BMP signaling and expression of BMP type I receptors in early postimplantation mouse embryos. (A–C) Activated (C-terminal phosphorylated) BMP receptor-regulated Smads were detected by immunohistochemistry in the VE and proximal epiblast (arrow) of E5.5 (A), E6.0 (B), and E6.25 (C) embryos. (D,E) Whole mount (D) and section (E) of E6.5 BRE:LacZ transgenic embryos. (EE) Epiblast; (epc) ectoplacental cone; (ExE) extraembryonic ectoderm; (VE) visceral endoderm. Bar, 50 μm. (F) Percentage of embryos (% embryos) expressing mRNA for the different BMP type I receptors, ALK2, ALK3, and ALK6 in isolated VE at E5.5 (n = 7), E6.0 (n = 7), and E6.5 (n = 5) using RT–PCR. The primer combinations used had similar sensitivities (data not shown). GAPDH mRNA was detected in all samples, confirming that RNA isolation and reverse transcription were successful (data not shown).

STO cells support the formation of PGCs in Bmp4^-/- mouse embryos. (A) Genes expressed in E5.5 explants cultured whole or without VE (-VE) either on STO cells or FN were studied by RT–PCR independently in three to six explants per group. Oct4 mRNA was present in explants where PGCs were observed, but it was also detected in half of the E5.5-VE-derived explants analyzed cultured on FN (three out of six). PCG7/Stella mRNA was not detected in explants derived from E5.5-VE explants cultured on FN. Esx1 mRNA was detected in samples (40%–100%) of all explants groups analyzed. α-Fetoprotein (AFP) and hepatocyte nuclear factor 4α (Hnf4α) mRNA was only present in explants derived from intact embryos (containing VE). ALK2 and ALK3 mRNA was detected in all samples analyzed, including STO cells. (B) Expression of BMP4, BMP8b, BMP2, and Ifitm3/Fragilis/mil-1 mRNA in STO cells determined by RT–PCR. cDNA from an E8.0 embryo was used as positive control. Contamination by genomic DNA did not occur as indicated by amplification of each sample without reverse transcription (-RT). (L) DNA ladder. (C) The fraction (n) represents the number of PGC-containing explants derived from E5.5, E6.0, and E6.5 Bmp4^-/- mouse half embryos (cut longitudinally) over the total number examined, cultured on STO cells in the presence (black bars) or absence (gray bars) of exogenous BMP4. The number of PGCs is presented as a range for each type of explant.

Expression of caALK2 in Bmp4^-/- embryos rescues the PGC defects. (A) The efficiency of adenoviral infection was studied in intact or VE-stripped E5.5 and E6.5 embryos cultured overnight on FN using adenovirus containing a LacZ expression construct (LacZ-virus). X-gal staining was detected in intact embryos exclusively in the VE and in the majority of the cells of VE-stripped derived explants. Bar, 300 μm. (B) Western blot analysis of the C-phosphorylated form of BMP-responsive Smads (PS1/5/8) in HepG2 cells (-); HepG2 cells treated for 1 h with exogenous BMP4; infected with two concentrations of adenovirus containing a caAlk2 expression construct (caAlk2-virus); and two different concentrations of LacZ-virus. (C) Analysis of X-gal staining of BRE:lacZ heterozygous E6.5 embryos cultured overnight on FN in the presence or absence of caAlk2-virus. (D) The fraction (n) represents the number of PGC-containing explants derived from intact E5.5–E6.0 and E6.5 embryos (from crossings of Bmp4 heterozygous mice) infected with caAlk2-virus over the total number examined cultured on FN. The number of PGCs is presented as a range for each type of explant. (E) Number of PGCs detected in explants derived from intact or VE-stripped E5.5–E6.0 and E6.5 embryos (n = 12–18) infected with caAlk2-virus and cultured on FN. The Wilcoxon rank test was used for statistical analysis [(^**) P ≤ 0.01], and medians are depicted by a red stripe.

Formation of PGCs after culture of E5.5–E6.5 embryos either on STO cells or fibronectin. (A–C) Most AP-positive cells identified as PGCs (A), coexpressed Oct4 (B), and PGC7/Stella (C) as demonstrated for Oct4ΔPE:gfp heterozygous E6.0 embryos cultured without VE on STO cells. PGCs are indicated by arrowheads. Bar, 40 μm. (D) Numbers of PGCs detected in explants derived from intact or VE-stripped E5.5–E6.5 embryos (n = 15–19) cultured on STO cells. (E) Numbers of PGCs detected in explants derived from intact or VE-stripped E5.5–E6.5 embryos (n = 15–20) cultured on FN. The Wilcoxon rank test was used for statistical analysis [(^**) P ≤ 0.01; (^***) P ≤ 0.001], and medians are depicted by a red stripe. (F–I) Explants derived from VE-stripped E5.5 and E6.5 embryos cultured on FN and STO cells after AP staining. Arrowheads indicate PGCs. Bar, 150 μm.

ALK2 is necessary for PGC formation. E7.5–E8.0 embryos resulting from crossings of Alk2 heterozygous mice were isolated and genotyped, and after AP staining, the number of PGCs present in the base of the allantois was analyzed. Alk2 heterozygous embryos contained a reduced number of PGCs (black arrow), when compared with wild-type littermates (A,B), and Alk2 homozygous embryos contained no PGCs (C). Note that as the result of squashing the embryos to allow an accurate counting of PGCs, the Alk2 mutant depicted in C was inevitably ruptured, but the extent of somatic development was as described (Gu et al. 1999). (D) Number of PGCs detected in embryos of the three Alk2 genotypes. The Wilcoxon rank test was used for statistical analysis [(^***) P ≤ 0.001; (^*) P = 0.06], and medians are depicted by a red stripe. Bar, 100 μm.

BMP signaling and the formation of PGCs. At E5.5–E6.0, BMP4 secreted by the ExE binds to BMP receptor complexes consisting of BMP receptor type II and ALK2 in the VE, which results in Smad1 phosphorylation and transcriptional regulation of BMP target genes. Among these target genes, an unknown factor (or factors) directs proximal epiblast cells toward a PGC (and possibly allantoic) fate. BMP2 produced by the VE and BMP8b produced by the ExE possibly signal together with BMP4 in the VE. However, we cannot exclude that all three BMPs act directly on the proximal epiblast via ALK2 (red) or possibly ALK3 (green) and Smad1/5.