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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: 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.

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.

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In vitro culture of Uba3+ and Uba3−/− embryos. Blastocysts were flashed out from the uteri and cultured on gelatin-coated coverglass. Phase–contrast images were taken at indicated time of incubation (A, B, D, and E). Genotypes were determined subsequently by PCR. In contrast to Uba3+ blastocysts (A and B), Uba3−/−blastocysts displayed atrophic ICM (D and E; note the bar), and failed to expand after 72 h. TUNEL assay was examined in cultured Uba3+ (C) and Uba3−/−(F) blastocysts. Apoptotic cells (yellow or green) increased in the Uba3−/−ICM (F) but not in Uba3+ (C). Bars, 100 μm.
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fig5: In vitro culture of Uba3+ and Uba3−/− embryos. Blastocysts were flashed out from the uteri and cultured on gelatin-coated coverglass. Phase–contrast images were taken at indicated time of incubation (A, B, D, and E). Genotypes were determined subsequently by PCR. In contrast to Uba3+ blastocysts (A and B), Uba3−/−blastocysts displayed atrophic ICM (D and E; note the bar), and failed to expand after 72 h. TUNEL assay was examined in cultured Uba3+ (C) and Uba3−/−(F) blastocysts. Apoptotic cells (yellow or green) increased in the Uba3−/−ICM (F) but not in Uba3+ (C). Bars, 100 μm.

Mentions: To further analyze the cellular defects of Uba3 deficiency, we next transferred the blastocysts into in vitro cultures (Fig. 5, A–F). After hatching, the wild-type blastocysts grew on the gelatin-coated glass. Trophoblastic cells with large nuclei spread over the glass, whereas the inner cell mass (ICM), which forms the future embryonic ectoderm, increased in size at the center of the sheet of trophoblastic cells (Fig. 5, A and B). Whereas half of the Uba3−/− blastocysts could hatch and outgrow, neither could expand after 72 h to a size similar to that seen with Uba3+ cells (Fig. 5, D and E; note the bar). Specifically, Uba3−/− ICM (Fig. 5 F) underwent apoptosis and detached from the culture dish, consistent with the cell death noted in histological studies. In this regard, all efforts to obtain ES cell lines from homozygous embryos were unsuccessful.


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)

In vitro culture of Uba3+ and Uba3−/− embryos. Blastocysts were flashed out from the uteri and cultured on gelatin-coated coverglass. Phase–contrast images were taken at indicated time of incubation (A, B, D, and E). Genotypes were determined subsequently by PCR. In contrast to Uba3+ blastocysts (A and B), Uba3−/−blastocysts displayed atrophic ICM (D and E; note the bar), and failed to expand after 72 h. TUNEL assay was examined in cultured Uba3+ (C) and Uba3−/−(F) blastocysts. Apoptotic cells (yellow or green) increased in the Uba3−/−ICM (F) but not in Uba3+ (C). Bars, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: In vitro culture of Uba3+ and Uba3−/− embryos. Blastocysts were flashed out from the uteri and cultured on gelatin-coated coverglass. Phase–contrast images were taken at indicated time of incubation (A, B, D, and E). Genotypes were determined subsequently by PCR. In contrast to Uba3+ blastocysts (A and B), Uba3−/−blastocysts displayed atrophic ICM (D and E; note the bar), and failed to expand after 72 h. TUNEL assay was examined in cultured Uba3+ (C) and Uba3−/−(F) blastocysts. Apoptotic cells (yellow or green) increased in the Uba3−/−ICM (F) but not in Uba3+ (C). Bars, 100 μm.
Mentions: To further analyze the cellular defects of Uba3 deficiency, we next transferred the blastocysts into in vitro cultures (Fig. 5, A–F). After hatching, the wild-type blastocysts grew on the gelatin-coated glass. Trophoblastic cells with large nuclei spread over the glass, whereas the inner cell mass (ICM), which forms the future embryonic ectoderm, increased in size at the center of the sheet of trophoblastic cells (Fig. 5, A and B). Whereas half of the Uba3−/− blastocysts could hatch and outgrow, neither could expand after 72 h to a size similar to that seen with Uba3+ cells (Fig. 5, D and E; note the bar). Specifically, Uba3−/− ICM (Fig. 5 F) underwent apoptosis and detached from the culture dish, consistent with the cell death noted in histological studies. In this regard, all efforts to obtain ES cell lines from homozygous embryos were unsuccessful.

Bottom Line: 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.

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