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In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline-Mt). However, the role of germline-Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline-Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C-terminal mitochondrial transmembrane domain, inhibited the aggregation of germline-Mt during early development. In Rhot1-inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail-bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.
Figure 1.
Distribution of germline-Mt at the early blastula stage. (a) Schematic representation of the molecular structure of Rhot1. (b–d) Continuous observation of germline-Mt in Mito-EGFP embryos. EGFP fluorescence indicates the distribution of mitochondria. Germline-Mt was observed from st.2 to st.7 in control (b), Rhot1δC-injected (c) and Rhot1-injected embryo (d). Scale bar, 125 μm. (e) Distribution of germline-Mt at st.5. Distribution patterns were divided into the following three types: normal aggregation (green), abnormal aggregation-N (pink, narrow stripes), and abnormal aggregation-D (gray, diffusion of small islands). Biological replicates (N) are 4. (f–h) Size of the aggregated germline-Mt mass. Red line shows the length of largest germline-Mt mass from the crossing point of cleavage furrows at the vegetal pole of Rhot1- (f) or Rhot1 δC-injected embryo (g). Scale bar, 300 μm in (f, g). Average length of largest germline-Mt mass was calculated from the germline-Mt mass of more than 40 embryos (h). **P < 0.01 (Student's t-test). Biological replicates (n) = 4.
Figure 2.
Immunostaining of mitochondria-associated glutamate oxaloacetate transaminase 2 (GOT) in the vegetal side of st.5 embryos. Localization of germline-Mt was detected by using anti-GOT antibody in control (a), Rhot1δC-injected (b), and Rhot1-injected embryo (c). (a–c) Low magnification images of GOT distribution. White box regions are shown at higher magnification in the fluorescent field (a'–c') and bright field (a”–c”). Arrows indicate the aggregated germline-Mt mass. Arrowheads indicate dispersed germline-Mt mass. Scale bar, 250 μm (a–c) and 100 μm (a'–c', a”–c”). (d) The indicated homogenates were subjected to SDS–PAGE in duplicate. The transferred membranes were immunostained with anti-GOT (upper) and without anti-GOT (middle) antibodies. The other gel was stained with Coomassie Brilliant Blue (CBB) to confirm sample loading (lower). The arrowheads show GOT at about 47.6 kDa.
Figure 3.
Gene expression of germinal granule components. (a–f) Whole-mount in situ hybridization of Xpat and nanos1 in control (a,d), Rhot1δC-(b,e), and Rhot1-injected embryos (c,f) at st.5. a”–f” show EGFP images of germline-Mt in the same region of a'–f', which were taken before fixation. Scale bar, 500 μm. (g) RT–PCR analysis of Xpat and nanos1 expression in control, Rhot1δC- and Rhot1-injected embryos at st.5. odc, internal control. RT(−), without reverse transcriptase reaction.
Figure 4.
Effect of Rhot1δC on PGC formation. (a) Pimordial germ cell (PGC) alignment at the genital ridge at st.40 embryos. PGCs were visualized by co-injection of DsRed2-DEADSouth3′UTR (230 pg) (Magenta) in wild-type embryos. Degree of PGC formation were graded as +++ (normal PGC formation), ++, + (one-three PGCs) and–(no PGC). Scale bar, 200 μm. (b) Ratio of four-graded embryos for PGC formations at the genital ridge. Mann–Whitney U-test was used to test for significance. (c–e) Distribution of PGCs in st.35 embryos co-injected with mRNA of DsRed2-DEADSouth3′UTR (c), DsRed2-DEADSouth3′UTR plus Rhot1δC (d) or DsRed2-DEADSouth3′UTR plus Rhot1 (e). (c'–e') High magnification views of the white boxes in (c–e). Scale bar, 1 mm. (f) Total number of PGCs in a single embryo at st.35. Total number of PGCs was counted from both sides of embryo. **P < 0.01 (Student's t-test).
Figure 5.
Effects of Rhot1δC on aggregation of germline-Mt. (a–d) Mt-rich cells were dissociated from control (a, a'), Rhot1δC-injected (b,b',c,c'), and Rhot1-injected embryos (d,d') at st.10.5. a–d, bright field. a'–d', fluorescent field showing localization of germline-Mt. Arrows, multiple germline-Mt in a single cell. Arrowheads, small volume of germline-Mt. Scale bar, 100 μm. (e) Distribution patterns of germline-Mt in Mt-rich cells. Dissociated cells were classified into three types: cells containing germline-Mt at the perinucleus (blue), cortex (pink), and abnormal region (gray). The three types of dissociated cells were counted at st.10.5 and the percentages were calculated. In rescue experiments, 900 pg of full-length Rhot1 mRNA was co-injected with an equal volume of Rhot1δC mRNA. Mann–Whitney U-test was used to test for significance (**P < 0.01). N indicates injected embryo numbers. n indicates total Mt-rich cell numbers. (f–i) In situ hybridization of Xpat in Mt-rich cells containing germline-Mt from control embryos (f,f'), Rhot1δC-injected embryos (g,g'), Rhot1-injected embryos (h,h') and endodermal cells (Endo) from control embryos (i,i') at st.10.5. f'–i' show EGFP images of germline-Mt in the same cells as f–i, which were taken before fixation. Arrowheads show small volume of germline-Mt localized at cortex. Scale bar, 100 μm.
