The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis - PubMed (original) (raw)

Andrew Castillo et al. Biochem Biophys Res Commun. 2007.

Abstract

Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.

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Figures

Figure 1

Figure 1. Generation and detection of Eg5 genetrapped KO allele

Genetrapped ES cells with a genetrapping vector integrated in intronic sequence between exons 18 and 19 of the endogenous Eg5 locus (A). The genetrap vector consists of a portion of the engrailed 2 intronic sequence containing splice acceptor sequence, a Lac-Z/neomycin (βGeo) cassette, and a poly-adenylated tail. (B) Transcription of the genetrapped allele results in a truncated LacZ tagged fusion protein. Genotyping of genetrapped mice was performed by Southern and PCR analysis. (C) Integration of the genetrap vector in intron 18 introduces an alternate _Sst_I restriction site detectable by southern analysis using a probe to exon 17. (D) PCR analysis detected both wildtype (225 bp) and genetrapped (327 bp) alleles in founder mice and offspring. (E) RT-PCR was used to detect both endogenous Eg5 and mutant Eg5-LacZ fusion transcripts.

Figure 2

Figure 2. Loss of Eg5 results in early embryonic lethality due to a proliferation defect

(A) Genotyping of E3.5 embryos revealed that _Eg5_−/− embryo numbers were reduced and appear to arrest at the 8 cell stage, most of which fail to undergo compaction. No _Eg5_−/− blastocyst stage embryos were detected at this stage of development. At E2.5 _Eg5_−/− embryos approached near Mendelian ratios and these embryos were observed to reach the 4–8 cell stage. The presence of homozygous embryos at E2.5 and reduced numbers at E3.5, in addition to failure to undergo compaction and progression to the blastocyst stage suggest a proliferation defect at the 4–8 cell stage resulting in the embryonic lethality associated with the loss of Eg5. (B) Cells of the inner cell mass stain positive for β-galactosidase in heterozygous embryos, as observed by LacZ staining of cultured blastocysts.

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