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First published online 5 January 2006
doi: 10.1242/dev.02218

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Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development

¹ Unité de Biologie du Développement, UMR 7009, CNRS, Université Pierre et Marie Curie, Observatoire Océanologique, 06230 Villefranche-sur-Mer, France.
² Developmental, Cell and Molecular Biology Group, Box 91000, Duke University, Durham, NC 27708, USA.

View larger version (25K): [in a new window]   Fig. 1. Fz5/8 protein. (A) Schematic representation of Fz5/8. Blue, signal peptide; red, cysteine rich domain; green, transmembrane domain; black, KTXXXW motif. (B) Cladogram of representative members of the Frizzled family. Amino acid sequences of Frizzled proteins from sea urchin and other organisms were aligned using ClustalX and the tree was drawn with Tree View.

View larger version (66K): [in a new window]   Fig. 2. Spatiotemporal expression pattern of Fz5/8 during embryogenesis. (A) Temporal analysis by northern blotting with total RNA isolated from embryos at indicated stages. (B) Spatial distribution of Fz5/8 transcripts revealed by whole-mount in situ hybridization. All embryos were fixed at indicated stages and hybridized with Fz5/8 antisense probe, except for double labeling (where Fz5/8 probe was used simultaneously with ske-T or Brachyury probes). Lateral views with the animal pole at the top. E, unfertilized egg; 16, 16-cell stage; 60, 60-cell stage; B1-5, blastula stages; HB, hatched blastula; MB, mesenchyme blastula; EG, early gastrula; G, gastrula; Pr, prism; P, pluteus.

View larger version (72K): [in a new window]   Fig. 3. Expression of Fz5/8 during perturbed development. (A) Fz5/8 expression in animalized embryos obtained either by treatment with 0.1 mM zinc chloride or by overexpression of the wild-type form of GSK3ß. (B) Fz5/8 expression in embryos vegetalized with 30 mM lithium chloride. All embryos were fixed at indicated stages and hybridized with Fz5/8 antisense probe. HB, hatched blastula; MB, mesenchyme blastula; Pr, prism. Lateral views with the animal pole at the top.

View larger version (65K): [in a new window]   Fig. 4. Perturbation of Fz5/8 function during embryogenesis. (A) Structure wild-type or dominant-negative forms of Fz5/8 encoded by cDNA constructs. S, signal peptide; CRD, cysteine rich domain; TM, transmembrane domain. (B) Phenotypes associated with the overexpression of FzE or FzTM1. B1-B3, gastrula stages; B4-B6, pluteus. (C) Rescue of FzTM1 phenotype. Control (left), injection of FzTM1 alone (middle), and co-injection of FzTM1 and Fz in a 1:2 ratio (right).

View larger version (46K): [in a new window]   Fig. 5. Polarity of FzTM1-injected larvae. (A) DIC (left) and dark-field (right) images of FzTM1-injected larvae. (B) Determination of the morphological polarity of FzTM1-injected larvae by labeling one of the two blastomeres at the two-cell stage. (C) Confocal images of neurons immunostaining on FzTM1-injected larvae. Red, 1e11 antibody (Nakajima et al., 2004); green, counterstain. Control embryo, animal pole view (left); FzTM1-injected embryos, oral view (middle) and transverse view (right).

View larger version (122K): [in a new window]   Fig. 6. Molecular characterization of the phenotype associated with the disruption of Fz5/8 signaling. (A-T) Embryos injected with FzTM1 were fixed at the appropriated stages and processed for whole-mount in situ hybridization with probes for specific markers of embryonic territories, as indicated. (A,B) Early blastula (B3), (C,D,K-N,Q,R) mesenchyme blastula, (E,F) hatched blastula, (G-J,O,P,S,T) gastrula. Lateral views with the animal pole at the top except M,N,Q,R (viewed from the vegetal pole).

View larger version (57K): [in a new window]   Fig. 7. Requirement of Fz5/8 signaling in SMC for archenteron and skeleton formation. (A) Experimental design. At the 16-cell stage, animal and vegetal halves of FzTM1-injected embryos (red) were isolated and transplanted to the complementary half of a normal embryo (white). (B) Experimental results. B1-B3, control glycerol-injected embryo; B4-B6, control embryo injected with FzTM1 and a red lineage tracer; B7-B9, injected animal half recombined with control vegetal half; B10-B12, injected vegetal half transplanted with control animal half. (B1,B4,B7,B10) DIC images of resulting larvae. (B2,B5,B8,B11) Epifluorescence images of the same embryos, showing the contribution of the transplanted halves. (B3,B6,B9,B12) Confocal images of the larvae stained with EndoI (endoderm antibody) in red (Wessel and McClay, 1985) and with 1d5 (PMC marker) in green (Hardin et al., 1992).

View larger version (58K): [in a new window]   Fig. 8. Relationship between Fz5/8 and Notch signaling. (A) Confocal images of Notch protein localization revealed by antibodies against the intracellular domain of the receptor (Sherwood and McClay, 1999). Immunolocalization performed in control and FzTM1-injected embryos at mesenchyme blastula (MB) and gastrula (G) stages. (B) Larvae obtained after injection or co-injection of mRNA encoding FzTM1 and Notch activated form (Nact).

View larger version (71K): [in a new window]   Fig. 9. Relationship between Frizzled and the PCP pathway. (A) Representative phenotypes of larvae treated with inhibitors for PKC (Bisindolylmaleimide I), ROCK (Y27632) or JNK [JNK inhibitor I (L-form)], added at HB separately or in combination. (B) Larvae obtained after injection or co-injection of mRNA encoding FzTM1 and an activated form of LvRhoA protein (actRhoA).

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