Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo

Abstract

Critical to our understanding of the developmental potential of stem cells and subsequent control of their differentiation in vitro and in vivo is a thorough understanding of the genes that control stem cell fate. Here, we report that Foxd3, a member of the forkhead family of transcriptional regulators, is required for maintenance of embryonic cells of the early mouse embryo. Foxd3-/- embryos die after implantation at approximately 6.5 days postcoitum with a loss of epiblast cells, expansion of proximal extraembryonic tissues, and a distal, mislocalized anterior organizing center. Moreover, it has not been possible to establish Foxd3-/- ES cell lines or to generate Foxd3-/- teratocarcinomas. Chimera analysis reveals that Foxd3 function is required in the epiblast and that Foxd3-/- embryos can be rescued by a small number of wild-type cells. Foxd3-/- mutant blastocysts appear morphologically normal and express Oct4, Sox2, and Fgf4, but when placed in vitro the inner cell mass initially proliferates and then fails to expand even when Fgf4 is added. These results establish Foxd3 as a factor required for the maintenance of progenitor cells in the mammalian embryo.

Figure 1

Embryonic expression of Foxd3 and generation of Foxd3 mutant allele. (a) RT-PCR analysis of staged mouse embryos shows that Foxd3 is not expressed in unfertilized oocytes or one-cell embryos but can be detected in the blastocyst at 3.5 dpc. Foxd3 mRNA is also detected at 6.5 dpc in both the extraembryonic and the embryonic portions of the embryo. Expression is undetectable in a 6.5-dpc Foxd3−/− embryo. Negative controls with no reverse transcriptase added show no amplification of the Foxd3 message. RT-PCR for the hypoxanthine phosphoribosyl transferase gene (Hprt) was used to monitor the integrity of the cDNA produced by reverse transcription. (b) Whole mount in situ hybridization of a 6.5-dpc embryo shows that Foxd3 transcripts are present in the layer of epiblast cells, but not in the outer layer of endoderm (arrow). (c) The Foxd3 null mutation was generated using a targeting vector shown here. PCR primers for genotyping are shown as red boxes, and the external 3′ probe used to screen ES cells is shown in green. The loxP sites flanking the neo cassette are yellow triangles. (d) Southern analysis of ES cells was performed by digesting genomic DNAs with BamH1 as diagrammed in c. (e) PCR was used to genotype Foxd3 mice and embryos.

Figure 2

Gastrulation of Foxd3−/− embryos is disrupted. (a,b) Morphology of embryos dissected at 6.5 dpc shows that Foxd3−/− embryos have an abnormal distal epiblast region and no evidence of a primitive streak. (c) A sagittal section of a normal 6.5-dpc embryo from a Foxd3+/− intercross. Note the clear distinction between the extraembryonic (ex) and embryonic (em) regions. The proamnionic cavity (pac) extends the length of the embryonic region, while the exocoelomic cavity (xc) is restricted to the extraembryonic region. (d) A sagittal section of an abnormal 6.5-dpc embryo from the same cross. Remnants of a proamniotic cavity are visible, and visceral endoderm (ve), extraembryonic ectoderm (ee), and embryonic ectoderm (e) can be identified. (e,f) The normal and abnormal visceral endoderm in c and d are shown at higher magnification. The region of the section is indicated in c and d with yellow arrows. (g,h) Phosphohistone H3 is detected at 6.5 dpc, and although several epiblast cells of the normal embryo are strongly stained, no internal distal cells of a Foxd3−/− embryo react with the antibody for phosphohistone H3. Positive cells are indicated with red arrows. (i,j) TUNEL staining to detect cells undergoing apoptosis shows no appreciable cell death at 6.5 dpc in a normal embryo, with some cell death in a Foxd3−/− embryo. By 7.5 dpc (k,l), there is very little embryonic tissue remaining in the Foxd3−/− embryo. e, embryonic ectoderm; ec, ectoplacental cone; ee, extraembryonic ectoderm; em, embryonic region; ex, extraembryonic region; pac, proamnionic cavity; ps, primitive streak; ve, visceral endoderm; xc, exocoelomic cavity.

