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???displayArticle.abstract??? Germ plasm is found in germ-line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria-line). Germ plasm with EGFP-labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ-line cells in Dria-line females. Using the Dria-line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.
Fig. 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 photo- graphed, 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) Tailbudembryo. (L′, L′′) Dissociated cells from the endodermal area of a tailbudembryo. PGCs with strong EGFP expression elongated for proba- ble migration (arrows). Scale bars represent 200 lm (A-E, H, A′-E′) or 50 lm (F-F′′, G-G′′, J-J′′, L, L′) or 100 lm (I, I′) or 1 mm (K). EGFP signals in Figure (a) are dotted and EGFP signals in Figure (c) are four separated signals.
Fig. 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 lm (A), 100 lm (B-H, J, B′-H′, J′) or 1 mm (I, I′).
Fig. 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 het- erozygous and wild-type offspring. In germ-line cells, EGFP expression was observed until St. 56 and then disap- peared. The EGFP protein of heterozy- gous individuals was again expressed in females at St. 60, and in males at sexual maturation. (B) Mating of trans- genic 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 heterozy- gous individuals in females and males is indicated by red line and blue dotted lines, respectively.
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. (CâCâ³, DâDâ³) Confocal images show that the germ plasm near the cell membrane was situated separately from nucleus before reorganization (C, Câ², Câ³) and surrounded the nucleus after reorganization (D, Dâ², Dâ³). Arrows indicate the nucleus. Scale bars represent 50 um.
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 5â10 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. (BâBâ³,CâCâ³, DâDâ³) 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 (BâBâ³). α-tubulin expanded toward the nucleus ahead of the germ plasm during reorganization (CâCâ³). α-tubulin and germ plasm were colocalized in the perinuclear space after reorganization (DâDâ³). (EâEâ³, FâFâ³) 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 (EâEâ³). γ-tubulin and germ plasm were colocalized in perinuclear space after reorganization (FâFâ³). Scale bars represent 50 μm.
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 (Aâ²) and nanos1 (Bâ²) mRNAs, and Xdazl protein (Câ²) were reorganized together with EGFP (AâC) in the absence of inhibitor. The germ plasm-specific molecules (Dâ²âFâ²) remained at the cell membrane as well as EGFP (DâF) in the presence of Nocodazole. Scale bars represents 50 μm.
