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Fig. 1. Expression and protein sequence of XGadd45-γ. (A) Vegetal view; (B–F,I) dorsal views; (G) ventral view; (H,J,K) lateral views. (A) XGadd45-γ is expressed in the dorsal mesoderm (arrowhead) at early gastrula stages (stg). Throughout gastrulation, XGadd45-γ is also expressed in scattered cells of the animal region (not shown). (B) At mid-late gastrula, three rows of cells at each side of the midline start to accumulate XGadd45-γ mRNA (arrowheads). (C) These rows of XGadd45-γ expressing cells are clearly visible in the early neurula (black arrowheads). XGadd45-γ is also expressed in forming somites (arrows) (Jen et al., 1999) and in a horseshoe-shaped domain within the anterior neural plate (white arrowhead). This pattern is similar to that of XDl1 (inset). (D) A similar expression profile is observed at mid neurula stages. (D, inset) Transverse section of a mid neurula revealing XGadd45-γ expression in three neuroectodermal rows of cells (black arrowheads), in the notocord (white arrowhead) and in somites (arrow). (E) Mid neurula doubly hybridized for XGadd45-γ and N-tubulin and shown after the first chromogenic reaction to detect XGadd45-γ mRNA (cian). (F) After second chromogenic reaction to detect N-tubulin (purple). Insets in (E) and (F): high magnifications views of the lateral neural stripe pointed at with an arrowhead in their corresponding panels. During neurula stages, XGadd45-γ is also expressed in the prospective cement gland (G, arrowhead), trigeminal placode (H, arrowhead) and in scattered cells in the non-neural ectoderm (H, arrow). XGadd45-γ expression is detected in the same sites at later stages (I). In late neurula (J) and tailbud stages (K), XGadd45-γ is expressed in territories in which secondary neurogenesis takes place, such as the eye (arrows) and brain (arrowheads). (L–M) Hindbrain transverse sections of tailbud embryos showing XGadd45-γ (L) and XDl1 (M) domains of expression (arrowheads). (N) Amino acid sequence alignment of Xenopus and human Gadd45-γ proteins (accession numbers AJ414384 and AF078078, respectively). Identical residues are indicated by dashes.
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Fig. 2. Proneural genes activate and Notch signaling represses XGadd45-γ. Dorsal (A–D) and lateral (E–G) views of midneurula embryos. Embryos were injected with 1 ng of the indicated mRNAs in one blastomere at the two-cell stage together with 0.3 ng of lacZ mRNA. The injection side was determined by X-Gal staining. Xngnr1 (A) and XNeuroD (B) mRNA promote ectopic XGadd45-γ expression (arrowheads). Increased (C) or decreased (D) Notch signaling, respectively, represses or activates XGadd45-γ expression (compare uninjected and injected sides, pointed at with white and black arrowheads, respectively). XGadd45-γ expression in scattered cells of the non-neural ectoderm (E) is also repressed (F) or activated (G) when Notch signaling is increased or decreased, respectively (arrowheads).
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Fig. 5. Xiro1 or -3 mRNAs similarly affect neural markers. All panels show dorsal views of midneurula embryos. (A,D,G,J,M,P) Wild type embryos. (B,E,H,K,N,Q) MT-Xiro3 mRNA (100 pg) injected embryos. This mRNA was coinjected with lacZ mRNA (300 pg) and the injection side was determined by X-Gal staining. (C,F,I,L,O,R) MT-Xiro1 mRNA (0.5–1 ng) injected embryos. In these embryos, the injected site was determined by anti-myc staining. (A) Xiro1 (blue) and Xngnr1 (purple) expression domains overlap (arrowhead). (B,C) Misexpression of MT-Xiro3 (B) or MT-Xiro1 (C) mRNAs promoted ectopic Xngnr1 expression (arrowheads). (D) The medial (M) and intermediate (I) N-tubulin stripes (purple) are located at the border of the Xiro1 domain (blue). Inset: transverse section (arrowheads point at the M and I rows of neurons). (E,F) Ectopic expression of MT-Xiro3 or MT-Xiro1 mRNAs repressed N-tubulin (uninjected side, white arrowhead; injected side, black arrowhead). (G) The intermediate stripe of XHairy2A (purple, arrowhead) is within the Xiro1 domain (blue). The overlapping domains of these genes are verified in transverse sections (inset, arrowhead). (H,I) XHairy2A is overexpressed in embryos injected with MT-Xiro3 or MT-Xiro1 mRNAs (arrowheads). (J) The intermediate stripe of XZic2 (purple) is also within the Xiro1 territory (blue) (arrowheads in whole mount and section, inset). (K,L) MT-Xiro3 or MT-Xiro1 mRNA promote ectopic XZic2 (arrowheads). (M) The medial and intermediate XDl1 stripes (purple) are located at the border of the Xiro1 domain (blue). (N,O) XDl1 is repressed by MT-Xiro3 or MT-Xiro1 misexpression (the stripes pointed at with a white arrowhead in the uninjected side are absent in the injected side, black arrowhead). In these embryos, XDl1 is also activated in other regions (arrow in N). (P) Similarly to XDl1, XGadd45-γ (purple) medial and intermediate stripes are located at the border of Xiro1 territories (blue). (Q,R) MT-Xiro3 or MT-Xiro1 mRNAs repress XGadd45-γ in the stripes territory (black arrowheads, compare with the stripes in the non-injected side, white arrowheads) and activate it in other regions (arrows).