Figure 3

Molecular marker analysis of Foxd3 mutant embryos. Whole mount in situ analysis of normal littermates is shown on the left side of each set of embryos. All embryos are 6.5 dpc except where noted. Anterior (when discernible) is to the left. (a) Brachyury (or T) is expressed in the proximal epiblast of early 6.5-dpc embryos and in all cells of the primitive streak at later stages. T expression is never detected in Foxd3−/− embryos. (b) Wnt3, also normally expressed in the proximal epiblast and later in the primitive streak, is not expressed in mutants. (c–e) Mm1, Eomes, and Fgf8, markers of mesodermal cells of the primitive streak, are not expressed in Foxd3−/− embryos. (f,g) Extraembryonic mesodermal markers Bmp4 and Mesp1 are not detected in mutant embryos. (h) Pou5f1/Oct4 expression is detected throughout the epiblast in a normal embryo at 5.5 dpc, but in Foxd3−/− embryos, expression is missing at 5.5 dpc. At 6.5 dpc, Pou5f1/Oct4 is expressed robustly in normal embryos (middle embryo) but is either missing or only weakly expressed in scattered cells (the two embryos on the right). (i) Otx2 expression is normally detected throughout the epiblast of the early streak-stage embryo, but is missing in mutant littermates (first pair of embryos). Otx2 expression in an early headfold-stage embryo (∼7 dpc) is concentrated in the anterior half of the embryo (third embryo). A small amount of Otx2 expression is detected in the distal tip of the mutant littermate (far right embryo). (j) Nodal-lacZ expression is detected in the proximal epiblast of the early streak-stage embryo (left embryo). However, Nodal-lacZ expression was not detected in most Foxd3−/− embryos (far right embryo). An exception is shown here with a low level of β-galactosidase activity restricted to the distal tip (arrow, middle embryo). (k) The Nodal cofactor Cripto is not expressed in mutant embryos. (l,m) Extraembryonic visceral endoderm, as indicated by Pem and Amn, is distally expanded in the mutants. (n–p) Extraembryonic ectoderm is also distally expanded as shown by expression of Bmp8b, Fgfr2, and Errβ. (q,r) The AVE signaling center, indicated by the arrows, is located distally in Foxd3−/− embryos as shown by the expression of Lim1 and Hex. The posterior Lim1 expression in q corresponds to the mesodermal expression domain of this gene. (s) Schematic illustrating the morphological changes in Foxd3−/− embryos.

Figure 4

Expression of Oct4, Sox2, and Fgf4 is maintained in Foxd3−/− blastocysts. (a) RT-PCR from single blastocysts shows that Oct4, Sox2, and Fgf4 mRNAs are expressed in Foxd3−/− blastocysts. Lanes 2 and 8 are from Foxd3−/− embryos, and all other lanes are from either Foxd3+/+ or +/− blastocysts. (b) Whole mount in situ hybridization for Oct4 mRNA demonstrated that blastocysts from intercrosses (18 of 18 total) have Oct4 mRNA localized to the ICM. (c) Immunofluorescent detection of Oct4 protein in blastocysts from an intercross show that all blastocysts (23 of 23 total) have robust nuclear staining of Oct4 protein.

Figure 5

Developmental and proliferative potential of Foxd3−/− embryos. (a,b) Two examples of chimeras obtained by injecting Rosa26.1 ES cells into blastocysts from Foxd3+/− intercrosses. Extraembryonic endoderm was used to genotype the recipient blastocysts, and chimeras were stained with Xgal to detect the wild-type Rosa26.1 cells. Wild-type ES cells rescued the mutant phenotype completely, indicating that Foxd3 function is required in the epiblast and that a low contribution of wild-type cells is sufficient to rescue the epiblast defect. (c,d) Histological analysis of chimera shown in panel a shows that Foxd3−/− cells (not stained blue) can differentiate into notochord, somites, and neural tube. (e,f) Teratocarcinomas derived from Foxd3+/− (e) and −/− (f) embryos. The portion of the embryo transferred is diagrammed. The heterozygous tumor extends far beyond the field shown and contains tissues derived from all three germ layers, including fat (f), skeletal muscle (m), neural tissue, and cartilage (data not shown). The Foxd3−/− embryonic cells do not produce any differentiated cell types and do not proliferate. The entire extent of the Foxd3−/− transplanted embryonic tissue is outlined with a white dashed line, and panels e and f are shown at the same magnification. Coagulated blood (orange) and calcium deposits (blue) are results of the surgery. f, fat; m, muscle; n, notochord; nt, neural tube; s, somite.

Figure 6

Developmental potential of the Foxd3−/− ICM in vitro. (a) Blastocysts were harvested from Foxd3+/− intercrosses and placed on gelatin-coated dishes in ES cell medium. Blastocysts attached and formed robust outgrowths within the first 3 d of culture. After prolonged culture (b), the Foxd3−/− cultures failed to maintain an ICM. Arrows in a and b indicate trophoblast giant cells. (c) TUNEL staining demonstrates that in Foxd3−/− outgrowths, attached cells were undergoing programmed cell death by day 6 in culture. Arrows in c point to TUNEL-positive cells.