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First published online 12 December 2007
doi: 10.1242/dev.014282

Development 135, 353-365 (2008)
Published by The Company of Biologists 2008
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FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis and regulate gastrulation during sea urchin development

Eric Röttinger1, Alexandra Saudemont1, Véronique Duboc1, Lydia Besnardeau1, David McClay2 and Thierry Lepage1,*

1 UMR 7009 CNRS, Université Pierre et Marie Curie (Paris 6) Observatoire Océanologique, 06230 Villefranche sur mer, France.
2 Department of Biology, French Family Science Center, Duke University Durham, NC 27708, USA.


Figure 1
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  		Fig. 1. Temporal and spatial expression of fgfA and FGFR2 in sea urchin. (A,B) Northern blot analysis of fgfA and FGFR2 expression. (C) Scheme of early development of the sea urchin embryo describing morphogenesis of the skeleton. Spatial distribution of fgfA (D-M) and fgfr2 (N-W) transcripts during normal development analysed by in situ hybridization. Insets in E and M show the same embryo in a different focal plane. (N) Egg, (O) 16-cell stage, (P) 120-cell stage, (D,E) swimming blastula, (F,G,Q) mesenchyme blastula, (I,J,R,S) early gastrula, (K,T) late gastrula, (L,M,U,V) prisme/early pluteus (36 hours), (W) pluteus (48 hours). Ectodermal and mesodermal expression domains are indicated by arrows in K.

 

Figure 2
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  		Fig. 2. The fgfA synexpression group and effects of treatments with lithium chloride or nickel chloride on expression of fgfA and pax2/5/8. (A-D) Schematic representation of fgfA expression in sea urchin. (E-P) Spatial expression of sprouty (E-H), pea3 (I-L), pax2/5/8 (M-P). (Q-Zb) Morphology and effects on fgfA and pax2/5/8 expression of treatments that perturb animal-vegetal or oral-aboral polarity; (Q-T) 60 hour embryos; (U-Zb) 24 hour embryos.

 

Figure 3
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  		Fig. 3. Patterning defects caused by overexpression of fgfA in sea urchin. Embryos overexpressing fgfA or treated with bFGF were observed at the early gastrula stage (A,B) or at 48 hours (C-H) or fixed at the mesenchyme blastula stage for in situ hybridization with a pax2/5/8 probe (I-L). fgfA overexpression induces radial (K) or broadened (L) expression of pax2/5/8. See text for details. vv, vegetal view. Arrows indicate the positions of the spicule rudiments; red square indicates the presence of an ectopic spicule.

 

Figure 4
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  		Fig. 4. Loss of FGFA function disrupts skeletogenesis and gut morphogenesis in sea urchin. (A) Controls of the efficiency of the fgfA-splice and fgfA-ATG morpholinos. (a) Scheme of the intron-exon organization of the fgfA pre-mRNA, indicating the positions of the target sequence and of the primers used to characterize the mRNA products generated in the presence of this morpholino. (b) RT-PCR analysis of control uninjected and embryos injected with the fgfA-splice morpholino at 0.5, 1 or 1.5 mM. The PCR product in control embryos had the expected size (101 codons, 303 bp) and predicted sequence. The PCR product amplified from the fgfA-splice morphants had the expected size (66 codons, 198 bp) and sequence for a transcript deleted from exon 2. (c) fgfA-ATG-Mo but not fgfA-splice-Mo, specifically blocks in vitro translation of a synthetic fgfA transcript. PCS2-fgfA was in vitro translated in the presence of fgf9A-ATG-Mo or fgfA-splice at the indicated concentrations (1.6, 8, 40 µM) and the products were analysed by PAGE and autoradiography. (Ba-o) Phenotypes caused by microinjection of the fgfA-splice-Mo (f-j) or fgfA-ATG-Mo (k-o) in the egg. (Ca-l) Rescue experiment. Eggs were first injected with fgfA-splice-Mo together with RLDX then subsequently re-injected at the one- or two-cell stage with a synthetic mRNA encoding FGFA together with an FLDX.

 

Figure 5
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  		Fig. 5. FGFA function is required in the vegetally derived tissues for skeleton formation and morphogenesis of the gut in sea urchin. (A) Experimental design. The animal and vegetal halves of wild-type (gray) and fgfA-splice morpholino-injected embryos (red) were recombined as shown. (Ba,b) Control uninjected embryos. Side views. (c-f) Chimeras in which fgfA function is inhibited in the animal half develop into pluteus larvae with reduced oral lobes and arms (arrow in e). (g-j) Inhibition of fgfA function in the vegetal half prevents skeletogenesis and disrupts gut morphogenesis.

 

Figure 6
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  		Fig. 6. Molecular analysis of FGFA loss of function in sea urchin. (A-X) The effects of loss of FGFA function on the gene expression program of the endoderm SMCs, ectoderm or PMCs were analysed by in situ hybridization with the indicated probes. See text for details.

 

Figure 7
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  		Fig. 7. FGFA is required for activation of ERK in lateral ectodermal regions whereas ERK is required for expression of sprouty and pax2/5/8 in sea urchin. (A-D) Control or MoFGF-injected embryos were processed for whole-mount immunolocalization of activated ERK using an anti MAPK-P antibody (A,C) or for in situ hybridization using a sprouty probe (B,D). (E-J) Embryos at the mesenchyme blastula stage were treated or not with the MEK inhibitor U0126 for 6 hours and processed for in situ hybridization using the indicated probes.

 

Figure 8
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  		Fig. 8. Roles of FGFR1 and FGFR2 in gastrulation and skeletogenesis in sea urchin. (A-D) Control embryos. (E-X) Embryos injected with antisense morpholino oligonucleotides directed against FGFR1 (E-H) or mRNA encoding a dominant-negative form of FGFR1 (I-L) or with antisense morpholino oligonucleotides against FGFR2 (M-T) or co-injected with both FGFR1-Mo and FGFR2-Mo (U-X).

 

Figure 9
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  		Fig. 9. Molecular analysis of FGFR1 and FGFR2 loss of function in sea urchin. (A-D) Mesenchyme blastula, (E-L) mid gastrula, (M-P) early pluteus. In situ hybridization using fz58 (A-D), bhmt (E-H), pax258 (I-L) and SM30 (M-P) probes.

 

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© The Company of Biologists Ltd 2008