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First published online November 7, 2008
doi: 10.1242/10.1242/dev.026229

Development 135, 3903-3910 (2008)
Published by The Company of Biologists 2008
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Fgf8a induces neural crest indirectly through the activation of Wnt8 in the paraxial mesoderm

Chang-Soo Hong1,2, Byung-Yong Park2 and Jean-Pierre Saint-Jeannet2,*

1 Department of Biological Science, College of Natural Sciences, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 712-714, South Korea.
2 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.


Figure 1
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  		Fig. 1. Wnt8 and Fgf8a are necessary for NC formation. (A) Embryos injected with Fgf8a (Fgf8aMO; 50 ng) or Wnt8 (Wnt8MO; 40 ng) morpholino antisense oligonucleotides exhibit a strong reduction of Pax3, Snail2, Sox8 and Sox10 expression at the neurula stage, while the expression domain of the pan-neural marker Sox2 is expanded. Embryos are viewed from the dorsal side anterior to the top. Injected side is on the right. (B) At the gastrula stage, Fgf8aMO- and Wnt8MO-injected embryos show normal expression of the mesoderm marker Xbra. Embryos are viewed from the vegetal pole. (C) At the tailbud stage the migration pattern of cranial NC cells is severely perturbed in both Fgf8aMO- and Wnt8MO-injected embryos, as revealed by expression of the cranial NC marker Ap2. Lateral views, dorsal to top. Left panels, anterior to the right (injected side); right panels, anterior to the left (control side). (D) TUNEL staining shows a similar increase in apoptotic cells in the cranial region of Fgf8aMO- and Wnt8MO-injected embryos at the tailbud stage (arrows). Embryos are viewed from the dorsal side, anterior to the right. The dotted lines indicate the position of the midline. (E) In animal explants, Wnt8 (W, 25 pg) or Fgf8a (C, 5 pg) share the same ability to induce NC markers (Pax3, Snail2 and Sox8) when co-expressed with the Bmp antagonist Chordin (C, 10 pg; C+W and C+F, respectively). In these explants the induction of NC fate occurs in the absence of mesoderm formation (mActin and Col2a1). Fgf8a also synergizes with Chordin to induce neural tissue (Sox2). Values (n=3) are presented as mean±s.e.m.; *P<0.05, versus uninjected animal explant (U). (F) The dual requirement of Fgf8a and Wnt8 suggests that these factors are acting either in parallel (1), or in the same pathway, one upstream of the other (2,3), to generate the NC.

 

Figure 2
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  		Fig. 2. Fgf8a and Wnt8 differ in their ability to restore NC progenitors in Wnt8- and Fgf8a-deficient embryos. (A) Fgf8a mRNA injection fails to rescue Snail2 and Sox8 expression at the neural plate border of embryos injected with Wnt8MO (25ng) or β-CatMO (25 ng). A single injection of Fgf8a mRNA (2.5 pg) expands Snail2 and Sox8 expression domains. (B) Conversely, Wnt8 (100 pg) or β-catenin (200 pg) plasmid DNA injection restores Snail2 and Sox8 expression in embryos injected with Fgf8aMO (50 ng). Injection of Wnt8 or β-catenin in sibling embryos expanded Snail2 and Sox8 expression domains. In all panels, embryos are viewed from the dorsal side with anterior to the top. The injected side is to the right.

 

Figure 3
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  		Fig. 3. NC induction by Fgf8a requires active canonical Wnt signaling in animal explants. (A) In animal explants the induction of NC markers (Snail2 and Sox8) by the co-expression of Chordin (10 pg) and Wnt8 (25 pg; C+W), or Chordin (10 pg) and Fgf8a (5 pg; C+F), is dramatically reduced in the context of embryos injected with Wnt8MO (W8MO, 50 ng) or β-CatMO (βCMO, 50 ng). Interference with the Wnt signaling pathway did not affect (C+F) or restore (C+W) the neuralization of these explants (Sox2). Values (n=3) are presented as mean±s.e.m.; *P<0.05, versus C+W (upper graphs) or C+F (lower graphs) samples. U, uninjected animal explant. (B) The expression of Snail2 detected by whole-mount in situ hybridization in Chordin and Wnt8 (C+W8), or Chordin and Fgf8a (C+F8a), treated animal explants is abolished by the co-injection of Wnt8MO (W8MO; 50 ng).

