Figure 9. <strong>noggin</strong> In Situ Hybridization Whole embryos were hybridized with antisense <strong>noggin</strong> RNA probes. Hybridization was visualized by an alkaline phosphatase chromogenic reaction. Arrowheads in (C) and (D) indicate dorsal lip <strong>of</strong> the blastopore. (A) Stage 9, vegetal pole view. Staining restricted to wedge on dorsal side <strong>of</strong> embryo. (6) Stage 9, side view. Staining exclusively in dorsal marginal zone. (C) Stage 10.5, side view. Hybridizing cells found in dorsal lip <strong>of</strong> the blastopore.
<strong>noggin</strong> Rescues Dorsal Development 837 Xwnt-8 (Smith and Harland, 1991); here we have described a novel gene that is equally effective in dorsal rescue. Members <strong>of</strong> the wnt family and <strong>noggin</strong> are the only two types <strong>of</strong> molecules known that have the ability to rescue complete dorsal development in ventralized embryos. Preliminary results indicate that at least one additional dorsalizing activity may be present in our library. However, the identities <strong>of</strong> any additional dorsalizing activities remain to be determined. Our preliminary experiments eliminated only the contributions from <strong>noggin</strong> and Xwnt-8 to the dorsalizing RNA transcribed from the library and did not exclude possible activities from other members <strong>of</strong> the wnt family or from <strong>noggin</strong>-related clones (if they exist). Although the results from the screening <strong>of</strong> our library clearly indicate that axis-rescuing activity is not restricted to only one type <strong>of</strong> molecule, the time and location <strong>of</strong> <strong>noggin</strong> expression suggest that it participates in the normal process <strong>of</strong> axis formation at blastula and gastrula stages. In addition, as would be expected for a molecule that is involved in cell-cell signaling, the <strong>noggin</strong> sequence prediets a secreted polypeptide. In contrast to Xwnt-8, <strong>noggin</strong> mRNA is present both maternally and zygotically, and is present in tissues that are known to be sources <strong>of</strong> dorsalinducing factors. Although maternal <strong>noggin</strong> transcripts do not appear to be localized within the cleaving embryo, zygotic transcription is localized to the dorsal mesoderm. Zygotic transcripts are detected in the dorsal marginal zone; this tissue involutes as part <strong>of</strong> the dorsal lip <strong>of</strong> the blastopore and becomes the notochord and head mesoderm (prechordal plate). Thus, <strong>noggin</strong> is expressed in the Spemann organizer and its descendant cells. A spatially separate and later phase <strong>of</strong> <strong>noggin</strong> expression initiates after the pattern <strong>of</strong> the tadpole is established; in the swimming tadpole, <strong>noggin</strong> transcripts are found in the ro<strong>of</strong> plate <strong>of</strong> the neural tube and in a variety <strong>of</strong> probable neural crest derivatives. (reviewed by Gerhart et al., 1989; Smith and Harland, 1991). Thus, <strong>noggin</strong> mRNA is most effective at rescue when injected into vegetal blastomeres <strong>of</strong> the cleaving embryo, and lineage tracing confirms that injected vegetal blastomeres populate the endoderm <strong>of</strong> the rescued embryo. Because the injection can be shown to confer an early-acting rescue, these experiments do not address the possibility that <strong>noggin</strong> protein may also act later at the gastrula and neurula stages. The location <strong>of</strong> zygotic <strong>noggin</strong> expression suggests that it participates in activities <strong>of</strong> the Spemann organizer, namely, neural induction and dorsalization <strong>of</strong> ventral mesoderm. The development <strong>of</strong> the organizer is dependent upon zygotic transcription initiating after the midblastula transition. A number <strong>of</strong> other genes have recently been characterized that are specifically turned on in the organizer following the start <strong>of</strong> zygotic transcription, ineluding goosecoid (Blumberg et al., 1991; Cho et al., 1991), Xlim-7 (Taira et al., 1992), and