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Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

Kunwar PS, Starz-Gaiano M, Bainton RJ, Heberlein U, Lehmann R - PLoS Biol. (2003)

Bottom Line: In tre1 mutant embryos, most germ cells do not exit the PMG.Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells.Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Developmental Genetics Program, New York University School of Medicine, New York, New York, USA.

ABSTRACT
In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

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tre1 Gene Structure, Genomic Rescue, and tre1 Phenotypic Series(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).
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pbio.0000080-g004: tre1 Gene Structure, Genomic Rescue, and tre1 Phenotypic Series(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).

Mentions: The tre1 gene is located in polytene band 5A10 on the X chromosome and, as mentioned above, was initially identified as a GPCR thought to act as a taste receptor for Trehalose. Subsequently, however, a second GPCR, Gr5a, which maps adjacent to tre1, was shown to be the actual receptor of Trehalose, leaving the function of tre1 and the nature of its ligand unknown (Ishimoto et al. 2000; Dahanukar et al. 2001; Ueno et al. 2001). The predicted transcription start sites of tre1 and Gr5a are about 900 basepairs apart (Figure 4A). The deletion mutant ΔEP5 extends from the first exon of tre1 to the start of the Gr5a transcription unit. ΔEP5 homozygous mutants are adult viable and were reported to lack both tre1 and Gr5a transcripts (Ueno et al. 2001). To confirm that indeed loss of tre1 and not loss of Gr5a gene function was responsible for the observed germ cell migration defect, we introduced into the deletion mutant genomic rescue constructs that contained a 10-kb genomic region, which covers both tre1 and Gr5a (Dahanukar et al. 2001) (Figure 4B). In addition to the transgene that is wild-type for both genes (T+ G+), we tested two other transgenes, T− G+ and T+ G− that carry a stop codon mutation near the N-terminus of tre1 or Gr5a, respectively, and therefore supply a functional gene product for only one of the two genes (see Materials and Methods). The wild-type construct for both genes (T+ G+) and the construct carrying the wild-type copy for tre1 (T+ G−) rescued completely the migration phenotype of embryos from ΔEP5 homozygous mothers (Figure 4C and 4E). In contrast, embryos from ΔEP5 mothers carrying a nonfunctional copy of the tre1 gene (T− G+) produced a strong migration phenotype, demonstrating that indeed tre1, and not Gr5a, is required for the migration of germ cells through the PMG (Figure 4D).


Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

Kunwar PS, Starz-Gaiano M, Bainton RJ, Heberlein U, Lehmann R - PLoS Biol. (2003)

tre1 Gene Structure, Genomic Rescue, and tre1 Phenotypic Series(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).
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Related In: Results  -  Collection

Show All Figures
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pbio.0000080-g004: tre1 Gene Structure, Genomic Rescue, and tre1 Phenotypic Series(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).
Mentions: The tre1 gene is located in polytene band 5A10 on the X chromosome and, as mentioned above, was initially identified as a GPCR thought to act as a taste receptor for Trehalose. Subsequently, however, a second GPCR, Gr5a, which maps adjacent to tre1, was shown to be the actual receptor of Trehalose, leaving the function of tre1 and the nature of its ligand unknown (Ishimoto et al. 2000; Dahanukar et al. 2001; Ueno et al. 2001). The predicted transcription start sites of tre1 and Gr5a are about 900 basepairs apart (Figure 4A). The deletion mutant ΔEP5 extends from the first exon of tre1 to the start of the Gr5a transcription unit. ΔEP5 homozygous mutants are adult viable and were reported to lack both tre1 and Gr5a transcripts (Ueno et al. 2001). To confirm that indeed loss of tre1 and not loss of Gr5a gene function was responsible for the observed germ cell migration defect, we introduced into the deletion mutant genomic rescue constructs that contained a 10-kb genomic region, which covers both tre1 and Gr5a (Dahanukar et al. 2001) (Figure 4B). In addition to the transgene that is wild-type for both genes (T+ G+), we tested two other transgenes, T− G+ and T+ G− that carry a stop codon mutation near the N-terminus of tre1 or Gr5a, respectively, and therefore supply a functional gene product for only one of the two genes (see Materials and Methods). The wild-type construct for both genes (T+ G+) and the construct carrying the wild-type copy for tre1 (T+ G−) rescued completely the migration phenotype of embryos from ΔEP5 homozygous mothers (Figure 4C and 4E). In contrast, embryos from ΔEP5 mothers carrying a nonfunctional copy of the tre1 gene (T− G+) produced a strong migration phenotype, demonstrating that indeed tre1, and not Gr5a, is required for the migration of germ cells through the PMG (Figure 4D).

Bottom Line: In tre1 mutant embryos, most germ cells do not exit the PMG.Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells.Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Developmental Genetics Program, New York University School of Medicine, New York, New York, USA.

ABSTRACT
In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

Show MeSH