XB-ART-19855Cell April 7, 1995; 81 (1): 85-94.
Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.
Using an expression cloning strategy that relies on a functional assay, we have cloned a novel Xenopus homeobox-containing gene, Siamois. Embryos injected in a ventral-vegetal blastomere with as little as 5 pg of Siamois mRNA develop a complete secondary axis, but the progeny of the injected cells do not participate in the secondary axis formation. In normal development, Siamois mRNA is first detected shortly after the midblastula transition, which is earlier than mRNAs for goosecoid or Xbrachyury, and is present most abundantly in the dorsal endoderm of early gastrulae. The activation of this gene can be obtained cell autonomously in dispersed embryo cells. These results indicate that Siamois may play an important role in the formation of the Nieuwkoop center.
PubMed ID: 7720076
Article link: Cell
Species referenced: Xenopus
Genes referenced: drosha fgfr1 gsc hdac1 lhx1 mixer nog sia1 tbxt
Article Images: [+] show captions
|Figure 1. Cloning of a cDNA That Can Create a Complete Secondary Axis (A) Schematic map of the pBluescript RN3 vector. (13) Embryo injected with 5 ng of synthetic mRNA from the whole dorsalized gastrula library; note that the secondary axis lacks the most anterior structures. (C) Embryo injected with 50 pg of synthetic mRNA from Siamois cDNA; the secondary axis is now complete with cement gland. Similar embryos were cultured further, until it was possible to see that the secondary heads were morphologically normal (data not shown).|
|Figure 2. Ventral Injection of noggin mRNA Generates Partial Secondary Axes Normal embryos at the 4- to 8-cell stages were injected in a ventralvegetal position with 10 pg of noggin mRNA (A), 200 pg of noggin mRNA (B), or 5 pg of Xwnt-8 mRNA (C). Embryos were fixed at the late tailbud stage. The secondary axes generated by noggin (arrows in A and B) lack a cement gland; those in (C) possess well-developed cement glands (arrows). Note that embryos in (B) and (C) lack posterior structures.|
|Figure 3. Siamois Encodes a Homeobox Protein Similar to mix. 1 and HD1 (A) DNA and deduced protein sequence of the coding region of Siatools. The homeodomain is boxed, and a potential glycosylation site is underlined. (8) Comparison of the homeodomains of Siamois and the two most closely related genes found in the GenEMBL data base, Xenopus mix. 1 and human HD1. The three helices typical of homeodomains are underlined. Percentages of amino acid identity (conservation) are given on the right hand side of the panel. Colons indicate conservative amino acid changes; asterisks indicate amino acids conserved in all homeodomains.|
|Figure 4. Siamois Is Expressed Transiently in the Dorsal Cells of Early Gastrulae (A), (B), and (C) display results of RNase protection assays. In all panels, the fibroblast growth factor receptor (FGF-R) is used as a loading control. (A) Analysis of the temporal expression of Siamois. (B) Expression of Siamois and Xwnt-8 along the dorsoventral axis of early gastrulae (stage 10); D, dorsal tissue; V, ventral tissue. To the right, a diagram shows the regions taken for analysis. (C) Siamois is enriched in the vegetal regions of stage 10.25 embryos: W, whole embryo; M, marginal zone explant; V, vegetal pole explant; A, animal cap explant. The two bottom lines present a quantitation of the signals. Sia, Siamois.|
|Figure 5. Comparison of the Patterns of Expression of Siamois and Xbra on Sagital Sections from Normal or Dorsalized Early Gastrulae Sections of normal (left panels) or dorsalized with lithium (right panels) stage 10 embryos were probed for Siamois (A and D) or Xbra (B and E) transcripts. (C) and (F) show computer generated diagrams of the expression domains of Siamois (green) or Xbra (open red). The position of the blastopore lip is indicated by an arrow in (D) and (F).|
|Figure 6. Siamois mRNA Accumulates Very Rapidly after the Midblastula Transition and Is Activated in Dispersed Embryonic Cells (A) Comparison by RNase protection of the rate of accumulation of mRNAs for the early genes Siamois, mix.l, Xlim-1, gsc, and Xbrs. The constant content of the fibroblast growth factor receptor (FGF-R) mRNA was used as a loading control. The fast development of the embryos (stage 10 was obtained 7 hr after fertilization) was due to incubation at 25°C. p.f., postfertilization. (B) Comparison by RNase protection of the expression of Siamois and Xbra in eggs and in dispersed (D) or whole ON) stage 10 animal caps. Embryos cultured in calcium- and magnesium-free medium from fertilization onward were dissociated from stage 6 to stage 10. The expression of Xbra, Siamois, and the FGF-R gene was assayed. The two bottom lines present a quantitation of the signals obtained. Sia, Siamois.|
|Figure 7. Siamois-Expressing Ventral-Vegetal Cells Induce an Ectopic Spemann Organizer Embryos were injected ventrally at the 4-cell stage with 1 ng of mRNA coding for NLS-~-Gal either alone (A) or in combination with 50 pg of Siamois mRNA (B and C). They were fixed at the tadpole (A and B) or early gastrula (C) stages, stained for I~-Gal activity with X-Gal, and cleared to reveal the position of the stained cells. The diffuse staining throughout the endoderm is due to endogenous ~-Gal activity. The specific NLS-~-Gal staining is nuclear. (A) shows that the progeny of NLS-I~-Gal mRNA injected ventral-vegetal cells is found in the posterior endoderm. (B) shows that the progeny of ventral-vegetal cells injected with Siamois and NLS-I~-Gal mRNA is found in the anterior endoderm. (D) Vegetal view of an early gastrula showing the primary (1 o) and secondary (2 °) dorsal blastopore lips and the position of the progeny of a ventral-vegetal blastomere injected with Siamois and NLS-13-Gal (blue cells underneath the secondary dorsal lip).|