Perturbation of Notch/Suppressor of Hairless pathway disturbs migration of primordial germ cells in Xenopus embryo

Authors

  • Keisuke Morichika,

    1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Akou-gun, Hyogo 678-1297
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  • Kensuke Kataoka,

    1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Akou-gun, Hyogo 678-1297
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    • Present address: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, A-1030 Vienna, Austria.

  • Kohei Terayama,

    1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Akou-gun, Hyogo 678-1297
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  • Akira Tazaki,

    1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Akou-gun, Hyogo 678-1297
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    • Present address: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

  • Tsutomu Kinoshita,

    1. Department of Bioscience, Faculty of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
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  • Kenji Watanabe,

    1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Akou-gun, Hyogo 678-1297
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  • Makoto Mochii

    Corresponding author
    1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Akou-gun, Hyogo 678-1297
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*Author to whom all correspondence should be addressed.
Email: mmochii@sci.u-hyogo.ac.jp

Abstract

Primordial germ cells (PGCs) in Xenopus embryo are specified in the endodermal cell mass and migrate dorsally toward the future gonads. The role of the signal mediated by Notch and Suppressor of Hairless [Su(H)] was analyzed on the migrating PGCs at the tailbud stage. X-Notch-1 and X-Delta-1 are expressed in the migrating PGCs and surrounding endodermal cells, whereas X-Delta-2 and X-Serrate-1 are expressed preferentially in the PGCs. Suppression and constitutive activation of the Notch/Su(H) signaling in the whole endoderm region or selectively in the PGCs resulted in an increase in ectopic PGCs located in lateral or ventral regions. Knocking down of the Notch ligands by morpholino oligonucleotides revealed that X-Delta-2 was indispensable for the correct PGC migration. The ectopic PGCs seemed to have lost their motility in the Notch/Su(H) signal-manipulated embryos. Our results suggest that a cell-to-cell interaction via the Notch/Su(H) pathway has a significant role in the PGC migration by regulating cell motility.

Introduction

Primordial germ cells (PGCs) are the embryonic cells that are destined to form gametes. They are specified at a distance from the future gonadal region at early embryonic stages in many species, and migrate from their original site to the gonadal anlagen via multiple pathways (Molyneaux & Wylie 2004). For example, mouse PGCs specified by cellular interactions appear at the base of the allantois, migrate into the hindgut, and finally arrive at the genital ridges, whereas zebrafish PGCs specified by maternal components migrate along the lateral mesoderm. In Xenopus embryo, cells that inherit the germplasm, which is a specific cytoplasm at the vegetal cortex of oocytes, are specified to be PGCs after the cleavage stage (Whitington & Dixon 1975). Several proteins and mRNAs have been identified as germplasm components (Zhou & King 1996; Houston et al. 1998; Hudson & Woodland 1998; MacArthur et al. 2000; Berekelya et al. 2003; Kaneshiro et al. 2007). Xenopus PGCs are assumed to migrate passively with surrounding endodermal cells until stage 23, start to migrate actively toward the dorsal-most region of the endoderm after stage 24, and finally arrive at the future genital ridge region in the mesentery by stage 46 (Nishiumi et al. 2005). One of the most characteristic features of the Xenopus PGCs is the long distance that they travel through the large endodermal cell mass in the tailbud embryo. Functional analysis has revealed that PGC migration requires several components of the germplasm, including X-dazl, Germes and glutamate receptor interacting protein 2 (Houston & King 2000; Berekelya et al. 2007; Kirilenko et al. 2008). PGCs interact directly or indirectly with surrounding somatic cells during the migration process. The chemokine receptor 4 (CXCR4)-mediated signaling is involved in the mechanism that underlies PGC migration (Takeuchi et al. 2009).