 

Figure 4
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  		Fig. 4. Developmental expression of Wnt8 and Fgf8. (A) Comparison of Wnt8 and Fgf8 expression at the gastrula stage. At the mid-gastrula stage (11.5), Wnt8 and Fgf8 have a complementary expression pattern in the ventrolateral and dorsolateral mesoderm, respectively. The embryos are oriented dorsal (D) to the top. Hemi-sections (red lines in the left panels) of these embryos along the animal-vegetal axis reveal that both genes are co-expressed in the lateral mesoderm. The arrowheads indicate the position of the lateral lip of the blastopore (bl, blastocoel). At stage 12, the Wnt8 expression domain expands anteriorly into the involuting mesoderm, the future paraxial mesoderm, while Fgf8 remains confined to the posterior mesoderm. (B) Comparative expression of Wnt8 and Sox8 at stage 12. Sox8 expression in the ectoderm (arrow) is adjacent to Wnt8 expression in the mesoderm. The red lines indicate the level of the serial sections shown in C,D. (C) Expression of Sox8 and Wnt8 on adjacent sections of a stage 12 embryo highlights the mesoderm expression of Wnt8 underlying the first Sox8-positive cells in the NC-forming region (bracket). At stage 12.5, Sox8 expression is stronger in the NC domain, and Wnt8 becomes more broadly expressed in both the ectoderm and the mesoderm layers. The red dotted lines in the lower panels demarcate the separation between the ectoderm and the mesoderm layers. Lower panels are higher magnifications of the upper panels. (D) Comparative expression of Sox8, Wnt8 and Fgf8 on adjacent sections of a stage 12/12.5 embryo confirms that Fgf8 is not co-expressed with Wnt8 in the mesoderm underlying the NC-forming region. The red dotted lines indicate the separation between the ectoderm and the mesoderm layers. (E) In the posterior region of the same embryo, Fgf8 is detected in the dorsolateral mesoderm, around the yolk plug (yp), while Wnt8 is confined to the ventrolateral region. Dorsal to the top.

 

Figure 5
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  		Fig. 5. Fgf8a is a strong inducer of Wnt8 in animal explants and in whole embryos. (A) Animal explants derived from embryos injected with Fgf8a (F) or with a combination of Fgf8a and Chordin (C+F) show a strong upregulation of Wnt8 after 4 hours in culture. For comparison, Wnt8 (W) or Wnt8 and Chordin (C+W) co-injection had little effect on the expression levels of Fgf8. U, uninjected animal explant. Values (n=3) are presented as mean±s.e.m.; *P<0.05, versus uninjected animal explant (U). (B) In whole embryos, loss of Fgf function by injection (arrow) of Fgf8aMO (50 ng) or a dominant-negative Fgf receptor (XFD; 2 ng) results in a reduction of Wnt8 expression in the involuting mesoderm at stage 12. Conversely, Fgf8a (5 pg) mis-expression strongly upregulates Wnt8. For all injections, dorsal and lateral views (control and injected sides) of the same embryo are shown. Dorsal views, anterior to the top, injected side to the right (arrows). Lateral views, dorsal to the top; for the control side anterior is to the left, for the injected side anterior is to the right.

 

Figure 6
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  		Fig. 6. Fgf8a induces NC at the anterior neural fold indirectly. (A) The anterior neural plate (blue) is devoid of NC tissue (orange) as a result of the activity of a Wnt inhibitor, Dkk1 (Carmona-Fontaine et al., 2007Go). Snail2 expression is shown in a control embryo at stage 15. (B) β-catenin misexpression (200 pg) can overcome this inhibition to induce NC markers (Snail2) at the anterior neural fold (arrows). (C) Fgf8a (5 pg) misexpression can also induce NC markers (Snail2 and Sox8) at the anterior neural fold (arrows), an activity that is mediated by upregulation of Wnt8 in this region of the embryo (arrows). (D) Normal pattern of expression of Wnt8 in a control embryo at the same stage. In all panels the embryos are viewed from the dorsal side, anterior to the top.

 

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© The Company of Biologists Ltd 2008