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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02207

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Ras-dva, a member of novel family of small GTPases, is required for the anterior ectoderm patterning in the Xenopus laevis embryo

¹ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, Moscow, Russia.
² Moscow State University, Moscow, Russia.

View larger version (41K): [in a new window]   Fig. 1. Phylogenetic tree of the superfamily of small GTPases. Phylogenies were reconstructed using a neighbor-joining algorithm and p-distance model (p=nd/L, where nd=number of amino acids different between two aligned sequences and L=number of sites compared). The bar sets a value of p. Thus, p-distance value in the pairing of XlRas-dva: with another protein, e.g. with GgRas, is 0.65; with XlRab1A or XlRho is 0.69; with XlRan is 0.79; with GgArf1 is 0.85; with GgRbj is 0.78; with GgGem is 0.73; with GgRas-dva is 0.44; and with XtRas-dva is 0.06. Initials before the protein name represent respective organisms, as follows: Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Xt, Xenopus tropicalis; Dr, Danio rerio. Every colored area indicates an individual family of small GTPases.

View larger version (73K): [in a new window]   Fig. 2. Normal expression patterns of Ras-dva and its transcriptional regulators, the homeobox genes Otx2 and Xanf1, in the gastrula and neurula stage Xenopus embryos as revealed by whole-mount in situ hybridization. All embryos are shown from the anterior, dorsal side upwards. Broken line indicates the neural plate border. (A) At midgastrula (stage 11) the Ras-dva is diffusely expressed within a broad territory that includes the presumptive anterior neural and non-neural ectoderm. (B) At late gastrula (stage 12.5) the Ras-dva expression weakens in the area corresponding to the anterior neural plate but it increases in the surrounding area. (C) At the midneurula (stage 14) Ras-dva is expressed in cells of the non-neural anterior ectoderm and in the lateral neural folds. (D-F) Normal expression patterns of the homeobox genes Otx2 and Xanf1. Although Ras-dva and Xanf1 are expressed in mutually excluding domains, the expression domains of Ras-dva and Otx2 are largely overlapping. (G,H) The results of in situ hybridization in halves of Xenopus embryos on the 12.5 and 14 stages. Left halves show the expression of Otx2 gene, right halves show Ras-dva expression. (I) The Xenopus embryo (stage 13) in which the left half is stained for Xanf1 expression and the right half is stained for Ras-dva expression. (J) The scheme of expression patterns of Ras-dva, Otx2 and Xanf1 at the midneurula stage (stage 14).

View larger version (106K): [in a new window]   Fig. 3. Effects of Otx2 and Xanf1 over-expression on Ras-dva expression at the neurula stage (stage 14). (A,A') Microinjection of the Otx2 mRNA into the right side of the embryo results in lateral and ventral expansion (red arrow) of the Ras-dva expression area on this side. (B,B') Overexpression of Xanf1 leads to an inhibition of the Ras-dva expression (black arrows). (C,C') The dominant repressor version of Xanf1 (EnR-Xanf1) inhibits Ras-dva expression at the microinjected side (black arrows). (D,D') The dominant activator version of Xanf1 (VP16-Xanf1) induces expansion of the Ras-dva expression area at the microinjected side (red arrows). (E,E') The dexamethasone-inducible version of Otx2 (Otx2-BDGR) elicits lateral expansion (red arrows) of the Ras-dva expression area when protein synthesis was completely inhibited by cycloheximide and dexamethasone treatment. This indicates that Ras-dva is the direct target of Otx2 in cells of the anterior ectoderm. (F,F') No expansion of the Ras-dva expression domain is detected in embryos, microinjected with Otx2-BDGR and treated by the cycloheximide solution alone. (G,G') The dexamethasone-inducible version of activating Xanf1 variant (VP16-Xanf1-BDGR) stimulates lateral expansion (red arrow) of the Ras-dva expression area when protein synthesis was completely inhibited by cycloheximide and dexamethasone treatment. This indicates that Ras-dva is the direct target of Xanf1 in cells of the anterior ectoderm. (H,H') No expansion of the Ras-dva expression domain is detected in embryos microinjected with VP16-Xanf1-BDGR and treated by the cycloheximide solution alone.

