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First published online 5 December 2007
doi: 10.1242/dev.010389

Development 135, 207-216 (2008)
Published by The Company of Biologists 2008
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Programmed cell death of primordial germ cells in Drosophila is regulated by p53 and the Outsiders monocarboxylate transporter

Yukiko Yamada, Keri D. Davis and Clark R. Coffman*

Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011-3260, USA.


Figure 1
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  		Fig. 1. Programmed cell death of migrating germ cells in wild-type, p53 and out mutant embryos. Total numbers of PGCs in embryos between stages 10-14 were determined. In wild-type embryos, germ cell death was essentially complete by stage 12. In p53 and out embryos, germ cell death was disrupted. Similar numbers of PGCs were observed with a gradual loss of germ cells between stages 10 and 14. Error bars represent s.d.

 

Figure 2
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  		Fig. 2. Mutants initiate migration and form bilateral clusters in a similar manner to wild-type embryos. (A-O) Dorsal views of wild-type, p53 and outsiders mutant PGC development at stages 10-14. Anterior is left in all figures. PGCs are labeled with an anti-Vasa antibody. (A) At stage 10, wild-type PGCs moved out of the posterior midgut. (F,K) The initial PGC movements in p53 and out appear normal. (B) At stage 11, bilateral segregation of PGCs occurred in the wild-type embryos. (G,L) PGCs in p53 and out mutants form bilateral clusters. However, there are occasionally PGCs left in the midline. (C) During stage 12, wild-type PGCs form clusters. (H,M) Bilateral clusters are also seen in p53 and out mutants. Note that some PGCs remained in the midline of some p53 embryos. (D) At stage 13, PGCs form tightly associated linear arrays of cells. (I,N) In both p53 and out embryos, most PGCs were aligned. However, isolated PGCs were observed. (E) At stage 14, PGCs in wild-type embryos coalesce with SGPs. (J,O) p53 and out mutant embryos exhibit very similar phenotypes. PGCs are able to migrate to the gonads; however, many PGCs persisted ectopic to the gonads.

 

Figure 3
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  		Fig. 3. Germ cells initiate migration, but those ectopic to the gonads fail to die. (A-I) Lateral views of wild-type, p53 and outsiders mutant PGC development at stages 12-14. Anterior is left and dorsal up in all figures. PGCs are labeled with an anti-Vasa antibody. (A,D,G) PGCs in stage 12 embryos migrate into the mesoderm. (B) At stage 13, PGCs form a band of cells and are associated with one another in wild-type embryos. (E,H) In p53 and out embryos, some PGCs form clusters, but isolation of several PGCs becomes apparent. (C) At stage 14, wild-type PGCs reach the gonads. (F,I) In the mutants, PGCs successfully migrated to the gonads whereas errant PGCs are found ectopic to the gonads.

 

Figure 4
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  		Fig. 4. SGPs form normally in mutants, but some germ cells fail to associate with SGPs. (A) Stage 13 wild-type embryo. PGCs form elongated clusters. (B) Stage 13 wild-type embryo. SGPs align with the PGCs. (C) Stage 13 out embryo. Arrows indicate errant germ cells that failed to align with SGPs. (D) Stage 13 out embryo. SGP formation appears normal. (E) Stage 11 p53 embryo. PGCs migrated toward the SGPs. (F) Stage 11 p53 embryo. SGPs were specified in three clusters in parasegments 10-12 (arrows). (G) Stage 13 p53 embryo. Arrows indicate errant germ cells that failed to align with SGPs. (H) Stage 13 p53 embryo. SGPs appear normal. Anterior is left in A-F, and downwards in G and H. A, C, E and G are stained with a Vasa antibody, and B, D, F, and H are stained with an EYA antibody.

 

Figure 5
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  		Fig. 5. Germ cells ectopic to the gonads persist in p53 and out mutants. For each embryo examined, PGCs in the gonads (blue bars) and PGCs ectopic to the gonads (orange bars) were determined. PGC incorporation into the gonads was similar in wild-type, p53 and out embryos. In both p53 and out mutants, many ectopic PGCs were observed. In wild-type embryos, an average of 0.4 PGCs were found ectopic to the gonads. Error bars represent s.d.

 

Figure 6
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  		Fig. 6. The p53 germ cell phenotype is similar to the out; p53 double mutant. Dorsal views of the p53 mutants at stage 14 are shown. (A,B,C) p53 mutants displayed germ cells ectopic to the gonads. (D) A transheterozygous p535A-1-4/p53-ns mutant embryo is shown. (E) The out1;p53 double mutant appears very similar to the p53 mutants. The germ cells in the double mutant are labeled with an anti-β-gal antibody.

 

Figure 7
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  		Fig. 7. Molecular identification of out. (A) A P-element insertion in CG8062 disrupts germ cell PCD. This allele fails to complement other out mutants. The P-element lies between exons 1 and 2. (B) Amino acid sequence of OUT. Three out alleles introduce premature stop codons in the CG8062 transcript. The wild-type OUT peptide comprises 655 amino acids. The positions of the stop codons are indicated by the bold underlined letters. (C) Schematic of the predicted OUT protein. Positions of the out nonsense mutations are indicated by arrows. The introduced stop codons would result in truncated proteins of 224, 276 and 310 amino acids for out2, out1 and out5, respectively.

 

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