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First published online 7 January 2004
doi: 10.1242/dev.00964

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Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations

Juliette Mathieu^1,*, Kevin Griffin², Philippe Herbomel³, Thomas Dickmeis⁴, Uwe Strähle⁴, David Kimelman², Frédéric M. Rosa¹ and Nadine Peyriéras^1,5,

¹ U 368 INSERM, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris cedex 5, France
² Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195-7350, USA
³ Unité `Macrophages et Développement de l'Immunité' Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
⁴ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, Strasbourg, France
⁵ UMR CNRS 7138, Université Pierre et Marie Curie, Batiment A, 4eme etage, case 5, 7 quai Saint Bernard, 75252 Paris Cedex 05, France

View larger version (91K): [in a new window]   Fig. 1. Fgf signalling pathway serves as a relay for the cell-nonautonomous induction of oep by the Tar*/Nodal pathway. (A-Z) Flat mounts of chimeric embryos at the shield stage showing the expression of tar, oep, cyc, sqt, ntl, fgf3 and fgf8 in blue (the probe is indicated in the bottom left of each panel). The progeny of transplanted cells expressing Tar* is revealed by immunocytochemistry (red fluorescence staining the green fluorescent protein, except in C,D,J,K where brown staining reveals nls-ß galactosidase). The genotype of the recipient embryo is shown in the bottom right of each panel. +SU, embryos treated with 10 µM SU5402 after transplantation. In some experiments, the recipient embryo was injected with a morpholino oligonucleotide directed against fgf8 (8MO). All views are of the animal pole. For all panels except C,D,J,K, a Normarski image and an epifluorescence image were overlayed.

View larger version (60K): [in a new window]   Fig. 2. Fgf3 and Fgf8 might account for the involvement of Fgf signalling pathway in the Nodal positive-feedback loop. (A-U) Whole mount embryos showing expression of fgf3, fgf8, oep and tar in blue (the probe is indicated in the bottom left and the stage at the bottom right of each panel: sh, shield stage: 80%, 80% epiboly). (A-L) Views from the animal pole, dorsal to the bottom of embryos uninjected (uninj) or injected with sqt, cyc, fgf3 and fgf8 synthetic mRNA (indicated top right). (M-R) Expression pattern of fgf3 (M-O) and fgf8 (P-R) throughout epiboly in the MZoep background. Arrows in R point to the dorsal limit of the marginal staining. (S-U) Lateral views, dorsal to the right, of untreated (control) embryos or embryos treated with SU5402 (top right). 10 µM SU5402 (T) phenocopies the ace mutant phenotype and 30 µM SU5402 (U) abolishes the expression of oep at the blastoderm margin. Note that oep is still expressed on the dorsal side, consistent with the involvement of other pathways.

View larger version (101K): [in a new window]   Fig. 3. oep and fgf8 interact in vivo in the formation of mesoderm. (A,D,G,J) Lateral views of live embryos at 30 hours of development, taken from the progeny of Zoep;ace double-heterozygous parents. The genotype, which is inferred from the phenotype and statistical analysis, is indicated at the top right. (B-C,E-F,H-I,K-L) Lateral views of fixed embryos at 30 hours of development taken from the progeny of Zoep;ace double-heterozygous parents. The genotype, inferred from the phenotype and statistical analysis, is indicated top right. myoD or myosin heavy-chain (myhz1) staining in blue (the probe is indicated bottom left). (M,P) Dorsal view of embryos in K,L, respectively, note the bilateral staining. (N,O) Higher magnification of the embryos in K,L, respectively.