Notch receptors and its ligands, Delta and Jagged/Serrate, are single-pass transmembrane proteins and interact between adjacent cells. Binding of the ligand to Notch triggers the release of the Notch intracellular domain (NICD), which then translocates into the nucleus and forms a complex with Suppressor of Hairless [Su(H)] to regulate gene expression (Fiúza & Arias 2007). The Notch/Su(H) pathway regulates a wide variety of developmental events, including cell fate determination, cell proliferation, border formation and cell migration (De Bellard et al. 2002; Fuss et al. 2004; Fiúza & Arias 2007). Notch signaling is required for maintenance of germ line stem cells in Drosophila gonads (Terry et al. 2006; Song et al. 2007), and has been suggested to have a role in mouse spermatogenesis (Dirami et al. 2001). However, the role of Notch signaling on PGC development before gonad formation has not been elucidated.

In this study we revealed that Notch signal-related genes were expressed in migrating PGCs and surrounding somatic cells, and that hyperactivation and inactivation of Notch/Su(H) signaling impaired the dorsal migration of PGCs in the endodermal cell mass. Therefore, cell-to-cell interaction via Notch/Su(H) signaling is involved in the migration mechanism of Xenopus PGCs.

Materials and methods

DNA constructs, mRNAs and Morpholino oligonucleotides

Venus-DEADSouth 3′untranslated region (UTR) (Venus-DS)/pCS2 has been described previously (Kataoka et al. 2006). CS2+ plasmids that encode cDNAs for glucocorticoid receptor (GR) hormone binding domain and dominant negative form of Su(H), Su(H)DBM, have been described by Kiyota et al. (2001). Full-length cDNAs of X-Delta-1and X-Delta-2 have been cloned by polymerase chain reaction (PCR) using degenerated primers designed from the consensus epidermal growth factor motif and 5′- and 3′-rapid amplification of cDNA ends method (Kiyota et al. 2001). X-Notch-1 cDNA clone was isolated from a pBluescript SK(−) library, as described previously (Tazaki et al. 2005), by a PCR-based selection using primers 5′-ATCATCTTCGTCTTCATGATGGTCATCGTT-3′ and 5′-GAGCCATCTGTCATATTCTTAATGGGCTTT-3′. The NICD of the X-Notch-1 cDNA was subcloned into GR/pCS2 at a Cla1/EcoRV site to make NICD-GR/pCS2 using primers 5′-GGTCATCGATGAATAAGAAGCGTCGCCGTG-3′ and 5′-CTTGAAAGCTTCAGGTATGTGGGTGCGCTG-3′. Su(H)DBM-GR/pCS2 was made by subcloning the Su(H) DBM fragment into GR/pCS2 using primers 5′-GCGATCGATGCAACCTGGCATTCCTAAATA-3′ and 5′-GGACACTACTGCTGCAGTGGATGATGTGAC-3′. To overexpress cloned genes specifically in PGCs, the 3′ UTR sequence of the DEAD South cDNA (Kataoka et al. 2006) was amplified using primers 5′-ACGCCTAGGCAGTATGGACATGGATGAGAT-3′ and 5′-ACGCCTAGGAATACGACTCACTATAGGGCG-3′, digested with Sty1, and subcloned into the above plasmids at an Xba1 site to make plasmids, Su(H)DBM-GR-DS/pCS2 and NICD-GR-DS/pCS2. mRNAs were synthesized with plasmids linearized by Not-1 using the mMESSAGE mMACHNE kit (Ambion), and purified with S-400 Spin Column (GE Healthcare) for microinjection.

The morpholino oligonucleotide (MO) for X-delta-2 has been described previously (Peres & Durston 2006). MO for X-delta-1 (5′-GCTCTGCTGTCCCATGTTGTCTGAT-3′) was designed in this study (Gene Tools, LLC). Standard control MO (Gene Tools, LLC) was used as the negative control. Effectiveness of X-delta-1 MO was confirmed by injecting it into the animal region of the embryo. MO injection caused defects, including an enlarged head region and disordered somitogenesis (data not shown), that were expected from the reported phenotypes of Notch signal suppression (Wettstein et al. 1997; Umbhauer et al. 2001).

