SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development

Abstract

Small ubiquitin-like modifier (SUMO1-3) conjugation plays a critical role in embryogenesis. Embryos deficient in the SUMO-conjugating enzyme Ubc9 die at the early postimplantation stage. Sumo1(-/-) mice are viable, as SUMO2/3 can compensate for most SUMO1 functions. To uncover the role of SUMO2/3 in embryogenesis, we generated Sumo2- and Sumo3-null mutant mice. Here, we report that Sumo3(-/-) mice were viable, while Sumo2(-/-) embryos exhibited severe developmental delay and died at approximately embryonic day 10.5 (E10.5). We also provide evidence that SUMO2 is the predominantly expressed SUMO isoform. Furthermore, although Sumo2(+/-) and Sumo2(+/-);Sumo3(+/-) mice lacked any overt phenotype, only 2 Sumo2(+/-);Sumo3(-/-) mice were found at birth in 35 litters after crossing Sumo2(+/-);Sumo3(+/-) with Sumo3(-/-) mice, and these rare mice were considerably smaller than littermates of the other genotypes. Thus, our findings suggest that expression levels and not functional differences between SUMO2 and SUMO3 are critical for normal embryogenesis.

Keywords: Embryonic development; Knockout; SUMO conjugation; SUMO2; SUMO3.

Figure 1. Targeted disruption of Sumo2 but not Sumo3 is lethal to embryos

A Ubiquitous expression of Sumo1, Sumo2, and Sumo3 in E7.5, E8.5, and E9.5 wild-type embryos. Whole-mount in situ hybridization analysis of Sumo1, Sumo2, and Sumo3 expression was performed in C57BL/6 wild-type mouse embryos. The specificity of all probes was validated by comparing whole-mount in situ hybridization in wild-type to Sumo1-, Sumo2-, and Sumo3-null embryos, as demonstrated in Supplementary Fig S1. Scale bars: 100 μm (left panel), 500 μm (right panel). B Targeting strategy for Sumo2 and Sumo3. The targeting vectors were designed to disrupt exon 1 by in-frame insertion of 2 stop codons and a neomycin (NEO) cassette downstream of the ATG codon. Arrowheads mark the location of primers for genotyping. Gray boxes show exons. TK, thymidine kinase cassette. C Representative genotyping results of embryos obtained from timed Sumo2^+/− or Sumo3^+/− intercrosses. Genomic DNA prepared from extraembryonic tissue was used for PCR amplification with primers as described in (B) and Supplementary Table S1. D Normal Mendelian distribution of newborn pups from Sumo3^+/− intercrosses. E Summary of genotyping analysis of staged embryos from Sumo2^+/− intercrosses. F Representative image of Sumo2 expression by whole-mount in situ hybridization in a wild-type embryo (control, left) and a homozygous null embryo (Sumo2^−/−, right) at E7.5. Scale bar: 100 μm.

Figure 2. Developmental arrest of Sumo2^−/− embryos

A Severe growth retardation of Sumo2^−/− embryos. Representative gross morphology of wild-type (left) and Sumo2^−/− littermate (right) embryos at different developmental stages (E7.5, E8.5, E9.5, E10.5, and E11.5). Sumo2^−/− embryo at E9.5 was enlarged to show somites (arrow). Scale bar: 500 μm. B Representative images of hematoxylin and eosin (H&E) staining of control and Sumo2^−/− embryos at E7.5. ee, embryonic ectoderm; me, mesoderm; ve, visceral endoderm; ch, chorion; ac, amniotic cavity; ex, exocoelomic cavity; am, amnion. Scale bar: 20 μm. C Representative images of whole-mount in situ hybridization analysis of Hnf4 (endoderm marker), Brachyury (mesoderm marker), and Otx2 (ectoderm marker) of wild-type and Sumo2^−/− littermate embryos at E7.5. ps, primitive streak.

Figure 3. Compromised cell proliferation and enhanced apoptotic cell death in Sumo2^−/− embryos

A Representative images of EdU (green) and TUNEL (red) staining of control and Sumo2^−/− embryos at E7.5. Nuclei were stained with DAPI. Scale bar: 20 μm. B, C Quantitative analysis of EdU and TUNEL staining. EdU-positive, TUNEL-positive, and total cells (DAPI staining) were counted, and percentages were calculated (n = 8–10 sections from 3 embryos/group). Data were presented as means ± SD. Student’s t-test was used to calculate statistical significance; * P < 0.05.

Figure 4. SUMO2 is the dominant SUMO isoform

A Relative expression levels of Sumo1, Sumo2, and Sumo3 in C57BL/6 wild-type embryos at E7.5 and E8.5, and in brains, hearts, and kidneys at postnatal day 0 (P0) and the adult state. Data are presented as means ± SD (n = 3). B Quantitative Western blot analysis results showed the dramatic decrease in levels of SUMO2/3-conjugated proteins in Sumo2^−/− E8.5 embryos compared to Sumo2^+/− and Sumo2^+/+ E8.5 embryos (***P < 0.001). Notably, SUMO1 conjugation did not increase in the absence of SUMO2. The high molecular weight regions marked by brackets were used to quantify SUMO1 or SUMO2/3 conjugation. Data are presented as means ± SD (n = 3). NS, not significant. C Quantitative Western blot analysis results showed no major difference in levels of SUMO2/3- or SUMO1-conjugated proteins in Sumo3^+/+, Sumo3^+/−, and Sumo3^−/− E8.5 embryos. The high molecular weight regions marked by brackets were used to quantify SUMO1 or SUMO2/3 conjugation. Data are presented as means ± SD (n = 3). NS, not significant. Source data are available online for this figure.