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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00898

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The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo

¹ Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH 45229, USA
² Department of Pathology, The University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA

View larger version (71K): [in a new window]   Fig. 1. Bax is expressed in migratory and post-migratory PGCs. Lane 1, E10.5 PGCs (gfp+); lane 2, E12.5 PGCs (gfp+); lane 3, E12.5 PGC cDNA RT-; lane 4, E12.5 whole embryo cDNA (+ control); lane 5, E11.5 PGCs (gfp+); lane 6, E11.5 somatic tissue from the genital ridge (gfp-); lane 7, E11.5 somatic tissue RT-. PGCs were prepared by flow cytometry and the purity of the sort was confirmed by RT-PCR for germ cell markers (Kit, Stag3) and somatic markers (Steel, cystatin 3 and Sparc) (data not shown).

View larger version (114K): [in a new window]   Fig. 2. The survival of PGCs in the midaxial region of the embryo in Bax-/- embryos at E10.5. (A,B) PGCs at 0 (A) and 700 (B) minutes in a slice from an E10.5 Bax+/+ embryo. a, aorta; m, mesonephric duct; mes, mesentery of the gut. PGCs in the mesentery and midaxial region near the aorta fragment and die during the culture period. (C-F) Slices from Bax-/- embryos, showing that after 700 minutes of culture, germ cells distant from the genital ridges do not fragment and die. C and E show slices at 0 minutes; D and F show the same slices at 700 minutes. Scale bar: 50 µm.

View larger version (111K): [in a new window]   Fig. 3. Positions of ectopic PGCs in frozen sections or whole isolated gonads at E13.5. (A) Cryostat section through a Bax+/+ embryo. SC, spinal cord; ML, midline mesenchyme; GM, gonad mesentery; G, gut. Almost all the germ cells are in the gonad, which can be seen as a GFP+ mass of cells at the end of the gonad mesentery. Occasional cells are found in the midline mesenchyme (arrow), and around the junction of the gonad and its mesentery (right hand side of whole gonad shown in the inset). (B,C) Whole isolated gonads from female (B) and male (C) Bax-/- embryos, showing ectopic PGCs at the hilus of the gonad, similar to the Bax+/+ embryos. In addition, Bax-/- embryos have groups of ectopic PGCs at other locations, including the gut mesentery (D), gonad mesentery and midline mesenchyme (E) and spinal cord (F). In each location, PGCs were also positive for alkaline phosphatase staining, shown for a group of cells in the spinal cord (G-I; enlargement of boxed area in F; green, GFP; red, alkaline phosphatase; yellow, merged images). Scale bars: 160 µm in A (inset 174 µm); 215 µm in B,C; 115 µm in D-F; 163 µm in E; 53 µm in G-I.

View larger version (76K): [in a new window]   Fig. 4. Whole gonads (G), together with their mesenteries (GM) dissected from E14.5 embryos. In the Bax+/+ embryos (A), no ectopic germ cells were found in the gonad mesenteries. Alkaline phosphatase staining was seen in blood vessels in the gonad mesentery (arrow), but not in the PGCs. In Bax-/- embryos (B-F), large numbers of ectopic PGCs were found in the gonad mesenteries. These retain alkaline phosphatase staining (C, overlaid in D) and SSEA1 staining (E,F; green, GFP; red, anti-SSEA1 stain). In D, the arrow indicates alkaline phosphatase staining in blood vessels. In E and F, the plane of section is through the hilus of the gonad. The body of the gonad is out of focus (arrow in E). Scale bars: 145 µm in A; 163 µm in B-F.

View larger version (28K): [in a new window]   Fig. 5. Forty-eight hour survival of PGCs isolated from E10.5 (A,B), and E12.5 (C) embryos, and cultured on MEFs. The data are shown as 48 hour survival of PGCs from each embryo of one litter (left hand side) and the means (±s.e.m.) for embryos of the same genotypes in the litter (right hand panels). There is a significant increase in 48 hour survival of PGCs that are Bax-/- (KO), compared with Bax+/+ (WT) and Bax+/- (HET) PGCs at 48 hours. Survival is measured as the % of germ cells counted in each sample at T0. See text for details.

View larger version (30K): [in a new window]   Fig. 6. Shows the effect of an anti-Kit antibody or an isotype matched control antibody (J1.2) on 48 hour survival of either E10.5 (A) or E12.5 (B) germ cells. Survival is shown first as % of the number of PGCs counted in each embryo sample from a single litter at T0, and second, as means (±s.e.m.) for all embryos of the same genotype in the litter. In the E10.5 sample (A), 15 µg/ml of control (blue bars) and anti-Kit (red bars) were used. In the E12.5 sample (B), two concentrations of antibody were used; 15 µg/ml and 50 µg/ml. The effects of each dose are shown separately on the charts. Antibody concentrations of 15 µg/ml are represented by blue (control antibody) and red (anti-Kit antibody) bars (as in A), while the effects of 50 µg/ml of control or anti-Kit are shown as yellow and green bars, respectively. The anti-Kit antibody causes a significant reduction in germ cell survival at 48 hours in both E10.5 and E12.5 Bax+/+ and Bax+/- embryos, but not in Bax-/- embryos. In the E12.5 litter, there was only one Bax+/+ embryo, so there is no s.e.m.

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