Maternal Cdx2 is dispensable for mouse development

Abstract

In many invertebrate and vertebrate species, cell fates are assigned through the cellular inheritance of differentially localized maternal determinants. Whether mammalian embryogenesis is also regulated by deterministic mechanisms is highly controversial. The caudal domain transcription factor CDX2 has been reported to act as a maternal determinant regulating cell fate decisions in mouse development. However, this finding is contentious because of reports that maternal Cdx2 is not essential for development. Notably, all of the previously published studies of maternal Cdx2 relied on injected RNA interference constructs, which could introduce experimental variation. Only deletion of the maternal gene can unambiguously resolve its requirement in mouse development. Here, we genetically ablated maternal Cdx2 using a Cre/lox strategy, and we definitively establish that maternal Cdx2 is not essential for mouse development.

Fig. 1.

Relative quantification of Cdx2 levels by qPCR. (A) Average Cdx2 levels, normalized to β-actin (Actb), in E3.5 blastocysts, oocytes and ES cells. Averages were calculated from three biological replicate measurements: three wild-type (wt) blastocysts, three ES cell lines (R1, E14 and G4), and oocytes from three mice. (B) Levels of Cdx2, relative to Actb mRNA, in wild-type and Cdx2 M null oocytes (average of three biological replicates for each genotype). (C) Average levels of Cdx2, relative to Actb mRNA, in single wild-type blastocysts (n=4) and Cdx2 MZ null blastocysts (n=5) at E3.5. Error bars indicate s.d. of biological replicates.

Fig. 2.

Loss of maternal Cdx2 does not worsen the Cdx2 zygotic null phenotype. (A) Expression of KRT8 (red) and NANOG (green) in confocal transverse sections of preimplantation mouse blastocysts at E3.75 (nuclei, blue). Images are representative of n=20 control (Cdx2^+/− or wild type), n=6 Z null, n=5 MZ null blastocysts. In control blastocysts, KRT8 is restricted to the trophectoderm (TE) and NANOG is restricted to the inner cell mass (ICM). In Cdx2 Z null and MZ null blastocysts, KRT8 is still expressed in the TE and NANOG is ectopically expressed in the TE (arrows). (B) Expression of CDH1 (red) and NANOG (green) in implantation stage blastocysts at E4.25 (nuclei, blue). Control blastocysts are expanded and Cdx2 Z null and MZ null blastocysts are collapsed. CDH1 and NANOG are detectable in Cdx2 Z null and MZ null blastocysts and NANOG is ectopically expressed in the TE of both mutants (arrows). Representative of n=7 control, n=7 Z null, n=9 MZ null blastocysts. (C) Average numbers of inside, outside and total cells in control (Zp3-Cre/+; n=28), Cdx2 Z null (n=4) and Cdx2 MZ null (n=22) blastocysts at E3.5. Inside and outside cells were counted on the basis of morphological position in the blastocyst. (D) Data from C showing the average proportion of outside cells per embryo, indicating no difference in the proportion of TE cells for any genotype (P>0.05, t-tests). Error bars indicate s.d. Scale bars: 20 μm.