EGFP expression in germ plasm and germ-line cells
To trace the behavior of germ plasm in development, we generated transgenic Xenopus, Dria-line, harboring EGFP labeled mitochondria (see 'Materials and methods' for details). We examined EGFP expression during the life cycle, using F1 from crosses between Dria-line females and wild-type males. Enormous mitochondria exist in the animal hemisphere (Kogo et al. 2011), but EGFP expression of animal hemisphere was covered by the surface melanin pigments in St. 1 to St. 8 embryos. Whereas punctate EGFP signals were widely distributed around the vegetal pole of St.1 embryos and very weak signals were observed at most of the vegetal hemisphere (Fig. 1A). They were aggregated, forming strong fluorescent patches near the vegetal pole of St. 3, 6, 8 and 11 embryos (Fig. 1B–E). These strong EGFP signals displayed germ plasm, because they were colocalized with Xpat mRNA, a germ plasm-specific mRNA, at any stage examined (Fig. 1A′–E′). Other germ plasm-specific molecules were also colocalized with EGFP in germ plasm (cf. Fig. 6).
Figure 1. Enhanced green fluorescent protein (EGFP)-labeled mitochondria in germ plasm (A–E, A′–E′) Localization of EGFP and germ plasm specific molecule, Xpat mRNA, of St. 1, 3, 6, 8 and 11 embryos (a vegetal view). After EGFP signal of living embryos were photographed, whole-mount in situ hybrydization of Xpat mRNA was performed. Punctate EGFP signals of a St. 1 embryo are indicated by a black circle (A). The aggregated EGFP signals of germ plasm are indicated by black arrows (B–E). Xpat expression is associated with strong EGFP signals (A′–E′). (F–F″, G–G″) Sections of an unfertilized egg double-stained with antibodies against EGFP and mitochondria-specific glutamate oxaloacetate transaminase2 (GOT). Abundant mitochondria in animal hemisphere are stained similarly with anti EGFP (F) and anti-GOT antibodies (F′). Aggregated mitochondria in germ plasm are detected in the cell cortex with both antibodies (G, G′, G″). (H) Neurula. (I′, I″) Dissociated cells from the endodermal area of neurula. EGFP expression in primordial germ cells (PGCs) is very strong and that in somatic cells were very weak. (J–J″) A probably PGC with strong EGFP expression in the dissociated cells is positively stained with anti-Dazl antibody. Note the surrounding endodermal cells with weak EGFP expression are not stained with anti-Dazl antibody. (K) Tailbud embryo. (L′, L″) Dissociated cells from the endodermal area of a tailbud embryo. PGCs with strong EGFP expression elongated for probable migration (arrows). Scale bars represent 200 μm (A–E, H, A′–E′) or 50 μm (F–F″, G–G″, J–J″, L, L′) or 100 μm (I, I′) or 1 mm (K). EGFP signals in Figure (a) are dotted and EGFP signals in Figure (c) are four separated signals.
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EGFP was surely expressed in mitochondria, because EGFP signals in the unfertilized eggs were colocalized with mitochondrial signals detected by mitochondria-specific anti-GOT antibody. In the animal hemisphere, mitochondria were abundant (Fig. 1F–F″). In the vegetal hemisphere, aggregated mitochondria showing germ plasm at the cell cortex was contrasted with a paucity of mitochondria in the inner vegetal cytoplasm (Fig. 1G–G″).
After St. 11, the endoderm including the germ plasm was enclosed by surface ectoderm. The EGFP became stronger around this stage (Fig. 1E). Finally germ plasm-containing cells (PGCs; primordial germ cells) resided inside of embryos. The trunks of neurula and tailbud embryos were dissociated into cells. The PGCs were distinguishable from numerous endodermal cells by EGFP expression: the former with strong signals and the latter with weak signals throughout the cell. PGCs in neurula expressed EGFP strongly in the perinuclear space and weakly throughout the cytoplasm (Fig. 1H, I, I′). Probable PGCs in dissociated cells judged from EGFP expression were positively stained with anti-Dazl antibody (Fig. 1J–J″). In the tailbud stage, The PGCs with strong signals showed the migrating and elongated forms (Fig. 1K, L, L′ arrows).
