XB-ART-12017Development December 1, 1999; 126 (23): 5327-37.
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Spatial and temporal properties of ventral blood island induction in Xenopus laevis.
PubMed ID: 10556058
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a bmp2 bmp4 bmp7.1 chrd.1 eef1a2 hba3 kcnt1 myod1 nog otx2 post tal1 tbx2 ventx1.2 ventx2.2 wnt8a
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|Fig. 1. Revised Xenopus blastula fate map (Lane and Smith, 1999).|
|Fig. 2. aT3 globin expression in Xvent-1 or Xvent-2 RNA- or pCSKA-Xwnt-8-injected embryos. Fertilized eggs were microinjected at 1-cell stage with either (A) 1.0 ng of GFP RNA, (B) 1.0 ng of Xvent-1 RNA, (C) 4.0 ng of Xvent-2 RNA or (D) 100 pg of pCSKA-Xwnt-8. Injected embryos were cultured until control embryos reached stage 37/38. aT3 globin expression was detected by in situ hybridization. Representative embryos are shown. Anterior is to the left for all embryos.|
|Fig. 3. Xvent-1, Xvent-2 and Xwnt-8 have a posteriorizing, not ventralizing, effect. Eggs were microinjected with 1.0 ng of Xvent-1 RNA, 4.0 ng of Xvent-2 RNA, 1.0 ng of Xvent-1 plus 4.0 ng of Xvent-2 RNAs, or 100 pg of pCSKA-Xwnt-8, and cultured until uninjected control embryos reached stage 30/31 (A), or 37/38 (B). RNA was isolated and analyzed by northern blotting for the expression of Xpo, myoD, otx2, aT3 globin and muscle actin. EF1a serves as a loading control.|
|Fig. 4. Quantitative analysis of the northern blots. Relative expression levels of Xpo, myoD, otx2, muscle actin and aT3 globin in response to injection of Xvent-1 RNA (Xvent-1), Xvent-2 RNA (Xvent-2), Xvent-1 plus Xvent-2 RNAs (1+2), or pCSKA-Xwnt-8 (Xwnt-8) are shown. Optical densities of the bands from the northern blots were calculated using NIH Image software. The values, after adjusting for total RNA loading by EF1a hybridization intensity, are shown for two independent experiments. (A) Xpo, (B) otx2, (C) aT3 globin, (D) myoD and (E) muscle actin at stage 30/31. Expressions of (F) aT3 globin and (G) muscle actin at stage 37/38 are also shown.|
|Fig. 5. SCL expression in Xvent-1 RNA, Xvent-2 RNA or pCSKA-Xwnt-8-injected embryos. Fertilized eggs were microinjected with 1.0 ng of GFP RNA, 1.0 ng of Xvent-1 RNA, 4.0 ng of Xvent-2 RNA or 100 pg of pCSKA-Xwnt-8, and the injected embryos were assayed for SCL expression at stage 24 by (A) in situ hybridization, and (B) northern analysis. (A) Xvent-1-injected embryos showed the greatest degree of variation and examples of elevated (left), control-like (center) and reduced (right) levels of expression are shown. (C) For Xvent-1 injection, optical densities of bands from northern blots were calculated using NIH image software, and the values are shown for four independent experiments after adjusting for total RNA loading by EF1a hybridization intensity.|
|Fig. 6. aT3 globin expression in posterior marginal zone explants. (A) Procedure for labeling and isolation of posterior marginal zone explants. Both B4 blastomeres were injected with 50 pg each GFP and lacZ RNAs at the 32-cell stage. At stage 9 (late blastula) posterior marginal zones were dissected under fluorescence, using GFP expression to direct dissection of an explant containing B4 progeny. The dissected explants were cultured under coverslips until control embryos reached stage 32. (B) Representative posterior marginal zone explants were stained for b-galactosidase activity and aT3 globin expression by in situ hybridization.|
|Fig. 7. Effects of inhibitors of the BMP signaling on aT3 globin expression in whole embryos. 32-cell-stage Xenopus embryos were injected in a single B1, C1, C4 or A4 blastomere with 500 pgs of noggin and GFP RNAs, 500 pgs of chordin and GFP RNAs or 500 pg of GFP RNA alone as a control, and also injected in the single A4 blastomere with 50 pg of pCSKA-noggin and 500 pg of GFP RNA. Other embryos were injected in both C1, C4 or A4 blastomeres with 500 pgs of Cm-XBMP-7 and GFP RNAs or 500 pgs of dnBMP-R and GFP RNAs. The injected embryos were allowed to develop to stage 37/38, at which time RNAs were isolated and analyzed by northern blotting for aT3 globin expression. EF1a serves as a loading control. (A) noggin-injected embryos; (B) chordininjected embryos; (C) Cm-XBMP-7-injected embryos; (D) dnBMP-R-injected embryos.|
|either BMP-2, BMP-4 or BMP-7 to rescue the phenotype caused by Cm-XBMP-7 (Nishimatsu and Thomsen, 1998). Similarly, the dominant negative BMP-4 receptor has broad inhibitory activity (Suzuki et al., 1994). Thus, we cannot identify the particular molecules responsible for the epidermisderived BMP activity, although several reports indicate a role for heterodimers, particularly BMP-4/BMP-7, in Xenopus mesoderm induction and patterning (Nishimatsu and Thomsen, 1998; Suzuki et al., 1997). A prepattern in the posterior marginal zone? Previous experiments have shown that excised posterior marginal zones express globin (Kelley et al., 1994), and that they can be induced to express muscle actin if combined with a Spemann Organizer or exposed to Noggin or other BMP inhibitors (Lettice and Slack, 1993; Smith et al., 1993). Our result has shown that the globin expression in these explants is restricted to the vegetal pole, despite the fact that BMPs are widely expressed in the posterior marginal zone and ectoderm (Hemmati-Brivanlou and Thomsen, 1995; Schmidt et al., 1995). These results argue against the hypothesis that all|