Limits...
The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

Tateishi K, Omata M, Tanaka K, Chiba T - J. Cell Biol. (2001)

Bottom Line: Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells.However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle.This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-Ku, Tokyo 113-8613, Japan.

ABSTRACT
NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

Show MeSH

Related in: MedlinePlus

Loss of embryonic ectoderm in Uba3−/− embryos. Sections of E6.0 embryos were stained with TROMA-1 (A and C) and H&E (B and D). TROMA-1 stained the extraembryonic tissue of wild-type embryos but not embryonic ectoderm (A and B). The majority of Uba3−/− embryos at E6.0 consisted of extraembryonic tissue (TROMA-1 positive) (C) and lacked embryonic proper (D). EE, embryonic ectoderm; EXE, extraembryonic ectoderm. Bars, 100 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198877&req=5

fig3: Loss of embryonic ectoderm in Uba3−/− embryos. Sections of E6.0 embryos were stained with TROMA-1 (A and C) and H&E (B and D). TROMA-1 stained the extraembryonic tissue of wild-type embryos but not embryonic ectoderm (A and B). The majority of Uba3−/− embryos at E6.0 consisted of extraembryonic tissue (TROMA-1 positive) (C) and lacked embryonic proper (D). EE, embryonic ectoderm; EXE, extraembryonic ectoderm. Bars, 100 μm.

Mentions: Uba3 heterozygous mice were born healthy and fertile without any noticeable pathological phenotypes compared with wild-type littermates at least within the 2-yr observation period. However, subsequent intercrossing of heterozygous mice has so far failed to produce any viable homozygous (Uba3−/−) mice (Table I). To further characterize Uba3−/− mice, embryos in utero at various stages of development were dissected out and their genotypes analyzed by Southern blot or PCR (Fig. 1, B and C). Although Uba3−/− mice were evident at embryonic day (E)3.5 with normal appearance, we noticed that ∼13% of total embryos had defects in blastocyst formation and hatching. These embryos were homozygous mutants as revealed by PCR genotyping. Thus, it was likely that half of the Uba3−/− embryos die before implantation (Table I). The phenotype may vary according to the amount of Uba3 maternally provided because weak Uba3 immunoreactivity was observed in every blastocyst examined (unpublished data). We next examined the histology of embryos between E5.5 and E7.5 (Fig. 2, A–H). In E6.0–6.5 embryos, a cylinder-like two-layered cellular structure was observed in normal embryos (Fig. 2, A–D). However, Uba3-deficient embryos, which could be identified at this stage by the absence of Uba3 immunoreactivity (Fig. 2 E), lacked such cylinder-like structure (Fig. 2, F and G). By E7.5, these disorganized embryos could not be found; rather, only completely absorbed embryonic tissues could be identified in the decidua (Fig. 2 H and Table I). We next characterized the nature of disorganized embryos at E6.0 by immunohistochemistry using several tissue-specific markers. Cytokeratin-endoA antibody (TROMA-1) stains the trophophectoderm cell lineage (Brulet et al., 1980). TROMA-1 stained the extraembryonic tissue of wild-type embryos but not embryonic ectoderm (Fig. 3, A and B). In Uba3-deficient embryos, TROMA-1 reacted to most cells at E6.0, suggesting that these cells were composed mostly of extraembryonic tissue (Fig. 3, C and D), and thus these embryos lacked embryonic ectoderm.


The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

Tateishi K, Omata M, Tanaka K, Chiba T - J. Cell Biol. (2001)

Loss of embryonic ectoderm in Uba3−/− embryos. Sections of E6.0 embryos were stained with TROMA-1 (A and C) and H&E (B and D). TROMA-1 stained the extraembryonic tissue of wild-type embryos but not embryonic ectoderm (A and B). The majority of Uba3−/− embryos at E6.0 consisted of extraembryonic tissue (TROMA-1 positive) (C) and lacked embryonic proper (D). EE, embryonic ectoderm; EXE, extraembryonic ectoderm. Bars, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198877&req=5

fig3: Loss of embryonic ectoderm in Uba3−/− embryos. Sections of E6.0 embryos were stained with TROMA-1 (A and C) and H&E (B and D). TROMA-1 stained the extraembryonic tissue of wild-type embryos but not embryonic ectoderm (A and B). The majority of Uba3−/− embryos at E6.0 consisted of extraembryonic tissue (TROMA-1 positive) (C) and lacked embryonic proper (D). EE, embryonic ectoderm; EXE, extraembryonic ectoderm. Bars, 100 μm.
Mentions: Uba3 heterozygous mice were born healthy and fertile without any noticeable pathological phenotypes compared with wild-type littermates at least within the 2-yr observation period. However, subsequent intercrossing of heterozygous mice has so far failed to produce any viable homozygous (Uba3−/−) mice (Table I). To further characterize Uba3−/− mice, embryos in utero at various stages of development were dissected out and their genotypes analyzed by Southern blot or PCR (Fig. 1, B and C). Although Uba3−/− mice were evident at embryonic day (E)3.5 with normal appearance, we noticed that ∼13% of total embryos had defects in blastocyst formation and hatching. These embryos were homozygous mutants as revealed by PCR genotyping. Thus, it was likely that half of the Uba3−/− embryos die before implantation (Table I). The phenotype may vary according to the amount of Uba3 maternally provided because weak Uba3 immunoreactivity was observed in every blastocyst examined (unpublished data). We next examined the histology of embryos between E5.5 and E7.5 (Fig. 2, A–H). In E6.0–6.5 embryos, a cylinder-like two-layered cellular structure was observed in normal embryos (Fig. 2, A–D). However, Uba3-deficient embryos, which could be identified at this stage by the absence of Uba3 immunoreactivity (Fig. 2 E), lacked such cylinder-like structure (Fig. 2, F and G). By E7.5, these disorganized embryos could not be found; rather, only completely absorbed embryonic tissues could be identified in the decidua (Fig. 2 H and Table I). We next characterized the nature of disorganized embryos at E6.0 by immunohistochemistry using several tissue-specific markers. Cytokeratin-endoA antibody (TROMA-1) stains the trophophectoderm cell lineage (Brulet et al., 1980). TROMA-1 stained the extraembryonic tissue of wild-type embryos but not embryonic ectoderm (Fig. 3, A and B). In Uba3-deficient embryos, TROMA-1 reacted to most cells at E6.0, suggesting that these cells were composed mostly of extraembryonic tissue (Fig. 3, C and D), and thus these embryos lacked embryonic ectoderm.

Bottom Line: Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells.However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle.This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-Ku, Tokyo 113-8613, Japan.

ABSTRACT
NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

Show MeSH
Related in: MedlinePlus