Embryo preparation and microinjection

Xenopus laevis embryos were obtained by in vitro fertilization and de-jellied with 3% cysteine (pH 7.8) according to the standard method (Kataoka et al. 2006). Embryos were maintained in 0.1 × Mark’s modified ringer (MMR) at 14°C until the two-cell stage, and then cultured at 18°C. The developmental stages were determined according to Nieuwkoop & Faber (1994). mRNA synthesized in vitro was injected into the vegetal cortex region of one- to four-cell-stage embryos as described previously (Kataoka et al. 2006), together with dextran tetramethyl rhodamine (10 000 MW, Molecular Probes) for lineage tracing. At stage 18, the embryos were transferred into 0.1 × MMR that contained 25 μmol/L dexamethasone (Sigma), to induce nuclear translocation of the GR fusion protein (Wettstein et al. 1997).

Reverse transcription–PCR of isolated cells

A ventral trunk region that contained most of the endoderm was excised from stage 24 embryos and incubated in 80% Dulbecco’s phosphate-buffered saline (PBS) without Ca2+ and Mg2+ [PBS(−)] for 30 min, to be dissociated at the single-cell level. The PGCs with Venus fluorescence and endodermal cells without fluorescence were collected independently with micropipettes under the fluorescent microscope (Leica MZ16F). Total RNA was extracted with 1.5 mL of TRIzol reagent (Invitrogen) from 200 cells that were prepared from 10 to 15 embryos. cDNA was synthesized using Ready-To-Go You-Prime First-strand Beads (GE Healthcare). Semi-quantitative PCR was performed as described previously (Taniguchi et al. 2008; Sugiura et al. 2009) using the primers listed in Table S1. Each template synthesized without reverse transcriptase was used as a negative control, which gave rise to no amplified product. At least three independent experiments were performed to obtain each result.

Successive analysis of PGC migration

Each tailbud embryo injected with Venus-DS mRNA was mounted with 0.1 × MMR in a hollow that was created on an agarose plate, and covered with a cover slip. PGCs labeled with Venus fluorescence were observed under the fluorescent microscope (Leica MZ16F).

Immunological detection of Venus-expressing cells

Embryos were fixed with 4% paraformaldehyde in PBS(−) at 4°C overnight. The fixed embryos were dehydrated through a series of ethanol–butanol solutions, embedded in paraffin, serially sliced at 12 μm thickness with a microtome, and mounted on glass slides. The rehydrated sections were washed in PBS(−), blocked in PBS(−) that contained 0.1% Triton × 100 (TPBS) supplemented with 10% goat serum, and incubated with rabbit anti-green fluorescent protein antibody (A.v. peptide antibody; Clontech). After washing in TPBS, the sections were incubated with Alexa 488-conjugated anti-rabbit IgG (Molecular Probes). After further washing in TPBS, the sections were mounted in 50% glycerol and observed under the fluorescent microscope (Olympus BX60). To evaluate the correlation between the number of ectopic PGCs identified externally and the total one examined on sections, some embryos were divided into three pools according to the number of the externally counted ectopic PGCs.

Results

Notch receptor and ligand genes were expressed in PGCs and surrounding endodermal cells

X-Notch-1 and X-Serrate-1 are expressed maternally in the early embryo (Coffman et al. 1990; Kiyota et al. 2001). X-Delta-1 and X-Delta-2 are expressed zygotically in embryos later than the blastula stage (Fig. 1A). We analyzed spatial expression patterns of these genes by in situ hybridization in stage-28 embryos, however, no reliable hybridization signal was observed in the endoderm region (data not shown). To analyze gene expression with higher sensitivity, we performed a semi-quantitative reverse transcription (RT)–PCR using isolated cells. PGCs were labeled with fluorescence in a living tailbud embryo injected with Venus-DS mRNA at the one-cell stage. The DEADSouth 3′UTR flanking the Venus-coding sequence participates in anchoring the mRNA at the germplasm and degrading it in the somatic cells, leading to the PGC-specific expression of the Venus in tailbud embryos (Kataoka et al. 2006) (Fig. 1B). An endoderm fragment was dissected from the PGC-labeled embryo and was dissociated into single cells (Fig. 1C). Total RNAs were extracted from Venus-positive PGCs and Venus-negative endodermal cells and used for RT–PCR. Transcripts of X-Notch-1 and X-Delta-1 were detected in both types of cells at a similar level (Fig. 1D). Transcripts of X-Delta-2 and X-Serrate-1 were detected in the Venus-positive PGCs but hardly detectable in the Venus-negative endodermal cells. Therefore, the genes for Notch receptor and its ligands were expressed differentially in PGCs and the surrounding somatic cells at the PGC-migrating stage. This suggested that Notch-mediated cell-to-cell interaction regulates some crucial events in PGC development, for example, cell fate specification and/or directional migration.

