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First published online 12 October 2004
doi: 10.1242/jcs.01476

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Suppression of Nek2A in mouse early embryos confirms its requirement for chromosome segregation

School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

View larger version (46K): [in a new window]   Fig. 4. Specific reduction of the Nek2 isoform expression with RNAi in mouse early embryos. (A) Nek2 dsRNAs corresponding to parts of the kinase domain (Fragment 1, 501 bp) and the regulatory domain (Fragment 2, 501 bp) that are common to both Nek2A and Nek2B were constructed. In addition, the 3' untranslated sequence specific for Nek2A (Fragment 3, 412bp) and a part of C-terminal coding sequence and 3' untranslated sequence for Nek2B (Fragment 4, 480bp) were also prepared. (B) Double-stranded RNA for a common region of Nek2 (Fragment 2) or specific to Nek2A (Fragment 3) or Nek2B (Fragment 4) was injected into zygote mouse embryos. After culture for 3 days, the embryos were subjected to RT-PCR analysis for detection of Nek2, Nek2A and Nek2B mRNA levels. GAPDH mRNA was detected as a control for RT-PCR. (C) Double-stranded RNA specific to a common region of Nek2 (Fragment 2), to Nek2A (Fragment 3) or Nek2B (Fragment 4) was injected into mouse zygote embryos. The GFP dsRNA was also injected as a non-specific control. After culture for 3 days, the embryos were subjected to immunoblot analyses for detection of the Nek2 proteins. The testis lysate was used as a control and ß-tubulin was detected as a loading control. (D) One-cell embryos injected with dsRNA specific to GFP (d-f), E-Cadherin (g-i) or Nek2 (j-l) were cultured for 72 hours and immunostained with antibodies specific to E-Cadherin and Nek2. Nuclei were stained with DAPI and mitotic nuclei were marked with arrowheads. Uninjected embryos were used as a control (a-c). The E-Cadherin antibody stained the cytoplasmic membrane of the embryonic cells, whereas the Nek2 antibody stained nuclei of the mitotic cells as marked with arrowheads.

View larger version (28K): [in a new window]   Fig. 1. Nek2 expression in mouse early embryos. (A) RT-PCR analysis. Total RNA isolated from mouse testis, oocytes and embryos at indicated stages was reverse-transcribed followed by PCR amplification with primers specific to a common region of Nek2A and Nek2B (Nek2), to Nek2A or to Nek2B. GAPDH was detected as a control. (B) Detection of the Nek2A and Nek2B proteins. Immunoblot analysis was carried out with lysates from the mouse testis, the human 293T cell line and 293T cells transfected with pNek2RHA1, which encodes the HA-tagged Nek2A protein. Mouse Nek2A protein routinely appeared smaller than the human Nek2A protein. (C) Immunoblot analysis of Nek2 in mouse early embryos. Protein samples were prepared from the mouse testis, oocytes and embryos at indicated developmental stages and were subjected to immunoblot analysis with a polyclonal antibody specific to Nek2. ß-Tubulin was detected as a control.

View larger version (59K): [in a new window]   Fig. 2. Expression and localization of the Nek2 proteins during the first mitosis of the mouse embryo. (A) Mitotic stages of mouse embryos that underwent the first mitosis were determined based on their morphology under a dissecting microscope. I, interphase; P, prophase; M, metaphase; A, anaphase; T, telophase. (B) Mouse embryos were collected based on their mitotic stages and were subjected to immunoblot analysis with the Nek2 antibody. The Nek2A- and Nek2B-specific bands are indicated on the right side of the figure. ß-Tubulin was used as a loading control. (C) A zygote at metaphase was co-immunostained with antibodies specific to Nek2 and -tubulin. DNA was stained with DAPI. (D) Immunostaining of the Nek2 protein during the first mitosis of the mouse embryos. Zygotes that were about to undergo the first mitotic division were immunostained with the Nek2 antibody. DNA was stained with DAPI. Arrowheads indicate spindle poles and arrow indicates the mid body.

View larger version (76K): [in a new window]   Fig. 3. Suppression of Nek2 expression with RNAi in mouse early embryos. (A) Specific suppression of Nek2 and E-Cadherin mRNA with RNAi. Double-stranded RNAs specific to Nek2 or E-Cadherin were injected into zygote embryos. The GFP dsRNA was also injected as a non-specific control. After culture for the indicated times, the embryos were subjected to RT-PCR for detection of levels of Nek2 and E-Cadherin mRNA. GAPDH mRNA was detected as a control for RT-PCR. (B) Suppression of Nek2 and E-Cadherin expression with RNAi. Competitive PCR was used to determine suppression levels. A fixed amount of the insertional mutant DNA fragments (Mt) specific to Nek2 or E-Cadherin was added to the PCR reaction mixture along with a variable amount of the reverse-transcribed cDNA (Wt). GAPDH mRNA was detected as a control for the RT-PCR reaction.

View larger version (32K): [in a new window]   Fig. 5. Effects of dsRNA specific to Nek2 and E-Cadherin on mouse early embryo development in culture. (A) One-cell embryos injected with dsRNA specific to GFP, E-Cadherin or Nek2 were cultured for 4 days and the number of embryos reaching the blastocyst stage was counted. Uninjected and vehicle-injected groups were included as controls. Experiments were repeated five times. Data are presented as mean±s.e.m. The number of embryos used for each experimental group is indicated within the bar. (B) Four kinds of Nek2 dsRNAs (see Fig. 4A) were injected into mouse one-cell embryos. Four days later, the number of embryos reaching the blastocyst stage was counted. GFP dsRNA was used as a control. Experiments were repeated five times and data are presented as mean±s.e.m. The number of embryos used for each experimental group is indicated within the bar.

View larger version (108K): [in a new window]   Fig. 6. Morphological phenotypes of the Nek2-suppressed embryos. One-cell embryos that had been injected with dsRNA specific to GFP, E-Cadherin or Nek2 were cultured for up to 4 days, and embryos with typical morphologies were photographed at the indicated developmental stages.

View larger version (39K): [in a new window]   Fig. 7. Mitotic abnormalities in the Nek2-suppressed mouse embryos. (A) Control and Nek2-suppressed embryos were immunostained with antibodies specific to ß-tubulin and -tubulin. Nuclei were stained with DAPI. Arrowheads indicate typical MTOCs observed in the control embryo. (B) The number of mitotic blastomeres was counted in uninjected embryos and in embryos injected with dsRNA specific to GFP, Nek2A or Nek2B and the proportions calculated. The experiments were repeated five times and data are presented as mean±s.e.m. The number of embryos used for each experimental group is indicated within the bar. (C) Nuclei of the Nek2-suppressed embryos (b,c) were stained with DAPI and compared with those of the uninjected control embryos (a). In the Nek2-suppressed embryos, abnormal segregations were observed such as dumbbell-like nuclei (arrows) and micronuclei (arrowheads). Nuclei of the polar body are indicated with open arrowheads.