Mcl-1 deficiency results in peri-implantation embryonic lethality

Abstract

We disrupted the Mcl-1 locus in murine ES cells to determine the developmental roles of this Bcl-2 family member. Deletion of Mcl-1 resulted in peri-implantation embryonic lethality. Mcl-1(-/-) embryos do not implant in utero, but could be recovered at E3.5-4.0. Null blastocysts failed to hatch or attach in vitro, indicating a trophectoderm defect, although the inner cell mass could grow in culture. Of note, Mcl-1(-/-) blastocysts showed no evidence of increased apoptosis, but exhibited a delay in maturation beyond the precompaction stage. This model indicates that Mcl-1 is essential for preimplantation development and implantation, and suggests that it has a function beyond regulating apoptosis.

Figure 1

(a) Map of the Mcl-1 murine genomic locus and targeting vector. Exons are indicated by black boxes, and positions of internal and external probes are represented by gray boxes; PCR primers are denoted by arrows. The pgk–neo cassette is inserted in the opposite transcriptional orientation and replaces a SacII (S2) to SacI (S1) fragment of exon I containing the translation start site. EcoRI (R1), ScaI (Sc1). (b) Representative genomic blot probed with the external probe. A wild-type 12-kb EcoRI fragment is recognized; the rearranged homologous recombinant allele is 10-kb. (c) Representative nested PCR genotyping results from preimplantation embryos. The paired primers distinguished a 350-bp rearranged fragment of the homologous recombinant allele from a 168-bp fragment of the wild-type allele.

Figure 2

Representative Mcl-1 RNA in situ hybridization results on preimplantation embryos. (a) Light- and dark-field images of a morula stage wild-type embryo hybridized with the Mcl-1 anti-sense probe. (b) Light- and dark-field images of a blastocyst stage wild-type embryo with the Mcl-1 antisense probe revealing signal in both TE and ICM. (c) The Mcl-1 sense probe hybridized to wild-type blastocyst displayed no signal above background. (d) Light- and dark-field image of an Mcl-1 null blastocyst hybridized with the Mcl-1 antisense probe. (e) Representative noncompacted blastomere stage Mcl-1 null embryo that fails to hybridize with the Mcl-1 antisense probe. Magnification, 400×.

Figure 3

Mcl-1 RNA in situ hybridization of peri-implantation wild-type embryos. (a) Light- and dark-field images of E5.0 implanting blastocyst hybridized with the Mcl-1 antisense probe. (b) Light- and dark-field images of E5.5 implantation site. (c) Light- and dark-field images of E6.5 implantation site. Arrow points to ICM of implanting embryo. Maternal decidua is marked with an asterisk in b and c. Magnification, 100×.

Figure 4

Light microscope images of inner cell mass outgrowths 5 days after immunosurgery and culture on fibronectin. Outgrowths were genotyped as Mcl-1^+/+ (a), Mcl-1^+/− (b), and Mcl-1^−/− (c). Magnification, 200×.