Figure 1.

 Expression of genes for Notch receptor and ligands in tailbud embryo. (A) RNA prepared from whole embryos at indicated stage was used for reverse transcription–polymerase chain reaction (RT–PCR) analysis. (B) Stage 31/32 embryo injected with Venus-DS mRNA. Primordial germ cells (PGCs) were visible externally under fluorescent light. (C) Cells dissociated from the endodermal region of the PGC-labeled embryo. Arrows in (B) and (C) indicate PGCs. (D) RT–PCR using isolated PGCs and somatic endoderm cells. Bars in (B), 500 μm; (C), 100 μm. ODC, ornithine decarboxylase.

Perturbations of Notch/Su(H) signaling disturb proper migration of PGCs

To inhibit the Notch/Su(H) pathway in the endoderm region that contains PGCs, we injected an mRNA for Su(H)DBM (Wettstein et al. 1997) fused to a hormone-binding domain of the GR [termed Su(H)DBM-GR] into the vegetal pole of the one-cell-stage embryo. Treatment of the injected embryo with dexamethasone induced translocation of Su(H)DBM into the nucleus and inhibited endogenous Su(H) activity at the desired developmental stage. Dexamethasone was added to the culture medium at stage 18, when gastrulation was completed, because manipulation of Notch/Su(H) signaling during gastrulation affected the mesoderm and endoderm formation (Contakos et al. 2005).

Although the inhibition of Notch/Su(H) signaling in the ectoderm and mesoderm causes defects in neurogenesis and somitogenesis (Chitnis et al. 1995; Jen et al. 1997; Peres & Durston 2006), injection of Su(H)DBM-GR mRNA at the vegetal pole, the future endoderm, and subsequent treatment with dexamethasone did not cause any morphological defect in the endoderm. When the embryo was injected with Venus-DS mRNA, PGCs were observed clearly at stage 41 in control embryos as the aligned fluorescent signals around the dorsal body wall region, as described previously (Kataoka et al. 2006) (Fig. 2A). In embryos injected with Su(H)DBM-GR mRNA, several PGCs were located ectopically in the lateral or ventral region (Fig. 2B). The number of PGCs aligned on the dorsal body wall region was reduced in a significant number of the embryos. We regarded the PGCs at the dorsal body wall or in the dorsal third of the endoderm at stage 41 as correctly located PGCs, and the PGCs outside the correct region as ectopic PGCs. Although some control embryos injected with only the Venus-DS mRNA had a few ectopic PGCs, inhibition of Su(H) increased the number of ectopic PGCs per embryo (Fig. 2B; Table 1). The rate of embryos with many ectopic PGCs was also increased by Su(H) inhibition. For example, the rate of embryo with at least three ectopic PGCs was increased from 7.0% to 37.0%. The injection of Su(H)DBM-GR mRNA without dexamethasone treatment slightly increased the rate of embryos with ectopic PGCs. The GR-fusion protein may have leaky activity without dexamethasone treatment.

Figure 2.

