Expression Cloning of noggin, a New Dorsalizing Factor Localized ...

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Cell 634 4 4 LacZ F + noggin obtained with embryos that were tilted 90° immediately following fertilization and then marked with a vital dye on their uppermost side to indicate the future dorsal side (Peng, 1991). Older (32-cell stage) blastula embryos were also dissected into dorsal-ventral and animal-vegetal halves. No enrichment of noggin mRNA in any of the hemispheres was seen relative to the total embryo (data not shown). In addition, UV treatment did not alter the abundance of maternally deposited noggin RNA, indicating no preferential degradation in ventral tissues (Figure 8). Samples with known amounts of in vitro synthesized noggin mRNA were included in the RNAase protection assay (Figure 8). From these and other data we estimate that there is approximately0.1 pg of noggin mRNA per blastula stage embryo and 1 pg per gastrula stage embryo. Figure 5. Dorsal Rescue of 32-Cell Embryos with noggin RNA Ventralized 32-cell stage embryos were coinjected with 25 pg of noggin and 0.5 ng of 6-galactosidase RNAs into a single blastomere in either the animal (tier l), marginal (tiers 2 and 3) or vegetal (tier 4) regions. Embryos were grown to about stage 25 and scored for dorsal axis rescue. halves, as well as animal and vegetal halves. noggin transcripts were found to be distributed evenly between dorsal and ventral hemispheres as well as animal and vegetal hemispheres (Figure 8). The same result (not shown) was In Situ Hybridization; Zygotic Expression of noggin in the Spemann Organizer The localization of noggin transcripts in developing embryos was examined in greater detail using whole-mount in situ hybridization (Harland, 1991). Whole fixed embryos were hybridized with digoxigenin-containing RNA probes. Hybridized RNA probe was then visualized with an alkaline phosphatase-conjugated anti-digoxigenin antibody. The specificity of hybridization seen with antisense noggin probes was tested both by hybridizing embryos with sense Figure 6. Lineage Tracing of noggin-Injected Blastomeres Ventralized embryos (32~cell stage) were coinjected with 25 pg of noggin and 0.5 ng of 5-galactosidase RNAs. The embryos were then stained with X-gal at about stage 25. Diffuse background staining can be seen in the endoderm. Specific staining from injected f3-galactosidase RNA can be seen as darker and more discrete points of staining. Arrows indicate cement glands. Arrowheads indicate staining of lineage tracer.
noggin Rescues 835 Dorsal Development al 5 Stage A. 8 9 11 12 13 14 20 35 6. noggin c-src noggin c-src Control LiCl uv 8 9 10 8 9 10 8 9 10 Figure 7. Expression of noggin RNA in Development (A) A Northern blot of poly(A)’ RNA from embryos of the indicated stages hybridized with both noggin and c-src probes. The amount of c-src hybridization controls for RNA loading differences between samples. (B) A Northern blot of total RNA from late blastula and early gastrula (stage E-10) embryos hybridized with noggin and C-WC probes to assess the effects of UV (ventralization) and LiCl (dorsalization) treatment on noggin RNA expression. noggin probes and by using two nonoverlapping antisense probes (data not shown). Owing to both the low level of expression and background staining, noggin mRNA could not be detected unequivocally before the late blastula stage. The increased level of noggin mRNA that was detected by Northern blot following activation of zygotic transcription (Figure 7) was apparent in in situ hybridization at stage 9 as a patch of staining cells on the dorsal side of the embryo. Viewed from the vegetal pole (Figure 9A), this patch of cells was restricted to a sector of about 60°. A side view of the same embryo (Figure 96) shows that the staining cells were located within the marginal zone (i.e., between the animal and vegetal poles and within the presumptive dorsal mesoderm-forming region). As with other newly activated genes, transcripts are largely restricted to the nucleus at this stage (Smith and Harland, 1991; Frank and Harland, 1991). A side view of an early gastrula stage embryo (approximately stage 10.5) shows specific hybridization primarily noggin+ ‘ - EFla+ Figure 8. Distribution of Maternal noggin RNA in Oocytes and 4-Cell Stage Xenopus Embryos RNAase protection assay for noggin and EFlc in total RNA samples from dissected oocytes and 4-cell stage embryos. Also included in the assay is in vitro synthesized noggin RNA at the amounts indicated. c K in the involuting mesoderm at the dorsal lip (Figure 9C). A vegetal view of the same embryo (blastopore lip indicated with an arrowhead) shows that noggin mRNA is most abundant on the dorsal side, but expression extends at a lower level to the ventral side of the embryo. For reasons that we do not understand, this method of in situ hybridization does not detect transcripts in the most yolky endodermal region of embryos (Frank and Harland, 1992; M. E. Bolce and R. M. H., unpublished data); therefore, we cannot rule out expression in more vegetal regions than those seen in the figure. Treatments that are known to affect the size of the dorsal lip (LiCI treatment, UV irradiation) had a profound effect on the pattern of noggin in situ hybridization. Figures 9D, 9E, and 9F show vegetal pole views of three stage 10.5 embryos either untreated, LiCl treated at stage 6, or UV treated at stage 1, respectively. In LiCItreated embryos the staining is intense throughout the marginal zone. UV treatment reduced the hybridization signal to undetectable levels. This result is consistent with amounts of noggin mRNA seen by Northern blot analysis (see Figure 76). The UV-treated embryo also is a negative control for specificity of hybridization. Asgastrulation proceeds, noggin mRNAstaining follows the involuting dorsal mesoderm and is highest in the presumptive notochord. By the late neurula stage (approximately 18) noggin mRNA-expressing cells are clearest in the most dorsal mesoderm, primarily in the notochord, but also extend more anteriorly into the prechordal mesoderm (Figures 9G and 9H). The anterior tip of the notochord is indicated by an arrow. During tailbud stages, expression of noggin in the dorsal mesoderm declines, though expression in the notochord persists in the growing tailbud. Expression of noggin initiates at several new sites, which become progressively clearer as the tadpole matures. A discontinuous line of stained cells runs the length of the roof plate of the neural tube (Figures 9J and 91). Staining is also apparent in the head mesoderm, primarily in the mandibular and gill arches. We suspect that this expression corresponds to skeletogenic neural crest cells, since the same sites also express twist (Hopwood et al., 1989; R. M. H., unpublished data); furthermore, subsetsof these cells express homeobox genes that mark different anterior-posterior levels of the head neural crest; for example, En-2 in the mandibular arch is seen by antibody staining (Hemmati-Brivanlou et al., 1991) and in situ hybridization (R. M. H., unpublished data). Cells with stellate morphology stained for noggin mRNA in the tail fin. These stellate cells are also likely to be derived from the neural crest. None of these patterns were seen with the sense probe or with a number of other probes (e.g., Xwnt-8). Discussion noggin, a Novel Polypeptide with Dorsalizing Activity in Xenopus Embryos Dorsal structures can be rescued in ventralized embryos by injection of mRNA from dorsalized embryos. Using this assay we have isolated cDNAs that, when transcribed into mRNA and injected into the embryo, can rescue adorsally complete anterior-posterior axis. Previously, we isolated
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