Furthermore, EGFP expression was examined in the later stage. Gonads with mesonephros were dissected from larvae and froglets. In St. 46 larvae, positive cells were found in gonads on the mesonephros (Fig. 2A, B, B′). EGFP expression gradually weakened in PGCs after St. 50 (data not shown). At St. 56, when sexual difference was distinguishable by pattern of pigment distribution, EGFP disappeared completely in all PGCs of both sexes (Fig. 2C, C′, F, F′). On the other hand, in somatic cells, EGFP expression was observed (see mesonephros behind the gonad). Then, at St. 60, the time of metamorphic climax, punctate signals were again expressed strongly in ovaries (Fig. 2D, D′ arrow), but not in testes (Fig. 2G, G′). The ovary formed several corrugations at St. 63, and contained a number of EGFP-positive cells (Fig. 2E, E′). EGFP signal was not detected in testes (Fig. 2H, H′). In females at 8 months post-fertilization, EGFP expression was strong throughout the ovary (Fig. 2I, I′). Pre-stage I and stage I oocytes were present and they contained strong EGFP signals in the precloud or mitochondrial cloud (Fig. 2J, J′). In male froglets, the EGFP level in testes was much lower than that in the surrounding somatic cells. After sexual maturation, EGFP was expressed weakly in the periphery of the testicular lobules (data not shown).
Figure 2. Enhanced green fluorescent protein (EGFP) expression in germ-line cells during late development. (A) Low magnification image of a part of the gonads on mesonephros dissected from a St. 46 embryo. (B, B′) High magnification images of a region corresponding to the white square in (A). Mesonephros is characterized by heavy pigmentation (A). EGFP-positive cells (arrow in B′) were present in gonads (white tissue in the central line). (C–H, C′–H′) High magnification images of gonads dissected from St. 56, St. 60 and St. 63 embryos. Females (C, D, E) and males (F, G, H). EGFP disappeared completely in all primordial germ cells (PGCs) at St. 56 (C′, F′). The EGFP signal was again expressed strongly in ovaries at St. 60 (white arrows in D′), but not in testes (G′). White tissue of ovary including pigments (E) and testis (H) proliferated remarkably at St. 63. EGFP-positive cells were increased in ovary (E′), but were not detected in testis (H′). (I, I′) Strong EGFP expression in the ovary dissected from froglets at 8 months postfertilization. (J, J′) The strong signal in the mitochondrial cloud of pre-stage I and stage I oocytes isolated from the same ovary. Scale bars represent 200 μm (A), 100 μm (B–H, J, B′–H′, J′) or 1 mm (I, I′).
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Maternal expression of transgene in germ-line
All mature oocytes laid by a Dria-line heterozygous female expressed the EGFP protein, translated from the maternal mRNA, and then all Dria-line eggs were fertilized by a wild-type sperms (Fig. 3A). Distinction of embryos with and without zygotic expression was difficult because of the maternal expression shortly after mid-blastula transition (MBT), but became clear at St. 27, when half of the embryos began to express strong EGFP protein in mitochondria-rich cells of epidermis (Brown et al. 1981). The other half gradually lost the EGFP signal from outside view because of a lack of zygotic expression (Fig. 3A, left panel). However, all individuals with and without the transgene similarly kept EGFP expression in germ plasm, and gradually lost EGFP until St. 56. In the case of individuals with transgene, EGFP expression in germ-line cells again began in oocytes of female gonads from St. 60 (Fig. 3A, right panel).
