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Fig. 1. The vegetal localization element of XDE mediates germ-line GFP expression by somatic mRNA clearance and PGC-specific RNA accumulation. (A–E) Two-cell-stage embryos were injected either vegetally with reporter mRNAs, cultivated to stage 30–34 and monitored for GFP protein expression (Left), or fixed and analyzed for GFP reporter RNA distribution (Right). PGC-specific GFP protein expression (magnified views in insets) was observed upon injection of 1.2–1.8 ng GFP-XDE 3′UTR (A, 69%, n = 52, N = 2), 1.2 ng GFP-XDE-LE (B, 73%, n = 74, N = 2), and 1.1 ng GFP-XDE-LE-R (C, 71%, n = 28, N = 1). In contrast, ubiquitous GFP protein expression was exhibited by 100% of embryos injected with 1.5 ng GFP-XDEδLE (D, n = 28, N = 1) or 93% of embryos injected with 1.6 ng GFP-XDEδLE-R (E, n = 14, N = 1). GFP RNA was specifically detected in PGCs in 72% (A, n = 44, N = 1) for XDE 3′UTR-injected embryos, in 78% (B, n = 82, N = 2) for XDE-LE-injected embryos and 82% (C, n = 74, N = 1) for XDE-LE-R-injected embryos. (D and E) High endodermal levels of GFP RNA were detected in embryos injected with XDEδLE (D, 97%, n = 113, N = 3) and XDEδLE-R (E, 100%, n = 69, N = 3). (F) Aspect of a single embryo injected with GFP-XDE-LE and double-stained for GFP (red) and the PGC marker Xpat (purple), photographed after the first (Left) and the second (Right) staining reaction. Although the time for the Xpat staining reaction was reduced, the majority of mGFP5-positive cells also exhibit Xpat signals. n, no. of injected embryos; N, no. of experiments.
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Fig. 2. Somatic RNA clearance is mediated by LEs of other vegetally localized mRNAs expressed in the germ line. (A–F) GFP-tagged LE reporter mRNAs of Xcat2 (A, 800 pg), XDazl (B, 800 pg), Xpat (C, 800 pg), VegT (1.2 ng), Vg1 (1.2 ng), Dead end (XDE-LE, 1.2 ng), or lacZ-tagged LE mRNAs (2 ng each) of the DeadSouth isoform (D) Velo76, XNIF, Velo 40, and Velo7 were injected vegetally into both blastomeres of two-cell-stage embryos. Embryos (stage 30–32) were analyzed for reporter RNA distribution by in situ hybridization using antisense GFP or lacZ probes. (G) LEs of the majority of vegetally localized mRNAs expressed in the germ line were found to mediate germ-line restriction of reporter RNAs irrespective of their transport pathway. Values for PGC stabilization indicate the percentage of embryos exhibiting PGC-specific reporter RNA staining (Fig. S5). Asterisk indicates that for Vg1 (E), VegT (F), and Velo7, PGC staining was detectable in <10% of injected embryos and accompanied by high somatic levels or reporter RNA (100%, 98.6%, and 46.7% of injected embryos, respectively).
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Fig. 3. Antisense morpholino oligonucleotide-mediated protection of the nanos1 homology element strongly inhibits somatic clearance of XDE-LE. (A) Partial alignment of XDE-LE-R with the zebrafish nanos1 3′UTR reveals a homologous sequence element (XDE nucleotide positions indicated) covering the dre-miR-430b target site (orange box). Identical nucleotides of XDE-LE-R are highlighted in black. The XDE-LE-R is represented by a red rectangle; the position of the nos1 homology domain is marked by a blue box. (B and C) Vegetal coinjection of antisense MOs (1.5 pmol each) with GFP-XDE-LE-R reporter mRNA (1.1 ng) led to inhibition of somatic reporter RNA clearance to variable extent; the strongest effect was achieved by MO7 targeting the nos1 homology region (Table S1). (B) Lateral view of representative embryos stained with a GFP antisense probe. (C) Diagram representing average pixel quantification results of in situ hybridization signals from embryos analyzed on both sides. The statistical significance of the obtained series of values was verified by one-tailed Student's t-test (type 3, P < 0.01; P values: LE-R/MO1: 2.695E-50, LE-R/MO2: 1.299E-14, LE-R/MO3: 0.004, LE-R/MO4: 7.53E-06, LE-R/MO5: 1.133E-47, LE-R/MO6: 3.216E-45, LE-R/MO7: 3.949E-57). Values were adjusted to the score of GFP-XDE-LE alone, which was set to 1.