 Effect of Notch/Su(H) signal perturbation on primordial germ cell (PGC) localization in tailbud embryo. Embryo co-injected with Venus-DS mRNA (250 pg) and mRNA for Su(H)DBM-GR (500 pg), Notch intracellular domain (NICD)-GR (250 pg), Su(H)DBM-GR-DS (500 pg) or NICD-GR-DS (250 pg) at vegetal pole was treated with or without dexamethasone (25 μmol/L) from stage 18 to 41. (A–E) Lateral views of dexamethasone-treated embryo at stage 41. PGCs labeled with Venus fluorescence were observed externally under fluorescent light. Most of the PGCs were located at the dorsal body wall region in the control embryo (arrows), whereas a significant number of PGCs was seen in the lateral or ventral region in the Notch/Su(H) signal-perturbed embryos (arrowheads). Bar, 500 μm. (F–J) Transverse section of the manipulated embryo. PGCs were labeled with anti-green fluorescent protein (GFP) antibody that recognized Venus protein (green). Nuclei were labeled with Hoechst 33342 (magenta). Arrows indicate the PGCs located in the mesentery. Arrowheads indicate the ectopic PGCs. E, endoderm; NC, notochord; NT, neural tube; S, somite. Bar, 100 μm. A dotted line in (A) and (F) indicates the dorsal third of the endoderm.

Table 1.   Effect of Notch/Su(H)-signal perturbation on primordial germ cell (PGC) localization
Injected mRNADEX treatmentTotal embryosEmbryos with ectopic PGCsAverage number of ectopic PGCs
0123456788<
  1. Asterisks indicate that values differ from dexamethasone treatment (DEX) (−) controls at *< 0.05 and **< 0.01 using Welch’s t-test. Embryos were treated with or without dexamethasone (25 mmol/L) from stage 18 to 41. PGCs labeled with Venus fluorescence were counted externally from both sides at stage 41. Number of embryos with indicated number of the ectopic PGCs is shown. Each data includes results of three experiments.

Venus-DS (250 pg)585115       0.26
 +52371131      0.35
Su(H)DBM-GR (500 pg)7152952111   0.56
+Venus-DS (250 pg)+541897973 1  1.85**
NICD-GR (250 pg)614764211    0.48
+Venus-DS (250 pg)+6618158972511 2.17**
Su(H)DBM-GR-DS (500 pg)704688422    0.77
+Venus-DS (250 pg)+74361196542 1 1.45*
NICD-GR-DS (250 pg)61437631  1  0.64
+Venus-DS (250 pg)+8626131177635173.02**

We determined total number and localization of PGCs inside the embryo by serial sectioning and staining with an antibody that recognizes the Venus protein (Fig. 2F,G). Total number of PGCs in the embryo was not affected by Su(H) inhibition (Table 2). Next, we analyzed localization of PGCs. In the control embryos, most of the PGCs were located at the mesentery or at the dorsal-most region of the endoderm at stage 41 as described previously (Kataoka et al. 2006) (Fig. 2F; Table 2), whereas some PGCs were found in lateral or ventral regions. If the PGCs located in the ventral two-thirds of the endoderm were regarded as ectopic PGCs, 10.2–12.8% of the total PGCs were ectopic in control embryos. Inhibition of Su(H) in the endoderm region increased the rate of ectopic PGCs with the statistical significance (Fig. 2G; Table 2). The rate of ectopic PGCs was correlated roughly with the number found externally (Table 2).

Table 2.   Localization of primordial germ cells (PGCs) in Notch/Su(H) signal-manipulated embryos
Injected mRNADEX treatmentEctopic PGCs identified externallyNumber of embryosTotal number of PGCs per embryoRate of ectopic PGCs (%)§
  1. Asterisks indicate that values differ from control at *< 0.05 using Welch’s t-test. Embryos were treated with or without dexamethasone (DEX) (25 mmol/L) from stage 18 to 41. Fixed embryos were divided into three pools according to the number of the externally counted ectopic PGCs, serially sectioned and stained with anti-green fluorescent protein (GFP) antibody to identify PGCs. Mean number of total PGCs was determined in each pool and in combined pools. §PGCs located in the dorsal mesentery or the dorsal third of the endoderm were regarded as correctly localized but others were regarded as ectopic. Mean rate of ectopic PGCs was determined in each pool and in combined pools.

