Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice

Abstract

Background: In mice, germ cells are specified through signalling between layers of cells comprising the primitive embryo. The function of Dppa3 (also known as Pgc7 or stella), a gene expressed in primordial germ cells at the time of their emergence in gastrulating embryos, is unknown, but a recent study has claimed that it plays a central role in germ cell specification.

Results: To test Dppa3's role in germ cell development, we disrupted the gene in mouse embryonic stem cells and generated mutant animals. We were able to obtain viable and fertile Dppa3-deficient animals of both sexes. Examination of embryonic and adult germ cells and gonads in Dppa3-deficient animals did not reveal any defects. However, most embryos derived from Dppa3-deficient oocytes failed to develop normally beyond the four-cell stage.

Conclusion: We found that Dppa3 is an important maternal factor in the cleavage stages of mouse embryogenesis. However, it is not required for germ cell specification.

Figure 1

Generation of Dppa3-deficient animals. A, Schematic representation of genomic ablation of Dppa3. The gene's four exons are shown; non-coding regions of the first and last exons are shaded gray. The hygromycin-thymidine kinase (Hygro-TK) cassette replaces the entire open reading frame of the gene. Cre-mediated excision of the selection cassette leaves only the non-coding portions of the gene, together with a single loxP site (white triangle). Also shown are the locations of genotyping primers p1, p2 and p3 in wild-type and mutated Dppa3 alleles. B, PCR genotyping of the offspring of an intercross between Dppa3^tm1WHT /+ animals. Inferred genotypes are shown above the gel image. The wild type allele yields a PCR product of 304 bp with primers p1 and p2. The mutant allele (Dppa3^tm1WHT) yields a PCR product of 492 bp with primers p1 and p3. M, DNA molecular weight marker.

Figure 2

Normal germ cell development in the absence of Dppa3. A, Gonads from E12.5 embryos (above: wild type; below: Dppa3^tm1WHT/Dppa3^tm1WHT) stained for alkaline phosphatase to reveal primordial germ cells. B, RT-PCR analysis of gene expression in wild-type and Dppa3^tm1WHT/Dppa3^tm1WHT adult ovaries. C,D, Dppa3^tm1WHT/Dppa3^tm1WHT testis (C) and ovary (D) are histologically normal.

Figure 3

Abnormal pre-implantation development of embryos derived from Dppa3–deficient oocytes. A,C, Cultured 2-cell (A) and 4-cell (C) control embryos derived from wild-type matings. B,D, Cultured 2-cell (B) and 4-cell (D) embryos produced by crossing Dppa3^tm1WHT/Dppa3^tm1WHT females with wild-type males. E,F, E3.5 control embryos derived from wild-type matings have progressed to the blastocyst stage (E). By contrast, most E3.5 embryos produced by crossing Dppa3^tm1WHT/Dppa3^tm1WHT females with wild-type males have not progressed to the blastocyst stage and instead cleave abnormally and degenerate (F). G,H, Many embryos produced by crossing Dppa3^tm1WHT/Dppa3^tm1WHT females with Dppa3 +/+, Tg^{(Pou5f1 ΔPE-GFP)10WHT}/Tg^{(Pou5f1 ΔPE-GFP)10WHT} males fail to develop normally beyond the 4-cell stage (G) but nonetheless express the Oct4-GFP marker (H).