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Fig. 4. Somatic clearance of XDE-LE-R reporter RNA is achieved by a microRNA-mediated mechanism. (A) Schematic overview of mutated or deleted reporter constructs. A blue rectangle marks the nos1 homology region; altered positions are marked by asterisks; mutations within the miR target motif are represented in red letters. (B) Mutation of the miR-430 target motif resulted in decreased somatic clearance of the XDE-LE reporter. Upon vegetal injection (2/2 cells) of 1.2 ng XDE-LE mRNA or XDE-LE-seedmut mRNA, somatic levels of reporter mRNAs were ranked visually into four classes: 0 (not detectable, as exemplified in D, Upper Left), 1 (weak; example in D, Lower Right), 2 (medium; example in C, Right), and 3 (very high; example in D, Lower Left). (C) Embryos injected with 1.1 ng XDE-LE-R mRNA or XDE-LE-Rδ-1352–81 mRNA were scored similarly. Partial deletion of the nos1 homology domain also resulted in decreased somatic clearance. (D) Vegetal coinjection of antisense 2′OMeOs amiR-130b (α130b) or amiR-301 (α301) (6.4 pmol each) with 1.1 ng of XDE-LE-R reporter RNA resulted only in slight elevation of somatic reporter RNA levels, whereas coinjection of a mixture of the antisense 2′OMeOs amiR-18a and amiR-18b (α18a/b) (3.2 pmol each) led to strong somatic stabilization of coinjected reporter RNA (Table S1). (E) Comparative diagram of somatic reporter RNA levels (%) scored in the injected embryos; in all cases, embryos compared were stained in parallel for the same time.
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Fig. 5. Elr protein binding protects XDE-LE reporter RNA from somatic clearance. (A) UV cross-linking assay using radiolabeled XDE-LE-MO hybrids. 32P-labeled XDE-LE RNA was prehybridized with the antisense MOs indicated and incubated with fractionated oocyte extract before UV irradiation. Cross-linked proteins were separated by SDS/PAGE; the positions of molecular weight markers are indicated. (B–D′) Embryos were injected vegetally (2/2) with 800 pg of wild-type XDE-LE (B and B′, n = 78; N = 1) or mutant reporter mRNAs XDE-LE mut1 (C and C′, n = 71; N = 1) and XDE mut2 (D and D′, n = 88; N = 1). (B′, C′, and D′) Magnified aspects of single embryos marked in B, C, and D. Injection of mutant XDE-LE reporter mRNAs deficient for Elr binding resulted in decreased intensities (B′, C′, and D′) and frequencies of PGC staining (11.3% for XDE-LE mut1, 12.5% for XDE mut2) compared with the wild-type XDE-LE (83.3% PGC staining). (E) Both blastomeres of two-cell-stage embryos were injected vegetally with 1.1 ng of XDE-LE-R reporter mRNA alone or together with 200 pg of MT-ElrB1 mRNA or 400 pg of MT-ElrA mRNA and scored for somatic reporter RNA levels as described (Fig. 4). Coexpression of ElrB1 led to strong somatic stabilization of XDE-LE-R, whereas only slightly elevated levels of somatic reporter levels were detected upon coinjection of ElrA. (F) Diagram of somatic reporter RNA levels (%) scored in ElrB1/ElrA-coninjected embryos. n, no. of injected embryos; N, no. of experiments.
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Fig. 6. ElrB1 and Xenopus Dead end proteins act synergistically in the somatic stabilization of XDE-LE reporter mRNA. (A–E) Whereas vegetal coinjection of FL-XDE mRNA (100 pg, B) or low levels of MT-ElrB1 mRNA (50 pg, C) with GFP-XDE-LE-R reporter mRNA (1.1 ng) led to no traceable or moderate somatic protection of XDE-LE-R, respectively, coexpression of both proteins (same amounts of injected mRNAs) strongly enhanced the stabilizing effect (D and E). (E) Comparison of somatic reporter RNA levels (%) scored in the injected embryos.
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