View larger version (98K): [in a new window]   Fig. 4. The dorsal mesoderm of Zoep;ace embryos undergoes massive cell death at the end of gastrulation. (A-D) Lateral views (tail region) of live embryos at 30 hours of development stained with acridine orange. Dead cells stain green. The genotype, which is inferred from the phenotype and statistical analysis is indicated at the top right. (E,F) Views from the dorsoposterior region of live embryos at the shield stage (the inferred genotype is indicated top right). (G,H) Confocal sections of live embryos injected at the tail-bud stage with Annexin V Alexa 488 15 minutes before inspection (the inferred genotype is indicated top right). (I-M) Live Zoep/ace embryos inspected with Nomarski optics. (I) Focus on the dorsal (axial) and dorso-lateral hypoblast at tailbud stage, presented as three overlapping panels because of the curvature of the underlying yolk ball. Axially, notochord does not form; instead there is a large mass of dying cells that have morphology typical of apoptotic cells. Paraxially (left), 100-160 µm away from the axis, dead cell bodies of various sizes are also found (boxed region; enlarged in K) next to live cells of typical migrating (converging) non-axial hypoblast cell morphology (black arrowheads). (J) 4-somite stage, parasagittal view (caudal to the lower right) through the neuroectoderm, somitic mesoderm, yolk syncytial layer and yolk core. The inset shows the yolk syncytial layer and yolk portion appearing at a shallower focus. Both focal planes reveal numerous cell corpses (arrows) in the yolk syncytial layer, some of which abut and penetrate the underlying yolk core. (K-L) Higher-magnification views of cell corpses. (K) Dead cell bodies in the paraxial/lateral hypoblast at tailbud stage, from panel I inset. (L) Cell corpses (arrows) at 4-somite stage inside the yolk syncytial layer, one of them (left) indenting the underlying yolk mass. A yolk syncytial layer nucleus (white arrowhead), seen here slightly out of focus behind a dead cell body, is in focus in panel M. Abbreviations: n, neuroectoderm; sm, somitic mesoderm; y, yolk core; ysl, yolk syncytial layer. Scale bar: 20 µm.

View larger version (130K): [in a new window]   Fig. 5. Cooperation between oep and fgf is required before the onset of gastrulation for the maintenance of dorsal mesoderm. (A-D) Flat mounts of embryos taken from the progeny of Zoep;ace double-heterozygous parents (the genotype, which is inferred from the phenotype and statistical analysis, is indicated top right). (A-B) Embryos at 90% epiboly, ntl staining occurs at the notochord and presumptive mesoderm at the margin of the blastoderm and hgg1 staining at the presumptive hatching gland (red), myoD staining labels adaxial cells, and her5 staining the presumptive midbrain-hindbrain boundary (blue). (C-D) Embryos injected with caged dextran-fluorescein at the one-cell stage, laser irradiated at the shield stage at the level of the embryonic shield, fixed and stained at the end of gastrulation for ntl and hgg1 (blue) and fluorescein (brown). Arrow points to the anterior limit of migration of the labelled cells in D. (E-H) Flat mounts of embryos taken from the progeny of oep heterozygous parents (the genotype, inferred from the phenotype and statistical analysis, is indicated top right), treated with 10 µM SU5402 for various periods of time (as shown), fixed at 90% epiboly and stained for ntl (blue) and hgg1 (red). Wild-type embryos appear to be unaffected by SU5402 treatment.

View larger version (102K): [in a new window]   Fig. 6. T-box genes expression is down regulated in Zoep;ace mutants. (A-X,XX-XZ) All mount, in situ hybridization of embryos were taken from the progeny of double-heterozygous parents. The genotype (indicated top right) is inferred from either the phenotype (P-R,V-X,XX-XZ) or PCR-based genotyping. The embryonic stage is shown at bottom right (30%, 30% epiboly; sh, shield; 80%, 80% epiboly; 12s, 12 somites) and the probe (ntl, tbx6 and spt in blue) is indicated at bottom left. (A,G) Lateral views. (D,F,J,L,M-X,XX-XZ) Lateral views, dorsal to the right. (B,C,E,H,I,K) Animal pole views. (N,T) Arrowhead points to the prechordal plate, which is stained in N and lost in T. Note that the double-mutant phenotype is otherwise similar to wild-type. (Y-Z) PCR-based genotyping of embryos after in situ hybridization. Digested and nondigested (nd) PCR products were run on a 1.5% agarose gel.

View larger version (119K): [in a new window]   Fig. 7. Posterior mesoderm is still induced but not maintained in MZoep;ace mutants. (A-B) Live embryos at 30 hours of development taken from the progeny of oep–/–;ace–/+ parents (genotype top right). (C-V) Whole mount in situ hybridization of embryos taken from the progeny of oep–/–;ace–/+ parents and oep–/– parents (genotype top right). MZoep;ace embryos were genotyped for the ace allele. We systematically compared the phenotypes of the progeny of oep–/–;ace–/+ parents with those of the progeny of oep–/– parents. The stage is shown bottom right and the probe is indicated bottom left. Dorsal is to the right. Lateral views of embryos (C,E,H,J). Animal-pole views (D,F,G,I,K-N,R,S). Vegetal-pole views (O-Q,T-V).