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???displayArticle.abstract??? Xdazl is an RNA component of Xenopus germ plasm and encodes an RNA-binding protein that can act as a functional homologue of Drosophila boule. boule is required for entry into meiotic cell division during fly spermatogenesis. Both Xdazl and boule are related to the human DAZ and DAZL, and murine Dazl genes, which are also involved in gamete differentiation. As suggested from its germ plasm localization, we show here that Xdazl is critically involved in PGC development in Xenopus. Xdazl protein is expressed in the cytoplasm, specifically in the germ plasm, from blastula to early tailbud stages. Specific depletion of maternal Xdazl RNA results in tadpoles lacking, or severely deficient in, primordial germ cells (PGCs). In the absence of Xdazl, PGCs do not successfully migrate from the ventral to the dorsal endoderm and do not reach the dorsal mesentery. Germ plasm aggregation and intracellular movements are normal indicating that the defect occurs after PGC formation. We propose that Xdazl is required for early PGC differentiation and is indirectly necessary for the migration of PGCs through the endoderm. As an RNA-binding protein, Xdazl may regulate translation or expression of factors that mediate migration of PGCs.
Fig. 1. Xdazl protein is expressed in the germ plasm of Xenopus embryos. (A) Vegetal view of albino 4 cell embryos lacking Xdazl immunoreactivity. (B) Bisected (upper) and intact (lower) albino stage 10.5 embryos stained with purified antibodies to Xdazl. Arrows indicate Xdazl-stained PGCs below the blastocoel (bl, upper embryo) and in the yolk plug (lower embryo) (C) Bisected (upper) and intact (lower) albino stage 10.5 embryos stained with antiserum depleted of Xdazl antibodies. (D). Bisected (left) and intact (right) stage 13 embryos stained for Xdazl and cleared in Murray. Arrows indicate stained PGCs in the endoderm below the archenteron (ar). Anterior is toward the left. (E) Bisected stage 18 cleared embryo showing Xdazl-stained PGCs (arrows). Anterior is to the left. (F) Stage 7 section immunostained for Xdazl showing reactivity in the germ plasm (gp). The vegetal pole (vp) is toward the bottom. (G) Section through a stage 10.5 embryo showing perinuclear Xdazl staining (arrow), nucleus, n. (H) Control section from a stage 10.5 embryo incubated with antiserum depleted of Xdazl antibodies showing unlabelled germ plasm. (I) The same section in H stained with Heindenhain Azan confirming the presence of germ plasm (arrow, dark blue) around a nucleus (n, pale blue). (J) Stage 13 section with Xdazl-stained germ plasm. Scale bar, 50 μm.
Fig. 4. PGCs are reduced in number or absent in Xdazl-depleted early tadpoles (stage 39/40). (A) RT-PCR analysis of Xpat RNA levels in individual uninjected (Un), oligo injected (Oligo) or rescued (Oligo+Xdazl) stage 39 embryos. EF1-alpha was assayed as a loading control. (B-F) Whole-mount in situ hybridization for Xpat RNA in uninjected (B, D) and Xdazl-depleted (C,E,F) stage 39/40 embryos. (B) Uninjected control embryos showing numerous Xpat- labelled PGCs in the posterior dorsal endoderm (arrowheads).
(C) Xdazl-depleted embryos showing examples of reduced (arrowhead) or absent Xpat-labelled PGCs. (D) Close up view of PGCs in an uninjected tadpole. (E) Close up of an oligo-injected tadpole showing only four PGCs. (F) Example of an oligo-injected tadpole with no PGCs.
Fig. 5. PGC migration from the ventralendoderm is impaired in Xdazl-depleted, late tailbud (stage 35) embryos. (A-I) Whole-mount in situ hybridization for Xpat RNA. (A) uninjected control embryo. (B) Xdazl- depleted embryo. (C) Close up view of the embryo in A showing Xpat-labelled PGCs in the process of migration. Note that many are equal or dorsal to level of the gut tube (dashes). (D) Section of the embryo in C showing position of PGCs in the lateralendoderm (arrowhead). (E) Close up of the Xdazl-depleted embryo in B. Xpat-labelled cells are located exclusively below the level of the gut tube (dashes). (F) Section through the embryo in E demonstrating the ventral location of PGCs (arrowhead). (G) Uninjected embryo from another experiment showing distribution of Xpat- labelled PGCs (arrowhead). (H) Xdazl-depleted embryo in which PGCs are absent. (I) Xdazl-depleted embryo rescued with injected Xdazl RNA. Note the restoration of migration and Xpat expression (arrowhead). (J) RT- PCR analysis of Xpat expression in stage 35 embryos (3 embryos pooled for each lane). Scale bar, 150 μm.
Fig. 6. Germ plasm aggregation and perinuclear positioning occurs normally in Xdazl-depleted embryos. Uninjected (A,C,E) or Xdazl- depleted embryos (B,D,F) were fixed at stage 10, sectioned and stained with Heindenhain Azan (A-D), or were fixed at stage 12 for Xpat whole-mount in situ hybridization and subsequently sectioned (E,F). (A) Section of an uninjected stage 10 embryo showing PGCs (arrowhead) in the vegetal endoderm. (B) Section of an Xdazl- depleted stage 10 embryo with a PGC in the vegetal endoderm (arrowhead). (C) High magnification view of the germ plasm (gp) from one of the PGCs in A. (D) High magnification view of the PGC in (B) demonstrating perinuclear germ plasm accumulation in Xdazl- depleted embryos. (E) Xpat staining of germ plasm in an uninjected stage 12 embryo. (F) Xpat staining in the germ plasm of an Xdazl- depleted stage 12 embryo showing two adjacent PGCs. (G) RT-PCR analysis of Xpat expression in individual stage 12 and 26 embryos showing equal RNA abundance in uninjected and oligo-injected samples. Scale bars, 625 μm (A,B), 80 μm (C,D), 50 μm (E,F).
Fig. 7. Clustering of PGCs in Xdazl-depleted tailbud embryos (stage 26). Whole-mount in situ hybridization for Xpat in uninjected (A,C) and Xdazl-depleted embryos (B,D). (A) Uninjected embryos showing spaced distribution of Xpat-labelled PGCs (arrowheads) in the posteriorendoderm. (B) Xdazl-depleted embryos, arrows indicate groups of tightly clustered PGCs. (C) Section of an uninjected embryo showing position of a PGC in the endoderm. (D) Section of a Xdazl-depleted embryo. nt, neural tube, g, gut tube. Scale bar,
150 μm
