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doi: 10.1242/10.1242/dev.00531

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Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

Department of Molecular and Cellular Biology, University of California at Berkeley, CA 94720, USA

View larger version (43K): [in a new window]   Fig. 1. Neural crest marker induction after recombining ectoderm to mesoderm. (A) The DMZ (blue) or the DLMZ (yellow) are dissected at stage 10-10.5 as depicted. (B) Each type of mesoderm explant is recombined to the animal cap ectoderm (AC, red) of stage 8-9 embryos, to form the DLMZ-AC and DMZ-AC recombinants, respectively. (C) RT-PCR analysis of gene expression in the stage 18 recombinants shows that the DLMZ-AC recombinants express a whole range of neural crest markers at stage 18 (Slug, FoxD3, Sox9, Zic5, Snail and Twist, lane 5), whereas the DMZ-AC recombinants express only a subset of them (lane 7). Lanes 1, 2: controls (see Materials and Methods). Lanes 3, 4, 6: isolated AC, DLMZ and DMZ, respectively. (D) In situ hybridization for the four most specific neural crest markers studied (see text) on normal embryos around stage 18 seen in dorsal view, anterior is towards the bottom. Note that Slug and FoxD3 are restricted to the neural crest, whereas Zic5 and Sox9 are also expressed in other areas.

View larger version (49K): [in a new window]   Fig. 2. Wnt signaling is not required for neural crest induction by the DLMZ. (A) The activity of different Wnt antagonists is monitored by analyzing cement gland formation in stage 20 injected embryos: Wnt antagonism results in a striking enlargement of the cement gland [top panels, control embryos; middle panels, NFz8 mRNA treated (800 pg); bottom panel, GSK3 mRNA treated (800 pg)]. (B-E) Slug expression in explants. (B) DLMZ, (C) AC, (D) AC-DLMZ control recombinants, (E) NFz8 (800 pg) injected AC-DLMZ. The top four recombinants in E are also stained for ß-galactosidase activity. Strong Slug expression is found in the ectoderm of about half of the recombinants in D and E. (F) RT-PCR analysis after injecting increasing amounts of NFz8: lanes 3-6 and 7-10, 0-400-800-1600 pg of NFz8 mRNA injections in AC and AC-DLMZ, respectively. 400-800 pg injections do not block response to DLMZ signals. Lanes 1 and 2, controls (see Materials and Methods); n.i., non-injected. (G) GSK3 (800 pg) or dnTCF3 (1 ng) mRNA injections do not prevent the ectoderm to form neural crest in response to the DLMZ. (H) Xdd1 (1 ng) injections do not prevent neural crest marker induction either. (I) The AC+DLMZ recombinants elongate in the same way as controls even in presence of 400 pg of NFz8 (middle) but their elongation is abolished by injections of 1600 pg of NFz8 in the ectoderm (bottom).

View larger version (59K): [in a new window]   Fig. 3. FGF signaling is required for neural crest induction by the DLMZ. (A) RT-PCR analysis after SU5402 treatment of the recombinants shows the lack of Slug induction, as well as defective paraxial mesoderm development (MA). Lane 3, DMSO treatment; lane 4, SU5402 treatment. (B) XFD injections (500 pg) in the ectoderm prevent normal induction of the most specific neural crest markers Slug and FoxD3 by the DLMZ. Zic5 and Sox9 are still induced. (C) Similar injections with dnFGFR4a (500 pg) do not prevent neural crest marker induction by the DLMZ. (D) Using situ hybridization, the recombinants show a strong downregulation of Slug expression after XFD injections (third panel) but not after dnFGFR4a injections (fourth panel). First panel, XFD-injected animal caps; second panel, control recombinants.

View larger version (63K): [in a new window]   Fig. 4. FGF signaling in the recombinants. (A) FGF3, FGF4 and FGF8 are expressed in the DLMZ cultivated in isolation, from stage 10.25 to stage 14 (lanes 3-6; DLMZ dissected at stage 10.25 and cultivated up to the stage indicated). (B) FGFRs are differentially expressed in the DLMZ-AC (lane 5) and DMZ-AC (lane 7) recombinants. In particular, FGFR1 and FGFR4a expression is maintained at stage 18 in AC-DMLZ recombinants (lane 5). (C) Isolated animal caps do not express FGF3, FGF4 or FGF8. (D) SU5402 treatment does not suppress FGFR1 and FGFR4a expression in the recombinants.

