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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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Related in: MedlinePlus

The Phenotype of M− Z− tre1 Mutant EmbryosAnterior 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).
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pbio.0000080-g003: The Phenotype of M− Z− tre1 Mutant EmbryosAnterior 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).

Mentions: Expression analysis of tre1 RNA showed a dynamic expression pattern (Figure 2B–2E). tre1 RNA is provided maternally, localized to the germ plasm, and can be detected clearly in germ cells until stage 9, when they initiate their migration through the PMG epithelium. We did not detect tre1 expression in germ cells as they coalesced into the embryonic gonads. tre1 RNA is also expressed in a variety of other tissues, including the amnioserosa, the developing CNS, the cardiac mesoderm primordium, midline glia, and (very prominently) the cuprophilic cells. To determine tre1 function, we studied the mutant phenotype of ΔEP5, a tre1 mutation generated by imprecise excision of the EP line EP0496 and previously reported to lack tre1 RNA (Rorth 1996; Ishimoto et al. 2000; Dahanukar et al. 2001; Ueno et al. 2001) (see below). Embryos from ΔEP5 homozygous mothers that are also zygotically mutant (M−,Z−) show no specific tre1 RNA expression until stage 9 (Figure 2F), suggesting that the ΔEP5 deletion affects the regulation of maternal and early zygotic tre1 gene expression (Ueno et al. 2001). Embryos derived from ΔEP5 homozygous mothers (hereafter referred as tre1 mutant embryos) are defective in the first active step of germ cell migration, the transepithelial migration though the PMG (Figure 3A–3J). During stage 10 of embryogenesis, wild-type germ cells migrate from the apical side of the PMG epithelium to its basal side. In contrast, most germ cells in tre1 mutant embryos do not transmigrate the PMG, but remain clumped together within the midgut pocket (Figure 3A and 3B). To follow germ cell and gut development in mutant and wild-type embryos, we double-labeled embryos with the germ cell marker Vasa and midgut-specific markers, such as race, and Fasciclin III, a visceral mesodermal marker (Figure S1A–S1D and S1I–S1J) (Patel et al. 1987; Stein et al. 2002). While in wild-type embryos, germ cells migrate away from the gut during stage 11, associate with gonadal mesoderm, and eventually form two bilateral gonads, germ cells remained within the gut throughout development in tre1 mutants (see Figure 3C–3F; Figure S1A–S1D and S1I–S1J). At the end of embryogenesis, tre1 mutant embryos have on average one to two germ cells in either gonad, compared to about 12–15 germ cells per gonad in wild-type (see Figure 3E and 3F; see also Figure 7E and 7F). The overall number of germ cells seemed unaffected in the mutant. This phenotype is fully penetrant, and 100% of embryos derived from tre1 mutant mothers show a strong germ cell migration defect.


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)

The Phenotype of M− Z− tre1 Mutant EmbryosAnterior 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC300690&req=5

pbio.0000080-g003: The Phenotype of M− Z− tre1 Mutant EmbryosAnterior 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).
Mentions: Expression analysis of tre1 RNA showed a dynamic expression pattern (Figure 2B–2E). tre1 RNA is provided maternally, localized to the germ plasm, and can be detected clearly in germ cells until stage 9, when they initiate their migration through the PMG epithelium. We did not detect tre1 expression in germ cells as they coalesced into the embryonic gonads. tre1 RNA is also expressed in a variety of other tissues, including the amnioserosa, the developing CNS, the cardiac mesoderm primordium, midline glia, and (very prominently) the cuprophilic cells. To determine tre1 function, we studied the mutant phenotype of ΔEP5, a tre1 mutation generated by imprecise excision of the EP line EP0496 and previously reported to lack tre1 RNA (Rorth 1996; Ishimoto et al. 2000; Dahanukar et al. 2001; Ueno et al. 2001) (see below). Embryos from ΔEP5 homozygous mothers that are also zygotically mutant (M−,Z−) show no specific tre1 RNA expression until stage 9 (Figure 2F), suggesting that the ΔEP5 deletion affects the regulation of maternal and early zygotic tre1 gene expression (Ueno et al. 2001). Embryos derived from ΔEP5 homozygous mothers (hereafter referred as tre1 mutant embryos) are defective in the first active step of germ cell migration, the transepithelial migration though the PMG (Figure 3A–3J). During stage 10 of embryogenesis, wild-type germ cells migrate from the apical side of the PMG epithelium to its basal side. In contrast, most germ cells in tre1 mutant embryos do not transmigrate the PMG, but remain clumped together within the midgut pocket (Figure 3A and 3B). To follow germ cell and gut development in mutant and wild-type embryos, we double-labeled embryos with the germ cell marker Vasa and midgut-specific markers, such as race, and Fasciclin III, a visceral mesodermal marker (Figure S1A–S1D and S1I–S1J) (Patel et al. 1987; Stein et al. 2002). While in wild-type embryos, germ cells migrate away from the gut during stage 11, associate with gonadal mesoderm, and eventually form two bilateral gonads, germ cells remained within the gut throughout development in tre1 mutants (see Figure 3C–3F; Figure S1A–S1D and S1I–S1J). At the end of embryogenesis, tre1 mutant embryos have on average one to two germ cells in either gonad, compared to about 12–15 germ cells per gonad in wild-type (see Figure 3E and 3F; see also Figure 7E and 7F). The overall number of germ cells seemed unaffected in the mutant. This phenotype is fully penetrant, and 100% of embryos derived from tre1 mutant mothers show a strong germ cell migration defect.

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
Related in: MedlinePlus