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. 2016 Feb 17:6:20999.
doi: 10.1038/srep20999.

Extraembryonic but not embryonic SUMO-specific protease 2 is required for heart development

Eri O Maruyama  1 , Heng Lin  1 , Shang-Yi Chiu  1 , H-M Ivy Yu  1 , George A Porter  2 , Wei Hsu  1   3   4
Affiliations

Extraembryonic but not embryonic SUMO-specific protease 2 is required for heart development

Eri O Maruyama et al. Sci Rep. .

Abstract

SUMO-specific protease 2 (SENP2) activities to remove SUMO from its substrates is essential for development of trophoblast stem cells, niches and lineages. Global deletion of SENP2 leads to midgestation lethality, and causes severe defects in the placenta which is accompanied by embryonic brain and heart abnormalities. Because of the placental deficiencies, the role of SENP2 in development of the embryonic tissues has not been properly determined. The brain and heart abnormalities may be secondary to placental insufficiency. Here we have created a new mouse strain permitting conditional inactivation of SENP2. Mice homozygous for germline deletion of the conditional allele exhibit trophoblast defects and embryonic abnormalities resembling the global SENP2 knockout. However, tissue-specific disruptions of SENP2 demonstrate its dispensable role in embryogenesis. Placental expression of SENP2 is necessary and sufficient for embryonic heart and brain development. Using a protease deficient model, we further demonstrate the requirement of SENP2-dependent SUMO modification in development of all major trophoblast lineages. SENP2 regulates sumoylation of Mdm2 which controls p53 activities critical for G-S transition of mitotic division and endoreduplication in trophoblast proliferation and differentiation, respectively. The differentiation of trophoblasts is also dependent on SENP2-mediated activation of p57(Kip2), a CDK-specific inhibitor required for endoreduplication.

