(A) For each stage, the position of germ cells (yellow) and midgut (red) is indicated. Yellow arrows point in the direction of migration. Note that in wild-type embryos, few germ cells can be observed on the basal side of the midgut anlage (black arrow, stage 7). Genes known for their specific role in germ cell guidance are indicated next to the step where the activity of the respective gene product is needed. In addition to the genes listed, a role for hedgehog has been suggested in germ cell migration (Desphpande et al. 2001); however, the exact step affected is unclear.
(B–G) Overexpression of EP1631 in germ cells and expression pattern of CG4322. Anterior is to the left in all panels. (B), (C), (F), and (G) are lateral views; (D) and (E) are top views. Wild-type embryos (B and D) and embryos overexpressing EP1631 (CG4322 GPCR) in germ cells (C and E) were stained with anti-Vasa to mark germ cells. At stage 11 (B and C), germ cells in the wild-type (B) associate with the mesoderm, while germ cells expressing EP1631 (CG4322 GPCR) using the germ cell-specific nos-GAL4 driver are disorganized (C). When the gonads are normally coalescing at stage 13 in wild-type control embryos (D), many germ cells expressing the CG4322 GPCR remain lost and are found in ectopic locations (E; arrow points to few germ cells in gonad). (F and G) Expression pattern of CG4322 RNA. CG4322 RNA is detected in hemocytes (asterisk), in the caudal visceral mesoderm (arrow), and in the PMG at stage 9 (F) and in midgut (arrow) and glial cells (arrowhead) at stage 13 (G).

Anterior is left in all figures.
(A–F) Embryos are stained with anti-Vasa (brown) to mark germ cells. (A–D) Lateral views. (E–F) Top views. (A), (C), and (E) are wild-type embryos. (B), (D), and (F) are tre1 mutant embryos. Wild-type germ cells migrate out of the PMG at stage 10 (A) and migrate toward mesoderm at stage 11 (C) and finally to the gonad at stage 13 (E), but in tre1 mutant embryos, germ cells fail to leave the PMG ([B] shows stage 10 and [D] shows stage 11) and are mostly found “clumped” together in the middle of the gut at stage 13 (F).
(G–J) High magnification view of wild-type (G and H) and tre1 mutant (I and J) embryos stained with anti-Neurotactin (red) to mark cell membranes of midgut epithelium and germ cell-specific anti-Vasa (green). Wild-type germ cells are migrating out of the PMG at early stage 10 (G) and are outside of the PMG and thus at a different optical plane than PMG at late stage 10 (H). tre1 germ cells, in contrast, do not migrate out of the PMG at stage 9/10 (I) and are still left inside the PMG and thus at the same optical level as the PMG cells at late stage 10 (J). Punctate appearance of anti-Vasa staining in tre mutant germ cells is likely due to heat fixation protocol used as it can also be observed in wild-type germ cells (data not shown).

(A) Molecular structure of the tre1 region (adapted from Dahanukar et al. 2001). The exons of tre1 and Gr5a are shown as black boxes. Only two of seven exons are shown for Gr5a. The inverted triangle marks the insertion EP(X)0496. Deleted regions in ΔEP5 and ΔEP19 are shown by interrupted lines below.
(B) Genomic rescue constructs. Black filled boxes denote translated exons; white open boxes denote exons likely not translated because of stop codon mutation. T⁺ G⁺ contains both wild-type constructs for tre1 (T) and Gr5a (G); T⁻ G⁺ and T⁺ G⁻ contain a stop codon mutation (asterisk) for tre1 and Gr5a, respectively.
(C–K) Anterior is to the left in all embryos. All embryos are at stage 13, except (F), which is at stage 11. Embryos are labeled with anti-Vasa (brown) to mark germ cells. The embryo in (K) is also stained for anti-β-galactosidase activity.
(C–E) Genomic rescued tre1 embryos. Embryos from tre1 homozygous mothers that carried either the wild-type construct for both genes (T⁺ G⁺) or the construct with a wild-type copy for tre1 (T⁺ G⁻) rescued the tre1 migration phenotype completely (C and E). However, embryos from a tre1 mother carrying a nonfunctional copy of the tre1 gene (T⁻ G⁺) did not rescue the tre1 migration phenotype (D).
(F–J) tre1 phenotypic series. (F–H) M⁻ Z^−-sctt embryos. (F) Stage 11 sctt embryos with strong transgut migration defect (for wild-type control, refer to Figure 3C); note that more germ cells have exited the gut compared to strong ΔEP5 mutants (Figure 3D). (G and H) At stage 13, most germ cells remain inside the gut in sctt mutants, as judged by Fasciclin III staining (arrow in [H]; arrowhead points to germ cells), and more germ cells reach the gonad (arrows in [G]) compared to ΔEP5 mutants (for wild-type and ΔEP5, refer to Figure 3E and 3F and Figure S1I and S1J). The phenotype is enhanced in embryos from sctt/ΔEP5 females (I). ΔEP19 embryos have weak phenotype (J).
(K) tre1 phenotype can be rescued weakly by paternal zygotic copy. An increased number of germ cells migrates to the gonad (shown by arrowhead). The zygotic rescued embryos were identified by deformed–LacZ staining (arrow).

