5 liver sections stained with α-FP and α-SMA antibodies (red) and with TUNEL assay. Nuclei were counterstained with DAPI (blue). Arrows indicate double stained cells. Scale bar 50 µm. N = 3 embryos/genotype.

Figure 5

Impairment of blood cells maturation in PDE2A^−/− embryos. (a) Smear of blood from E14.5 PDE2A^+/+ and PDE2A^−/− embryos. Arrowhead indicates nucleated red blood cells originated in the yolk sac; arrow indicates mature non-nucleated cells derived from liver. Scale bars: 20 µm. In PDE2A^−/− embryos the immature nucleated blood cells are predominant compared to PDE2A^+/+. (b–h) Flow cytometry analysis of liver cells isolated from E14.5 PDE2A^+/+, PDE2A^+/−, PDE2A^−/− embryos, stained with antibodies against CD45, Ter119, F4/80, CD41/CD61, GR1 and LSK markers. Ter119, F4/80, CD41/CD61, GR1, that label mature erythrocytes, macrophages, megakaryoblasts and granulocytes respectively, show statistically significant reduction, whereas LSK and CD11b, detecting stem/progenitor cells, show a significant increase in PDE2A^−/− samples compared to PDE2A^+/+ and PDE2A^+/− embryos. N = 4 embryos/genotype. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 6

The maturation potential of hematopoietic progenitor/stem cells is preserved in PDE2A^−/− embryos. Colony-Forming-Cell (CFC) assay identifies an equivalent number of colonies in cells isolated from E14.5 PDE2A^+/+ and PDE2A^−/− livers. The different types of colonies are labeled as follows: GEMM colony forming unit granulocyte-erythrocyte-megakaryocyte-monocyte; CFU-GM granulocyte-monocyte; CFU-G granulocyte; CFU-M monocyte; BFU-E Burst forming unit erythroid. At least N = 3 embryos/genotype were analyzed in each experiment in triplicates.