Venus-DS (250 pg)0640.5 ± 4.310.2 ± 6.3
Su(H)DBM-GR (500 pg) + Venus-DS (250 pg)0639.0 ± 2.812.8 ± 6.6
+0633.5 ± 9.434.8 ± 8.319.7 ± 13.727.1 ± 15.1*
1–2537.2 ± 2.927.3 ± 17.3
3–4433.8 ± 12.137.9 ± 8.8*
NICD-GR (250 pg) + Venus-DS (250 pg)0535.8 ± 9.919.0 ± 16.2
+0433.5 ± 4.834.1 ± 8.019.6 ± 7.632.2 ± 18.7
1–2431.3 ± 11.025.7 ± 13.4
3–4439.0 ± 7.951.3 ± 17.2*

To analyze the effect of hyperactivated Notch signaling on PGC migration, we injected the NICD-GR mRNA into the vegetal pole and treated it with dexamethasone (Fig. 2C). Activation of NICD in the whole endoderm region affected PGC localization but not the embryonic morphology. External observation showed that the NICD activation increased the average number of ectopic PGCs per embryo (Table 1). The rate of the embryo with at least three ectopic PGCs was increased from 6.6% to 37.6%. Serial sectioning confirmed this result (Fig. 2H: Table 2), and showed that the total number of PGCs per embryo was not changed by NICD injection.

The ectopic PGCs described above did not have apoptotic nuclei, at least until stage 41 (data not shown). The above results showed that both inhibition and hyperactivation of Notch/Su(H) signaling in the whole endoderm region affected PGC migration.

PGC-targeted manipulations of Notch/Su(H) signaling affects PGC migration

As described above, perturbations of Notch/Su(H) signaling in the whole endoderm region affected PGC migration. However, it is unknown whether Notch/Su(H) signaling is required in the PGCs themselves or in the surrounding somatic cells. To answer this question, we injected mRNAs flanked by the DEADSouth 3′UTR [termed Su(H)DBM-GR-DS and NICD-GR-DS] to induce PGC-specific gene expression (Kataoka et al. 2006). External observation showed that inactivation of Su(H) and activation of NICD using the above constructs increased the rate of embryos with at least three ectopic PGCs from 11.4% to 24.3%, and from 8.2% to 41.9%, respectively (Fig. 2D,E; Table 1), and also increased the number of ectopic PGCs per embryo (Table 1), as in the experiments using the constructs without the DEADSouth 3′UTR. The most drastic effect was shown in the case of NICD activation, which gave rise to more than eight ectopic PGCs in several embryos, whereas the PGC-targeted inhibition of Su(H) was affected moderately (Table 1). Serial sectioning confirmed these results (Fig. 2I,J).

X-Delta-2 is required for correct migration of PGCs

To test the requirements for the Notch ligands in PGC migration, we injected MOs for X-delta-1 and X-delta-2, the zygotically expressed ligand genes, into the vegetal pole of the fertilized eggs. X-Delta-1 knockdown in the endoderm caused severe defects in embryogenesis, including a shortened body axis at stage 31/32 (Fig. 3C). Co-injection of the full-length X-delta-1 mRNA rescued the defects (Fig. 3D). Although some PGCs labeled with Venus fluorescence were observed in the abnormal embryo (data not shown), we could not evaluate the effect of X-Delta-1 knockdown on PGC migration because of the severe defect in endoderm morphogenesis.

Figure 3.