(A) and (C) depict the experimental rationale for the tissue-specific gene expression experiment. EP(X)0496, which drives expression of tre1 RNA, was expressed either in the germline by the germline-specific driver nos-GAL4 (A) or in the soma by somatic blastoderm cell-specific driver nullo-GAL4 (C). nos-GAL4 (yellow) is maternally localized to the posterior pole (yellow) and drives expression in germ cells (green), starting at stage 7 and persisting through embryogenesis and transiently in posterior somatic tissues at blastoderm stage (data not shown). nullo-GAL4 (C) (yellow) drives expression in all somatic cells at blastoderm stage except for germ cells (green). (B and D) Embryos at stage 13 (top view) stained with anti-Vasa. Anterior is left. Expression of tre1 only in the germ cells rescued the tre1 phenotype (B). Expression of tre1 in somatic tissues did not rescue the tre1 mutant phenotype (D).

(A) Phylogenetic tree of Tre1 protein with other closely related GPCRs (drawn using ClustalW of MegAlign program from DNA-STAR). Tre1 (indicated by arrow) is closely related to a group of fly, Anopheles, and vertebrate GPCRs. Among known ligand–receptor pairs, this novel receptor group is most closely related to melatonin, histamine, and serotonin receptors. Abbreviations: Ag, Anopheles gambiae; Dm, Drosophila melanogaster; Dr, Danio rerio; Fr, Fugu rubripes; Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis.
(B–G) RNA expression pattern of CG3171 (tre1). Anterior is to the left in all embryos. All embryos are in lateral views. (B–E) Expression pattern of tre1 (CG3171) RNA in wild-type embryos.
(B) In a stage 3 embryo, tre1 transcript is provided maternally and enriched at the posterior pole (arrow).
(C) At stage 6, tre1 transcript is degraded in somatic tissues, but protected in the germ cells (arrow).
(D) At stage 9, tre1 transcript is still detected in germ cells (arrow), but is also expressed broadly throughout the soma.
(E) By stage 13, tre1 transcript is highly expressed in several somatic tissues, including the midline glial, cuprophilic cells, glial cells, and CNS.
(F) In a stage 6 M⁻ Z⁻ tre1 embryo, no specific tre1 transcript is detected in germ cells (arrow). Weak staining in somatic tissues represents the background as it is also seen with sense control RNA probe. At stage 13, weak but specific tre1 expression is detected in cuprophilic cells and CNS (data not shown).
(G) M⁻ Z⁺ tre1 embryo. tre1 transcript is detected weakly in the germ cells at stage 9, indicating zygotic expression in the germ cells (arrow). Note the broad zygotic tre1 expression similar to (D).

(A–F) tre1 embryos labeled with anti-Vasa to mark germ cells. Anterior is left. Embryos in (A)–(D) are lateral views, and embryos in (E) and (F) are top views. Arrows point to the few germ cells that migrate correctly. Arrows in (A) and (B) point to a few germ cells that are clearly on the basal side of PMG anlage (on average, 1.27 germ cells per embryo). Arrow in (C) points to germ cells on the basal side of the PMG (on average, 1.47 germ cells per embryo). The arrow in (D) points to a germ cell that migrated successfully into the mesoderm. Arrows in (E) and (F) mark single germ cells that reached the embryonic gonad (on average, 1.2 germ cells per embryo).

(A), (C), and (E) depict the experimental scheme for germ cell transplantation. Germ cells (yellow) were transplanted from wild-type (A) or tre1 (C) stage 6 embryos to same stage embryos from tudor mothers, which do not have germ cells. In (E), germ cells (blue) labeled with LacZ (faf–LacZ) transgene were transplanted to the same stage tre1 embryos, to distinguish donor and host germ cells by β-galactosidase activity. (B), (D), and (F) are examples of transplanted, fixed, and stained embryos. Anterior is left in all embryos. Embryos in (B) and (F) are at stage 13; embryo in (D) is at stage 14. Embryos in (B) and (D) are stained with anti-Vasa (brown), and the embryo in (F) is stained with anti-β-galactosidase (brown). Arrow points to transplanted germ cells. (G–H) Summary of transplantation experiments. The bar graph in (G) summarizes the position of germ cells in embryos with successful transplantation (n = 36 for wild-type germ cells, n = 38 for tre1 mutant germ cells, and n = 48 for faf–LacZ-labeled wild-type germ cells). The bar graph in (H) summarizes the number of germ cells from successful transplantations at particular locations (n = 115 for wild-type germ cells, n = 87 for tre1 mutant germ cells, and n = 184 for faf–LacZ-labeled wild-type germ cells). Note that even in wild-type control transplantations, most germ cells, which do not reach the gonad, remain associated with the gut.

(A–F) Wild-type, constitutively active, and dominant-negative Rho1 constructs under UAS promoter control were expressed in germ cells using the nos-GAL4 driver. Embryos are stained with anti-Vasa (brown) to mark germ cells. Anterior is left. Embryos in (A), (C), and (E) are lateral views at stage 11. Embryos in (B), (D), and (F) are top views, stage 13.
(A and B) Germ cells expressing wild-type Rho1 (Rho1^wt) migrate normally.
(C and D) Germ cells expressing constitutively active Rho1 (Rho1^V14) successfully transmigrated the PMG (C), but some germ cells fail to move from the PMG into the mesoderm and remain associated with the PMG (D).
(E and F) Germ cells expressing dominant-negative Rho1 (Rho1^N19) are still inside the PMG at stage 11 (E), and most germ cells remain “clumped” inside the gut (F).