View larger version (98K): [in a new window]   Fig. 4. Ras-dva functioning is essential for the development of the head structures. (A) The head of control tadpole, as seen from the dorsal side. (B) The head of a tadpole developed from the embryo microinjected with the anti-Ras-dva morpholino oligonucleotides (MO) into the right blastomere at the two-cell stage. As a result of inhibition of translation of the Ras-dva mRNA by the morpholino, the tadpole has a reduced eye, telencephalon, olfactory pit and otic vesicle. (C) The head of the tadpole developed from the embryo microinjected by dnRas-dvaT22N mRNA into the right blastomere at the two-cell stage. The malformations are similar to those described above that were caused by anti-Ras-dva MO microinjections. This indicates that the dnRas-dvaT22N construct works. (D,E) The rescue experiment. Co-injection of anti-Ras-dva MO with synthetic Ras-dva mRNA lacking the MO binding sequence (E) is able to rescue the effects of anti-Ras-dva morpholino oligonucleotides (D).

View larger version (67K): [in a new window]   Fig. 5. Downregulation of Ras-dva functioning by the anti-Ras-dva morpholino or Ras-dva dominant-negative mutant, DN-Ras-dvaT22N, leads to an inhibition of anterior ectoderm markers expression. (A,B) The expression of the neural crest markers Sox9 and Slug is inhibited on the side of embryos microinjected with DN-Ras-dvaT22N mRNA. (C) By contrast, the microinjection of DN-Ras-dvaT22N mRNA resulted in a broadening of the expression domain of the neural border marker Msx1. (D) No effect on the expression of posterior neural marker HoxB9 was observed on the side microinjected with DN-Ras-dvaT22N mRNA. (E-J) The expression of markers of the neural plate (G, Otx2; H, Pax6), the anterior neural ridge (E, BF-1), the cranial placodes (G, Otx2;H, Pax6; F, Xag2) and the neural crest (J, Slug; I, Sox9) is inhibited on the side of embryos microinjected with the anti-Ras-dva morpholino. (K,L) Rescue of anti-Ras-dvaMO effects by co-injection of synthetic Ras-dva mRNA. The inhibited expression of the Slug marker, caused by a break of endogenous Ras-dva mRNA translation (K), can be restored by co-injection of a synthetic Ras-dva construct lacking the MO binding site (L). This result confirms the specificity of anti-Ras-dva MO effects. All embryos are shown from the anterior, dorsal side upwards. The `primed' counterpart pictures show the location of progenies of the microinjected blastomeres labeled by FLD tracer.

View larger version (33K): [in a new window]   Fig. 6. Representative RT-PCR analysis of marker gene expression in animal cap explants induced to anterior neural differentiation by microinjection of the Otx2 or Noggin mRNA in conditions of Ras-dva functioning downregulation. (A,B) Ras-dva downregulation by co-injection of the anti-Ras-dva MO or DN-Ras-dvaT22N mutant mRNA inhibits induction by Otx2 (A) or Noggin (B) of the neural plate and cranial placodal markers (BF-1, Otx2 and Xag-2) but promotes expression of the neural inhibitors (BMP-4, Vent-1, Vent-2B). Expression of the pan-neural marker NCAM does not change when Ras-dva is downregulated.

View larger version (51K): [in a new window]   Fig. 7. Ras-dva downregulation, in contrast to Ras, can interrupt FGF8a signaling in the anterior ectoderm. (A) Two crescent-shaped stripes of the FGF8a expression in the anterior ectoderm of the Xenopus neurula (left half) are exactly coincident with the anterior and posterior borders of the Ras-dva expression domain (right half). (B-D) Ras-dva downregulation, in contrast to Ras, interferes with FGF-8a signaling in anterior ectoderm in Xenopus embryos. (B,B') FGF-8a mRNA injection induces ectopic BF-1 expression (red arrow). (C,C') Co-injection of FGF-8a and DN-Ras-dvaT22N RNAs results in inhibition of BF-1 expression in the injected area (black arrow). (D,D') Co-injection of DN-RasS17N with FGF8a did not affect the BF-1 activating signal from FGF8a (red arrow). These results were revealed by whole-mount in situ hybridization in neurula stage Xenopus embryos. All embryos are shown from the anterior, dorsal side upwards.

View larger version (111K): [in a new window]   Fig. 8. Ras-dva downregulation, in contrast to Ras, is unable to interrupt FGF signaling in the posterior regions of the embryo. (A,A') Microinjection of DN-RasS17N results in inhibition of the mesoderm marker Brachyury (Bra) around the blastopore at the midgastrula stage (black arrowheads). (B,B') By contrast, DN-Ras-dvaT22N has no influence upon Bra expression.

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