View larger version (58K): [in a new window]   Fig. 5. FGF8 induces neural crest in vivo and in vitro. (A-D) In vivo injections of FGF8 mRNA in one of two-cell stage embryos, analyzed by in situ hybridization for Slug (A,B) or MyoD (C,D) at stage 18-20. (A) Control embryos. (B) FGF8 mRNA unilateral injections result in a strong overexpression of Slug on the injected side (yellow arrows) and sometimes in the contralateral side and the anterior neural fold (red arrowheads). (C) Control embryos. (D) FGF8 mRNA injections (injected side indicated by yellow arrowheads) do not expand paraxial mesoderm, they even reduce it in some embryos (embryo on the right) (red arrowhead). (E) FGF8 mRNA is expressed as a ring around the blastopore at stage 11 (top), reinforced dorsally (red arrows). Later on, FGF8 is expressed in the DLMZ and downregulated in the midline (bottom, red arrow). (F) FGF8 mRNA injections induce neural crest markers in animal caps. RT-PCR analysis shows the induction of FoxD3 and Zic5 by 100 pg of FGF8 mRNA, but not of paraxial mesoderm formation. (G) When the caps are analyzed earlier (stage 15), increased doses of FGF8 induce strongly FoxD3, Sox9 and Zic5. By stage 19, FoxD3 and Zic5 expression was not maintained.

View larger version (79K): [in a new window]   Fig. 6. In vivo analysis of neural crest and neural plate patterning after modification of Wnt signaling. (A,B) Control embryos stained for Slug and Krox20 at stage 19. (C,D) Wnt8 mRNA injections (50 pg) are followed by the extension of Slug and Krox 20 domains together, in the posterior parts of the embryos (arrows). (E,F) dnWnt8 mRNA injections (50 pg) result in reduction of both Slug and Krox 20 expression (arrows).

View larger version (73K): [in a new window]   Fig. 7. In vivo GSK3 injections perturb neural crest, neural plate and mesoderm development. (A-C) Injections in one of two-cell stage embryos of lacZ (A) or GSK3 (300 pg) plus lacZ mRNA (B,C). The GSK3 injected side (white arrow, pink lacZ staining) displays greatly reduced Slug expression (B, red arrow, blue staining), abnormal Krox20 expression (C, red arrow, blue staining) and reduction of the paraxial mesoderm marker 12-101 staining (B,C; black arrows, brown staining). Red and black arrows in A indicate normal staining for Slug (blue) and 12-101 (brown), respectively. (D-F) GSK3 (150 pg) was injected into one dorsoanimal blastomere at the 16-cell stage to target one neural fold and reduce the effect on adjacent tissues. (D) Control ß-galactosidase staining. (E) The injected area shows reduction in both Slug (red arrow) and 12-101 staining (black arrow). (F) Ectopic expression of the anterior neural plate marker Cpl-1 is induced ectopically (red arrow), showing that neural patterning is also affected.

View larger version (69K): [in a new window]   Fig. 8. Neural crest formation can be experimentally uncoupled from neural plate patterning. (A-D) Injected embryos were analyzed around stage 18 and stained for Slug expression. (A) Control embryo. (B) XFD injections result in gastrulation defects and loss of most Slug staining. (C) NFz8 injections most often produce an abnormally shaped neural plate, gastrulation defects and reduced Slug expression. (D) dnFGFR4a-injected embryos show severe gastrulation defects but still present a strong Slug staining (the right hand embryo is shown in side view). (E,F) Similar injections were analyzed at stage 11.5-12 for Otx2 expression. Otx2 labels the area anterior to the neural crest-forming regions. It is found further from the blastopore as development proceeds (blue bars measure the distance between the posterior part of the Otx2 domain and the blastopore; anterior is indicated by the red star). (E) Stage 11.5 (left) and stage 12 (right) control embryos. (F) NFz8-injected embryos show a strongly reduced posterior neural crest-forming domain. (G) dnFGFR4a injections result either in normal sized posterior domain (left) or strongly reduced ones (right). Both types of embryos will show a strong Slug expression at stage 18 (D). Red stars indicate anterior.