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Figures

Figure 1
Figure 1. Diagrams illustrate the targeting strategy and the creation of mice carrying SENP2Fx or SENP2 mutant allele.
(A) In the targeted allele, a loxP site and a pgk-neo cassette flanked by two loxP sites were inserted into intron 3 and intron 4, respectively. Mice carrying the SENP2 targeted allele were created, and crossed with the EIIa-Cre transgenic mice to generate progeny carrying the SENP2Fx or SENP2 mutant allele. (B–D) PCR analysis detected the presence of 5′ (PCR: P1–P2) and 3′ (PCR: P3–P4) loxP sites for genotyping the wild type (+/+) and heterozygous (Fx/+) mice, and examined the deletion of exon 4 (PCR: P1–P4). (E) RT-PCR analysis detected the transcripts generated from the wild type (+/+), heterozygous (+/−) and homozygous (−/−) embryos of SENP2lacZ (left panel) and SENP2 mutant (right panel). (F) Immunoblot analysis examines protein expression in SENP2+/+ and SENP2−/− embryos. Actin level is used as a loading control.
Figure 2
Figure 2. Heart development is deformed in the SENP2 homozygous embryos similar to those of SENP2lacZ.
Gross morphological evaluation of the wild type (A), SENP2lacZ−/− (D), SENP2+/− (G) and SENP2−/− (J) embryos at the specific somite stage (S) as indicated identifies growth restriction caused by the deletion of SENP2. Histology shows the atrioventricular (AV) cushion (B,E,H,K; asterisks) and myocardium (C,F,I,L) defective in the mutants (E,F,K,L). Scale bars, 1 mm (A,D,G,J); 500 μm (B,E,H,K); 100 μm (C,F,I,L).
Figure 3
Figure 3. The SENP2 homozygous mutant exhibits extraembryonic abnormalities.
Histology examines the placentas of SENP2+/+ (A–D) and SENP2−/− (E–H) in transverse sections at E10.5. Labyrinth (L), spongiotrophoblast (S) and trophoblast giant cell (G) layers were defined by blue, red and green broken lines, respectively. Scale bars, 500 μm (A,E); 50 μm (B–D,F–H).
Figure 4
Figure 4. SENP2 is expressed in developing embryonic heart.
The expression pattern of SENP2 is examined in control (A–C,G–I) and SENP2lacZ heterozygous (D–F,J–L) embryos by β-gal staining and in situ hybridization (I,L) in whole mounts (A–F) and sections (G–L). Asterisks indicate AV cushions. Scale bars, 1 mm (A–F); 100 μm (G–L).
Figure 5
Figure 5. Sox2-Cre permits loxP site-specific recombination in the embryonic but not extraembryonic tissues.
The efficiency of the Sox2-Cre mediated recombination in the epiblast, including myocardium (B,E) and AV cushion (C,F), and trophoblast (H,I) is analyzed by β-gal staining in whole mounts (A,D,G) and sections (B,C,E,F,H,I) of the R26RlacZ heterozygous embryo (A–F) and placenta (G–I), negative (A–C,G–H) or positive (D–F,G,I) for the Cre transgene. Note positive β-gal stains in the left placenta in panel G are attributed to presence of the residual embryonic tissue/yolk sac. Sections were counterstained by nuclear fast red. Labyrinth (L), spongiotrophoblast (S) and trophoblast giant cell (G) layers were defined by blue, red and green broken lines, respectively. (J) Immunoblot analysis examines protein expression in the control and SENP2 mutant epiblasts. Actin level is used as a loading control. Scale bars, 1 mm (A,D); 500 μm (B,C,E–I); 800 μm (G).
Figure 6
Figure 6. SENP2 is dispensable for embryogenesis.
The E10.5 (A–H), E14.5 (I–P) and E18.5 (Q–X) control (A–D,I–L,Q–T, genotype: SENP2Fx/Fx) and SENP2Sox2 mutant (E–H,M–P, genotype: Sox2-Cre+; SENP2Fx/Fx and U-X, genotype: Sox2-Cre+; SENP2Fx/−) embryos were examined in whole mounts and H&E stained sections. Asterisks indicate AV cushions and enlargements of the inset are shown in (C,D,G,H,K,L,O,P,S,T,W,X). Scale bars, 1 mm (A,E); 5 mm (I,M,Q,U); 500 μm (B,F,J,N,R,V); 100 μm (C,D,G,H,K,L,O,P,S,T,W,X).
Figure 7
Figure 7. The SUMO protease core domain of SENP2 is essential for normal placentation leading to development of a healthy embryo.
The E10.5 control (1st and 3rd rows, genotype: SENP2∆SUMO+/+ or +/∆) and protease core domain-deficient mutant (2nd and 4th rows, genotype: SENP2∆SUMO−/−) embryos (A–H) and placentas (I–P) were examined in whole mounts (A,B) and H&E stained sections (C–P). Enlargements of the insets (C,F) are shown in D, E, G and H. Labyrinth (L), spongiotrophoblast (S) and trophoblast giant cell (G) layers were defined by blue, red and green broken lines, respectively. M, MC and asterisk indicate maternal decidua, myocardium and AV cushion, respectively. Scale bars, 1 mm (A,B,I,M); 500 μm (C,F); 50 μm (D,E,G,H,J–L,N–P).
Figure 8
Figure 8. SENP2-mediated SUMO modification regulates subcellular distribution of Mdm2.
Immunostaining of Mdm2 was performed on the SENP2+/+ (A–C,G) and SENP2−/− (D–F,H) placentas at E7.5 (A–F) and E8.5 (G,H). Sections were counterstained with DAPI (B–C,E–F) or hematoxylin (G,H). (I) Statistical analysis indicates the percentage of nuclear (Nuc) vs. non-nuclear localizations of Mdm2 (data represent the mean ± SEM; *p < 0.000001, n = 3). Three samples were used and 3–4 sections from each sample were counted. GFP analysis of TS cells, transfected by the GFP tagged Mdm2 (J–L) or Mdm2-SUMO1∆GG (M–O), reveals their differential compartmentalization. Arrows indicate dislocation of Mdm2 from the cytoplasm to nucleus of TGC. Ch, chorion; G, TGC layer. Scale bars, 20 μm (A–F,J–O); 50 μm (G,H).
Figure 9
Figure 9. SENP2 regulation of cyclin-dependent kinase inhibitor 1C through SUMO modification of Mdm2 is essential for trophoblast stem cell development.
Immunostaining of Cyclin-dependent kinase inhibitor 1C (p57Kip2) was performed on E8.5 (A,B) and E9.5 (C,D) SENP2+/+ (A,C) and SENP2−/− (B,D) placentas. Positive staining of p57Kip2 identifies differentiated TGCs in four independent trophoblast stem cell lines (Wild type: TS1, TS12; Mutant: TS6, TS17) before (E,G,I,K) and after (F,H,J,L,M–O) induction of differentiation. (M–O) Wild type TS1 trophoblast stem cells transfected with a construct expressing GFP-tagged Mdm2-SUMO1∆GG were differentiated into TGCs, followed by immunostaining of p57Kip2, DAPI counterstaining and imaging analysis. G, TGC layer (primary TGC in A–B and secondary TGC in C–D); M, maternal decidua; Yc, yolk sac. Scale bars, 50 μm (A–D,M–O); 200 μm (E–L).
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