X-Delta-2 knock down affects primordial germ cell (PGC) local-ization. (A–C) Stage-28 embryos injected with control (20 ng), X-Delta-2 (20 ng) or X-Delta-1 (10 ng) morpholino oligonucleotide (MO) at vegetal pole region. X-Delta-2 MO caused no morphological defect, while X-Delta-1 MO caused severe defects in the body axis elongation and endoderm morphogenesis. (D) The defects caused by X-delta-1 MO were rescued by co-injection with X-delta-1 mRNA. (E–G) Stage-41 embryos injected with mRNAs for Venus-DS and indicated construct, and MO. (H–J) Transverse section of embryo shown in (E–G). PGCs and nuclei are labeled with anti-green fluorescent protein (GFP) (green) and Hoechst 33342 (magenta), respectively. Arrows indicate the PGCs in the normal region. Arrowheads indicate the ectopic PGCs. Bars in (A), 500 μm; (E), 500 μm; (H), 100 μm.

Embryos injected with X-Delta-2 MO had an increased number of ectopic PGCs but no morphological defect (Fig. 3B,F,I; Table 3). The MO injection increased the rate of embryo with at least three ectopic PGCs from 5.3% to 41.9%. Co-injection of the full-length X-delta-2 mRNA reduced the rate to 12.6% (Fig. 3G,J), which showed the specific effect of the MO.

Table 3.   Effect of X-Delta-2 morpholino oligonucleotide on primordial germ cell (PGC) localization
Injected MO and mRNATotal embryoEmbryos with ectopic PGCsAverage number of ectopic PGCs
0123456788<
  1. Asterisk indicates that value differs from control at *< 0.01 using Welch’s t-test. PGCs labeled with Venus fluorescence were counted externally from both sides at stage 41. Number of embryos with indicated number of the ectopic PGCs is shown. Each data includes results of three experiments. MO, morpholino oligonucleotide.

Control MO (20 ng) + Venus-DS mRNA (250 pg)946516841     0.51
X-Delta-2 MO (20 ng) + Venus-DS mRNA (250 pg)86291110148443 32.29*
X-Delta-2 MO (20 ng) + X-Delta-2 mRNA (1 ng) + Venus-DS mRNA (250 pg)8751141143211  0.95

Perturbation of Notch/Su(H) signaling affects motility of PGCs

Primordial germ cell migration might be regulated by various cellular mechanisms, such as cell motility, directionality, and adhesion to extracellular matrices or to other cells. Migratory behavior of PGCs in a PGC-labeled embryo was examined under the fluorescence microscope. Several PGCs were identified in a control embryo at stage 32 (Fig. 4A). Most of them migrated into deep regions of the endoderm and became undetectable within 4 h in all nine control embryos. In contrast, most of the PGCs identified from the outside did not change their relative positions in the embryo injected with Su(H)DBM-GR [9 out of 10 embryos (9/10)], NICD-GR (8/8), Su(H)DBM-GR-DS (7/10), NICD-GR-DS (11/11) or X-Delta-2 MO (6/10) (Fig. 4B–F). These data indicated that a significant number of PGCs, at least those located near the lateral surface, lost their migratory activity in the embryo.

Figure 4.

 Perturbation of Notch/Su(H) signaling affects primordial germ cell (PGC) migration. PGCs labeled with Venus were observed in a stage-31/32 embryo mounted on an agarose bed with a cover slip. Area indicated with dashed square in upper panel was photographed at indicated time points. Arrows indicate the PGCs detected at 0 h. Bars in upper panel of (F), 500 μm; lower panel of (F), 100 μm.

Notch/Su(H) signaling regulates expression of HES-family genes in PGCs and endodermal cells

The NICD/Su(H) complex induces expression of the direct target genes, such as HES and HES-related genes, which encode basic helix-loop-helix type transcriptional repressors and suppress expression of downstream target genes (Iso et al. 2003). In this context it is interesting to see whether perturbation of Notch/Su(H) signaling affects expression of the target genes in the PGCs and/or endodermal cells. We analyzed expression of 13 HES and HES-related genes, which were identified in the Genebank database, by RT–PCR using dissociated cells. At least five genes for the HES and HES-related proteins were expressed in PGCs isolated from the control embryos at stage 28, but none of them was downregulated by Su(H) inhibition (Fig. 5A). On the other hand, NICD activation upregulated expression of X-ESR1 and X-ESR10 in the PGCs, which suggests that the two genes are targets of Notch signaling in PGCs. Control endodermal cells expressed X-hairy1 and X-hairy2, neither of which was downregulated by the Su(H) inhibition (Fig. 5B), although NICD activation upregulated many genes, including X-hairy2, X-ESR1, X-ESR3/7b, X-ESR4, X-ESR5, X-ESR6e, X-ESR9, X-ESR10 and X-Hesr1. These results showed that activation of Notch/Su(H) signaling induced expression of several HES and HES-related genes, presumably direct targets, in the PGCs and endodermal cells, whereas we could not detect any genes that were downregulated by Su(H) inhibition.

