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Fig. 1. Localization of XDead end mRNA in Xenopus oocytes and embryos. The subcellular distribution of the XDead end mRNA was analyzed using albino oocytes or embryos of different stages for whole mount in situ hybridization. In stage I and II oocytes, XDead end RNA is homogeneously distributed (A). In later stages (early and late stage III, stage IV) (A) and stage V and VI (B), the XDead end RNA becomes enriched at the vegetal cortex. (D, E) Two-cell stage embryo exhibiting a granular structure at the vegetal tip of both blastomeres. (F) Vegetal view of an 8-cell stage embryo (G) Vegetal view of a gastrula embryo with XDead end positive cells in the center of the yolk plug (marked by an arrow). (H) Transverse section of a neurula stage embryo exhibiting XDead end expression in scattered cells linking the future gut. (I, I′) XDead end expression in migrating germ cells of a tailbud stage embryo. (J) Temporal expression during embryogenesis and tissue expression of XDead end. RT-PCR analysis with RNA preparations from staged embryos (according to Nieuwkoop and Faber, 1967) and adult tissue respectively (sc, spinal cord; mu, muscle; li, liver; he, heart; in, intestine; sk, skin; pa, pancreas; br, brain; ey, eye; lu, lung; te, testis; ov, ovary). Control RT-PCR reactions with primers specifically for Histone 4 (H4).
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Fig. 2. The 3′-UTR of XDead end contains a vegetal localization element. lacZ-tagged RNAs were injected into stage III–IV albino oocytes and visualized by whole mount in situ hybridization after culturing for 3 days. Vegetal localization was analyzed in oocytes injected with transcripts containing the complete RNA (A), with a fragment containing the 3′-UTR (B), with a fragment containing the open reading frame and 5′-UTR (C), a fragment containing nucleotides 1296–1547 (D), a fragment containing nucleotides 1274–1426 (E), a fragment containing nucleotides 1408–1547 (F) and a fragment containing an internal deletion spanning nucleotides 1296–1547 (G). Section (H) shows the cortical staining of a stage III oocyte that had been injected with a transcript containing the nucleotides 1296–1547 as shown in panel (D). (I) Schematic illustration of the constructs used for XDead end localization element mapping experiments. 5′-UTR, ORF and 3′-UTR are marked with light blue, dark blue and normal blue respectively. Corresponding nucleotide positions of the XDead end cDNA are indicated. The capability of vegetal localization is marked (+), absence of vegetal localization is indicated by (−).
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Fig. 5. XDead end protein is important for primordial germ cell development. (A) In the coupled in vitro transcription/translation system (Promega), XDead end and zDead end respectively were translated with or without different amounts of XDead end specific morpholino antisense oligonucleotide (DE-MO) or with control morpholino as indicated. Two-cell stage albino embryos were injected vegetally into both blastomeres with XDead end specific morpholino antisense oligonucleotide (DE-MO) or control morpholino (CO-MO). Stage 31/32 embryos were fixed and analyzed by whole mount in situ hybridization using a digoxigenin labeled antisense RNA probe for Xpat. Visible Xpat positive primordial germ cells (PGCs) were counted. (B) An uninjected stage 31 embryo showing an average number of Xpat expressing primordial germ cells. (C) Example of a stage 31 embryo injected with DE-MO resulting in a reduced number of Xpat containing primordial germ cells. (D) Example of a DE-MO injected stage 31 embryo with a total loss of Xpat positive primordial germ cells. (E) Percentage of embryos with a normal number of PGC marker positive cells (light blue), less or at least 4 PGC marker positive cells (blue) and no PGC marker expressing cells (dark blue) after DE-MO or control MO injection. Usage of Xpat or Xdazl respectively is indicated above the chart. Amount of injected DE-MO or control MO are indicated below the chart. (F) Partial rescue of the DE-MO effect by coinjection of increasing amounts of zebrafish Dead end-globinUTR mRNA.
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Fig. 6. Xpat expression in DE-MO injected embryos. (A) DE-MO injected embryos were analyzed for Xpat expression at the indicated stages. Data are shown as the number of embryos with the indicated expression out of the total number tested. (B, B′) Control and DE-MO injected stage 11 embryos analyzed for Xpat expression showing normal Xpat expression located in the posterior endoderm. (C, C′) Control and DE-MO injected stage 17 embryos showing Xpat stained PGC in the endoderm. (D) Control embryo (stage 24) showing normal PGC distribution and (D′) DE-MO injected embryos with tightly clustered PGCs. (E) Control stage 27 embryo with spaced distribution of Xpat-labeled PGCs and (E′) in the DE-MO injected embryo PGCs appear slightly clustered.
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pgat (primordial germ cell-associated transcript protein ) expression by in situ hybridization in NF stage 31 Xenopus laevis embryos, anterior left, dorsal up.
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dnd1 (dead end homolog 1) expression by in situ hybridization in NF stage 2 Xenopus laevis embryos. Vegetal view.
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dnd1 (dead end homolog 1) expression by in situ hybridization in NF stage 4 Xenopus laevis embryos. Vegetal view.
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dnd1 (dead end homolog 1) expression by in situ hybridization in NF stage 10 Xenopus laevis embryos. Vegetal view.
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dnd1 (dead end homolog 1) expression by in situ hybridization in stage V and VI oocytes of Xenopus laevis.
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pgat (primordial germ cell-associated transcript protein) gene expression in a Xenopus laevis embryo, assayed by in situ hybridization, NF stage 25/26, lateral view, anterior left, dorsal up
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pgat (primordial germ cell-associated transcript protein) gene expression in a Xenopus laevis embryo, assayed by in situ hybridization, NF stage 11.
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pgat (primordial germ cell-associated transcript protein) gene expression in a Xenopus laevis embryo, assayed by in situ hybridization, NF stage 17.
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pgat (primordial germ cell-associated transcript protein) gene expression in a Xenopus laevis embryo, assayed by in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
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