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First published online 15 November 2006
doi: 10.1242/dev.02688

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Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration

¹ Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH 45229, USA.
² Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
³ Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USA.

View larger version (6K): [in this window] [in a new window]   Fig. 1. Models of E10.5 midline germ cell death. (A) Midline expression of cell death factors (red arrows) and/or the activation of cell death factor receptors of midline germ cells may activate the extrinsic apoptotic pathway in PGCs (green). (B) Loss of survival signaling in the midline at E10.5 due to restricted expression of a survival factor (yellow arrowheads) or inactivation of a survival factor receptor in midline germ cells may activate the intrinsic apoptotic pathway.

View larger version (92K): [in this window] [in a new window]   Fig. 2. Spatial distribution of apoptosis among migratory germ cells. (A,B) Cleaved-PARP immunostaining (red) labels apoptotic cells, including PGCs (white arrows) and the mesonephroi (arrowheads), which are known to degenerate during this time period (Pole et al., 2002). (A) A transverse section showing the direction of imaging (blue arrows) of whole-mount embryos in B. (B) Apoptotic PGCs (yellow) occurred in greater numbers in the midline (area between white lines). (C) Quantification of PGC apoptosis in B, showing enhanced death amongst midline PGCs (n=8). Scale bars: 100 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Germ cell expression of apoptosis-related genes. (A) Real-time RT-PCR shows that pro-apoptotic genes, including Bcl2 superfamily members Bax, Bak, Bad and Bim, were downregulated in postmigratory PGCs. (B) Genes with gender-specific differential regulation include Fas and Bcl-x, which increased in postmigratory germ cells in the male, and Casp6, which was upregulated in the female from E13.5-14.5. E10.5 was considered baseline for statistical comparisons, except for Bcl-x, for which E11.5 was considered baseline. F, female; M, male. **P<0.001.

View larger version (109K): [in this window] [in a new window]   Fig. 4. Expression of c-Kit and Steel Factor. (A) Anti-c-Kit staining of E10.5 embryo slices shows that both midline (arrowheads) and lateral germ cells express c-Kit protein (observe colocalization in overlay). (B) Dissected midlines (M) have less than two-thirds steel expression compared with bilateral (L) regions as determined by real-time RT-PCR. (C) Isotype-matched primary antibody serves as a negative control for c-Kit and Steel antibodies. (D) Immunostaining of a Steel-/- embryo with an anti-Steel antibody demonstrates the antibody's specificity (note the lack of red signal). (E) A Steel wild-type embryo at E9.5 has Steel protein in the genital ridges and midline structures, including the coelomic angles and tissues ventral to the dorsal aorta. (F) At E10.75, Steel protein becomes restricted bilaterally to the genital ridges. Ao, aorta. Scale bars: 100 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Slice culture experiments. (A,C) Germ cells in E10.5 embryo slices survive in the midline in the presence of Steel factor in a dose-dependent manner. Midline PGCs scored as ectopic in (C) are marked with arrows in A. No significant change in total PGC numbers was observed in the different treatment groups in A. (B,D) Conversely, PGCs cultured with Ack2 died in a dose-dependent manner. Error bars represent means ± s.e.m. (n=8-12).

View larger version (11K): [in this window] [in a new window]   Fig. 6. Mapping the Steel (KitlSl) deletion. (A) The exact positions of the 5'- and 3'-breakpoints of KitlSl are indicated as boxed nucleotides, 1 (position 99,177,807) and 2 (position 100,151,173). The total deletion is 973,366 bp, not including an insert of four nucleotides. Nucleotide positions correspond to NCBI Map Viewer numbers. (B) A genotyping strategy was designed to identify wild-type and deleted alleles of Steel. (Top) The blue line represents DNA containing the Steel gene, and the dotted region represents the portion that is missing in the deleted allele. The red arrows represent primers (see Materials and methods). (Bottom) A gel showing a genotyping reaction using a mix of all three primers. Heterozygotes are identifiable by bands generated from both 1+2 and 1+3.

View larger version (59K): [in this window] [in a new window]   Fig. 7. Bax is required for E9.0 germ cell death in the absence of Steel. (A-D) Immunostaining of Bax/Steel E9.0 embryos for cleaved PARP shows that apoptosis occurs at a higher frequency in Steel-/- embryos, and is reduced by the absence of Bax. (A) Bax+/- Steel+/-, (B) Bax-/- Steel+/-, (C) Bax+/- Steel-/-, (D) Bax-/- Steel-/-. Scale bars: 100 µm. (E) Germ cell numbers were significantly reduced in Bax+/* Steel-/- embryos compared with Bax+/* Steel+/* littermates (* denotes + or -), but are rescued by the loss of Bax. (F) The percentage of cleaved-Parp+ PGCs is significantly increased in Bax+/* Steel-/- embryos compared with Bax+/* Steel+/* littermates, but is rescued by the loss of Bax. P<0.05, P<0.01. Error bars represent means ± s.e.m.

View larger version (68K): [in this window] [in a new window]   Fig. 8. Steel is required for proper germ cell localization and for proliferation at E10.5. (A-I) Bax/Steel embryos were immunostained for phospho-histone H3, cleaved Parp, and the PGC marker SSEA1. (A-C) Bax-/- Steel+/+, (D-F) Bax+/- Steel+/-, and (G-I) Bax-/- Steel-/-. Apoptotic PGCs (arrowheads) were enriched in Bax+/- embryos that lacked at least one allele of Steel (D). Proliferating germ cells (arrows) were enriched in embryos with an intact allele of Steel (C,F). Germ cells in Bax/Steel DKO embryos were mislocalized (G-I), and were found in the ventral aspect of the hindgut and further ventral structures (arrows), with few near the genital ridges (arrowhead). SSEA1 staining (B,E,H) confirmed the presence of PGCs, independent of Oct4. No differences in PGC numbers or locations were observed between SSEA1 stained and unstained slices. (J) Cleaved-PARP+ PGCs from Bax+/- Steel+/- (D) and Bax-/- Steel-/- (G) embryos were compared and found to be significantly reduced in the absence of Bax. (K) Phospho-histone H3-positive PGCs were significantly reduced in Steel-/- embryos (I) compared with Steel heterozygous (F) and wild-type (C) littermates. (L) PGCs were reduced in Steel heterozygous compared with wild-type embryos, and nearly absent in Steel-/- embryos. The additional loss of Bax rescues PGC numbers to E9.0 levels. P<0.01 for Bax+/- Steel-/- vs Bax-/- Steel-/- embryos. (M) The percentage of ectopic germ cells in Bax/Steel embryos is markedly higher in Steel-null embryos. Horizontal brackets indicate samples that were compared for statistical significance measurements. P<0.05, P<0.01, P<0.001. Error bars represent means ± s.e.m.