Figure 5.

 Activation of Notch signaling induces expression of target genes in primordial germ cells (PGCs) and endodermal cells. Expression of indicated genes was analyzed by reverse transcription–polymerase chain reaction (RT–PCR) with PGCs (A) and endodermal cells (B) isolated from stage-28 embryos injected with Venus-DS and indicated mRNA. Primer and PCR conditions are indicated in Table S1. DEX, dexamethasone treatment. No band was detected in any negative control without reverse transcription (data not shown). At least three independent experiments showed the same result.

Discussion

Primordial germ cells of Xenopus laevis are established as those that migrate actively through endodermal cells toward gonads from stage 24 to stage 41. We revealed that spatiotemporal perturbation of Notch/Su(H) signaling in PGCs and somatic cells affected PGC migration in Xenopus embryos, and that at least one of the Notch ligand genes, X-delta-2, is required for this process. Notch signaling has crucial roles in a wide range of developmental systems, including maintenance of germ line stem cells in developed gonad (Terry et al. 2006; Song et al. 2007). The present work is the first report of the role for Notch signaling on germ cell development before gonad formation.

Suppression or activation of Notch signaling was expected to cause the opposite effect as reported in neurogenesis (Wettstein et al. 1997); however, they resulted in similar defects in PGC migration. This may indicate that a certain level of Notch/Su(H) activity might be required. If so, too low and too much activity of the signal might interfere with normal migration. Alternatively, PGC migration might be regulated by a Notch/Su(H)-mediated feedback loop, which could be broken by inhibition or hyperactivation of Notch signaling.

Injection of X-Delta-1 MO caused severe defects in elongation and subsequent morphogenesis of the endoderm. X-delta-1 MO may work via an unidentified mechanism other than inhibition of the Notch/Su(H) pathway, because continuous inhibition of Su(H) in the vegetal region from the one-cell to tailbud stage never resulted in similar defects (data not shown).

Specific effects of X-Delta-2 MO on PGC migration is in accordance with the expression of X-delta-2 in PGCs. X-Delta-2 expressing PGCs probably interact with nearby endodermal cells that express Notch. The interaction may cause the change of interrelation between the two types of cells, finally leading to the PGC migration.

Venus-labeled PGCs in vivo can be observed and traced around stage 32, when the trunk of embryos becomes thin. As shown in Figure 4, PGCs in normal embryos migrate very rapidly, whereas PGCs in most embryos perturbed in Notch signaling cannot migrate or change their position. These findings suggest that the PGC migration mechanism may include directionality, motility and/or adhesion, based on nearby Notch-mediated interaction between PGCs and endodermal cells.

Constitutive activation of the NICD induced expression of several HES and HES-related genes, presumably direct targets, in the PGCs and endodermal cells, whereas we could not detect any genes that were clearly downregulated by Su(H) inhibition. Expression of the HES and HES-related genes activated by endogenous Notch/Su(H) signaling could have been below the detection level. As HES and HES-related genes have redundant activities in several systems (Iso et al. 2003), the NICD-regulated HES-family genes might have redundant roles in the regulation of PGC migration. It is conceivable that Notch/Su(H)-mediated cell interaction modulates gene expression in PGCs and somatic cells, which in turn regulates directly or indirectly the PGC motility required for directional migration.

Acknowledgments

This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan to MM. We thank Drs Orii, Sugiura and Taniguchi and other members of the laboratory for discussions and their support.

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