Figure 3. Developmental changes of enhanced green fluorescent protein (EGFP) expression in germ-line cells. (A) Mating of transgenic heterozygous females and wild-type males produced eggs with EGFP expression: both heterozygous and wild-type offspring. In germ-line cells, EGFP expression was observed until St. 56 and then disappeared. The EGFP protein of heterozygous individuals was again expressed in females at St. 60, and in males at sexual maturation. (B) Mating of transgenic heterozygous males and wild-type females produced fertilized eggs without EGFP expression. In germ-line cells of heterozygous females, the EGFP protein was expressed after St. 60. EGFP expression of heterozygous individuals in females and males is indicated by red line and blue dotted lines, respectively.
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Mating of a Dria-line heterozygous male and a wild-type female produced fertilized eggs all without EGFP expression (Fig. 3B left panel). In somatic cells, half of the embryos with transgene began to express strong EGFP from St. 27 as mentioned above and the other half without transgene did not express EGFP. In germ-line cells of embryos with transgene, it was at St. 60 that the EGFP first began to express in oocytes of female gonads (Fig. 3B right panel, data not shown).
In germ-line, cytomegalovirus promoter works limitedly during oogenesis, and very weakly throughout spermatogenesis. In somatic line, it may work after MBT, though obvious expression of EGFP was observed from neurula.
Reorganization of germ plasm at gastrulation
Histological observation reported that germ plasm is reorganized to the perinuclear space from the cell cortex shortly before beginning of gastrulation (Whitington & Dixon 1975; Akita & Wakahara 1985). The reorganization of germ plasm at this stage is of particular interest, because it may be the beginning of close interaction between the germ plasm and nucleus, leading to the formation of PGCs. By live imaging of germ plasm, we first analyzed the participation of cytoskeleton to the reorganization of germ plasm. To determine when germ plasm is reorganized, about 10 embryos were fixed around the gastrula stage with a 2% paraformaldehyde solution and dissociated into cells. The PGCs were collected (2–9 cells per embryo) and the intracellular distribution of germ plasm was examined. Cells with little or dispersed germ plasm was ruled out, and only the cells clearly distinguished as PGCs were used. The germ plasm was condensed near the cell membrane (M-pattern) or expanded towards the center of the cell (C-pattern). The ratios of cells of M- or C-pattern to the total cells were indicated (Fig. 4A). At St. 8, 93.9% of cells were M-pattern and 6.1% were C-pattern, at St. 9, 55.0% were M-pattern and 45.0% were C-pattern, and at St. 10, 11.8% were M-pattern and 88.2% were C-pattern. The reorganization of germ plasm was shown to occur at St. 9, just before gastrulation as was proposed by histological observation (Whitington & Dixon 1975; Akita & Wakahara 1985). Next, we dissociated St. 8 living embryos into cells and observed the behavior of the germ plasm. The germ plasm was somewhat expanded near the cell membrane just after dissociation. It then gradually expanded towards the center of the cell, and surrounded the nucleus after reorganization (Fig. 4B). Even in the dissociated cells, reorganization of germ plasm occurred at a similar timing and frequency, as in the intact embryo (data not shown). It was found that the reorganization of germ plasm proceeded in most cases without cell division, and in a few cases accompanying cell division. Confocal images showed that the germ plasm was situated near the cell membrane, separately from nucleus before reorganization, and surrounded the nucleus after reorganization (Fig. 4C–C″, D–D″).
Figure 4. The reorganization of germ plasm around St. 9. (A) Distribution pattern of germ plasm in the dissociated cells from the fixed embryos around St. 9. The cells containing germ plasm were classified into two types. M-pattern cells had germ plasm located near the cell membrane, and C-pattern cells had germ plasm expanded towards the cell center. The number of M- or C-pattern cells was counted and their ratios to the total cells were indicated. The germ plasm is reorganized frequently at St. 9. (B) Time-lapse images of germ plasm during the reorganization of a living dissociated cell. (C–C″, D–D″) Confocal images show that the germ plasm near the cell membrane was situated separately from nucleus before reorganization (C, C′, C″) and surrounded the nucleus after reorganization (D, D′, D″). Arrows indicate the nucleus. Scale bars represent 50 μm.