XFK/-/l (Dirksen and Jamrich, 1992; Ruiz i Altaba and Jessel, 1992). In contrast to these genes, which all encode nuclear transcription factors, <strong>noggin</strong> encodes a secreted protein and could potentially mediate the inductive activities<strong>of</strong> the Spemann organizer. The later localization in the notochord suggests that <strong>noggin</strong> could be involved in induction <strong>of</strong> the floor plate <strong>of</strong> the neural tube (Yamada et al., 1991). In the tadpole, expression in the neural ro<strong>of</strong> plate and neural crest suggests yet other trophic or differentiating activities. Production <strong>of</strong> recombinant <strong>noggin</strong> protein will allow us to address the question <strong>of</strong> whether responsiveness to <strong>noggin</strong> persists to the gastrula (or later) stages, and if so, how these older tissues respond to <strong>noggin</strong> treatment. Is <strong>noggin</strong> an Endogenous Inducer <strong>of</strong> Dorsal Development <strong>noggin</strong> mRNA is present throughout dorsal development and is expressed in tissues that have been identified by The Role <strong>of</strong> Zygotic <strong>noggin</strong> Transcripts dissection assources<strong>of</strong> inducing activity (i.e., dorsal vege- Because <strong>noggin</strong> was isolated from dorsalized gastrula tal cells in the blastula, dorsal lip in the gastrula, and noto- RNA (from LiCI-treated embryos), and because these em- chord in the neurula). We have demonstrated that exogebryos contain increased amounts <strong>of</strong> <strong>noggin</strong> transcript rela- nous <strong>noggin</strong> mRNA has dorsalizing activity when injected tive to controls, it is not surprising that gastrula transcripts into cleavage stage embryos. Injected <strong>noggin</strong> mRNA can are dorsally localized. However, injection assays into blas- substitute for the formation <strong>of</strong> the Nieuwkoop center in a tula embryos revealed dorsalizing activity <strong>of</strong> exogenous ventralized embryo, but it is not clear if endogenous<strong>noggin</strong> <strong>noggin</strong> via the Nieuwkoop center, the early-acting source transcript performs the same role in normal development. <strong>of</strong> dorsal signals that resides in the vegetal hemisphere The amount <strong>of</strong> maternal <strong>noggin</strong> mRNA is significantly (D) Stage 10.5, vegetal pole view. Staining is restricted primarily to the dorsal side <strong>of</strong> the embryo. Faint staining can also be seen extending around the lateral and ventral sides <strong>of</strong> the embryo. (E) Stage 10.5, LiCI-treated, vegetal pole view. Strong <strong>noggin</strong> hybridization encircles the embryo as a result <strong>of</strong> dorsalizing treatment (LiCI). (F) Stage 10.5, W-treated, vegetal pole view. Only background staining was detected in the ventralized embryo. The sharp circle is the outline <strong>of</strong> the blastocoel. (G) Stage 18, dorsal view. Detectable staining along dorsal midline in notochord and prechordal plate. The anterior end <strong>of</strong> the embryo is to the left. Arrow indicates the anterior limit <strong>of</strong> the notochord. (H) Stage 18, side view. Note the anterior extent <strong>of</strong> <strong>noggin</strong> expression (to the left). Staining cells extend beyond the anterior limit <strong>of</strong> the notochord into the presumptive head mesoderm. (I) stage 40, dorsal view. A line running along dorsal midline is stained as well as the mandibular and gill arches in the head, These appear as bilateral patches <strong>of</strong> stain anterior lo the eye (mandibular arch) and “fingers” <strong>of</strong> stain behind the eye. Staining in the lens and in the pharynx (between the eyes) is not specific. (J) Stage 40, side view. Broken line <strong>of</strong> staining dorsal cells extending along the anterior-posterior axis corresponds to the ro<strong>of</strong> plate <strong>of</strong> neural tube. Staining can still be detected in the posterior tip <strong>of</strong> the notochord. The speckled appearance <strong>of</strong> the tailfin is due to stained stellate cells.
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