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Fig. 3. XGadd45-γ overexpression antagonizes cell division. Animal views of stage 11 embryos injected with 0.5 ng of the indicated mRNAs and/or 10 ng of the indicated morpholino oligonucleotide and 0.3 ng of lacZ mRNA. Embryos were in situ hybridized for lacZ mRNA (blue) and stained for anti PH3 as a mitotic marker. (A) Distribution of mitotic cells in stage 11 control embryos injected with lacZ mRNA alone. (B,C) Expression of XGadd45-MT (B) or MT-XGadd45 (C) reduces the number of dividing cells. (D,E) Injection of MOGadd45 (D) or a control morpholino oligonucleotide (E) did not affect the number of dividing cells. (F,G) Reduction of mitotic cells promoted by XGadd45-MT mRNA is not affected by coinjection of a control morpholino (F) but is antagonized by coinjection of MOGadd45 (G). (H) MOGadd45 cannot interfere with MT-XGadd45 promoted cell cycle arrest.
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Fig. 4. XGadd45-γ overexpression causes a mild increase of cell death. Animal views of stage 11 (A–F) and dorsal views of stage 15 (G,H) embryos injected with 0.5 ng of the indicated mRNAs and/or 10 ng of the indicated morpholino oligonucleotide. (A–D) Tunel staining to label apoptotic cells. (A) Uninjected embryos showed no apoptotic cells. (B) In a few embryos injected with XGadd45-MT mRNA several dying cells were detected. (C,D) This effect was antagonized by coinjection of MOGadd45 (C), but not by a control morpholino (D). (E) The Myc epitope was detected in XGadd45-MT injected embryos. The XGadd45-MT protein was present both in the nucleous and in the cytoplasm (lower inset). Upper inset shows a western blot to detect Myc in XGadd45-MT injected embryos. Note the presence of both a control cross-reacting protein (upper band) and the XGadd45-MT protein (lower band). (F) In embryos coinjected with XGadd45-MT mRNA and MOGadd45 the Myc epitope was undetectable both in whole mount and in western blot (inset). However, the control cross-reacting protein (upper band, inset) is present.(G,H) N-tubulin expression is not affected in embryos injected with XGadd45-MT mRNA (G) or MOGadd45 (H).
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Fig. 6. Overexpression of HD-GR-E1A mRNA affects neural markers. Dorsal views (A–F) or lateral views (G–I) of neurula stage embryos injected with 0.5 ng of HD-GR-E1A mRNA with (G–I) or without (A–F) 0.3 ng of LacZ mRNA. Dexamethasone was added at stage 12.5 (A–F) or at stage 13.5 (G) to avoid suppression of neural plate formation caused by interference with Xiro function at gastrula stages ( Gómez-Skarmeta et al., 2001). The injected side was determined by anti-Myc staining (A–F) or XGal staining (G–I). (A) Sox2 is not affected. However, Xngnr1 (B), N-tubulin (C) and XGadd45-γ (D) expressions are suppressed (arrowheads). Coinjection with Xngnr1 mRNA (1 ng) rescued N-tubulin (not shown) and XGadd45-γ (E, arrowhead) expression. In embryos injected with 0.5 ng of HD-GR-E1A mRNA, XDl1 (F) is overexpressed. (G) This activation was also observed when protein synthesis was blocked. (H,I) No ectopic XDl1 was observed in injected embryos in the presence of CHX and in the absence of the hormone (H), or just in the absence of the hormone (I).
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Fig. 7. Model of Xiro function in primary neurogenesis. See text for details.
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