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Requirement of microtubule polymerization for the reorganization of germ plasm
In general, the cytoskeleton plays an important role in the subcellular transport of organelles and mRNA. We examined whether the reorganization of germ plasm is driven by microfilament or microtubule system. The cells were dissociated from about 5–10 embryos at St.8 and only the PGCs with M-pattern were isolated, and treated with inhibitors. After 5 h treatment, the ratios of cells of M- or C-pattern to the total cells were indicated (Fig. 5A). Germ plasm was hardly reorganized in the presence of nocodazole (89.8%), an inhibitor of microtubule polymerization. In the cells treated with cytochalasin D, an inhibitor of actin polymerization, germ plasm was reorganized at the same level as the control (cytochalasin D: 83.3%, methanol: 91.1%). Additionally, to examine localization of germ plasm and microtubule components, we performed immunocytochemistry staining for α-tubulin and γ-tubulin. α-tubulin was colocalized with germ plasm throughout the reorganization process (Fig. 5B–B″, C–C″, D–D″). γ-tubulin resided in two separate areas before the reorganization: two centrioles near the nucleus, and germ plasm (Fig. 5E–E″). After the reorganization, almost all γ-tubulin resided in the perinuclear space (Fig. 5F–F″).
Figure 5. The reorganization of germ plasm is dependent upon microtubules. (A) The ratio of reorganized cells after treatment with cytoskeleton inhibitors. The primordial germ cells (PGCs) with only M-pattern were isolated from about 5–10 embryos at St. 8, and treated with Nocodazole (Noc) or cytochalasin D (CytoD) for 5 h. The ratios of reorganized cells to the total cells were indicated. The ratio of dissociated cells with the reorganized germ plasm was decreased in the presence of Noc relative to control cells. The ratio was not affected by CytoD. (B–B″,C–C″, D–D″) The dissociated cells during reorganization were stained with antibody against α-tubulin. α-tubulin colocalized with germ plasm located near the cell membrane before reorganization (B–B″). α-tubulin expanded toward the nucleus ahead of the germ plasm during reorganization (C–C″). α-tubulin and germ plasm were colocalized in the perinuclear space after reorganization (D–D″). (E–E″, F–F″) The dissociated cells during reorganization were stained with antibody against γ-tubulin. Most of γ-tubulin was located together with germ plasm near the cell membrane and a little in the perinuclear space (E–E″). γ-tubulin and germ plasm were colocalized in perinuclear space after reorganization (F–F″). Scale bars represent 50 μm.
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Then, reorganization of other germ plasm specific molecules was studied. Together with mitochondria (Fig. 6A–C), germ plasm-specific molecules, Xpat mRNA (Fig. 6A′), nanos1 mRNA (Fig. 6B′) and XDazl protein (Fig. 6C′), were also reorganized. In cells treated with nocodazole, these molecules remained at the cell membrane (Fig. 6D–F, D′–F′). Mitochondria and germ plasm-specific molecules were retained by microtubules and transported towards the nucleus by the extending microtubules.
Figure 6. Localization of germ plasm-specific molecules in dissociated cells. After enhanced green fluorescent protein (EGFP) signal of living dissociated cells from St. 8 embryos were photographed, in situ hybrydization of Xpat and nanos mRNAs was performed or immunostained with antibody against XDazl. Germ plasm-specific Xpat (A′) and nanos1 (B′) mRNAs, and Xdazl protein (C′) were reorganized together with EGFP (A–C) in the absence of inhibitor. The germ plasm-specific molecules (D′–F′) remained at the cell membrane as well as EGFP (D–F) in the presence of Nocodazole. Scale bars represents 50 μm.
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