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First published online 14 May 2008
doi: 10.1242/dev.021543

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A maternally localised Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica

UMR7009 Laboratory of Developmental Biology, CNRS and Université Pierre et Marie Curie (Paris 6), Observatoire Océanologique, F-06234 Villefranche-sur-mer, France.

View larger version (63K): [in this window] [in a new window]   Fig. 1. CheWnt3 RNA is localised to the animal/oral pole of eggs and embryos. (A) The relationship between egg animal-vegetal polarity, gastrulation and embryo oral-aboral polarity in Clytia. The animal pole of the egg marks the site of cell ingression at gastrulation and the future oral pole of the embryo and planula larva. (B) In situ hybridisation analysis of Wnt3 expression in embryos from unfertilised eggs to mid-blastula stage (top row) and from early gastrula to 1-day-old planula (bottom row). Embryos are oriented with the animal/oral pole up. Scale bar: 40 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 2. CheWnt3 is required for the development of axial polarity and oral fates. (A) Gastrulation and elongation along the oral-aboral axis in a normal Clytia embryo (top row) was completely blocked in Wnt3-MO-injected embryos (bottom row), shown fixed at the early gastrula stage (15 hpf). (B) Complete loss of morphological axis in Wnt3-MO-injected embryos at late gastrula (20 hpf) stage. Cell contours were visualised by phalloidin (green) and nuclei by To-Pro3 (red). Gastrulation was severely delayed compared with uninjected embryos. The exact timing and site of residual cell ingression formation varied. (C) Equivalent 2-day-old planulae. No morphological oral-aboral axis is discernable in Wnt3-MO-injected embryos; however, endoderm formation has recovered. (D) Representative in situ hybridisation image of characteristic oral CheBra expression in early gastrulae (15 hpf) that is lost in Wnt3-MO-injected embryos. (E,F) Loss of CheBra expression in oral ectoderm, and of CheAxin in oral endoderm and ectoderm in Wnt3-MO-injected planulae (1.5 day). (G,H) Expansion of aboral FoxQ2A expression to nearly the entire body in Wnt3-MO-injected embryos fixed at early gastrula and planula stages (1.5 day). (I) Loss of expression of oral-ectoderm-expressed ligands CheWntX2, CheWnt9, CheWntX1A and CheWnt5 in Wnt3-MO-injected embryos fixed at the planula stage (1.5 day). Scale bars: 40 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 3. CheWnt3 is required first to activate the canonical Wnt pathway and then to restrict it to the oral pole. Activation of the canonical Wnt pathway in control Clytia embryos (A) and embryos derived from eggs injected with Wnt3-MO (B) or Wnt3 RNA (C) were visualised by injecting eggs prior to fertilisation with RNA encoding a β-catenin-Venus (GFP) fusion protein and visualised at early blastula (128-cell/4.5 hpf), mid-blastula (6.5 hpf) and early gastrula stages. Scale bars: 40 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 4. Participation of CheWnt3 in Wnt pathway regulatory interactions. (A) CheWnt3-MO injection into Clytia eggs did not affect the graded distribution of CheFz1 and CheFz3 RNAs at the early blastula stage (4.5 hpf; essentially maternally derived RNA). However, by the early gastrula stage (10 hpf), both RNAs were found strongly expressed throughout the embryo. This shows that CheWnt3 is required for mutual negative regulation of the receptors during the intervening period, probably at the transcriptional level. (B) The oral territory of zygotic Wnt3 expression was not greatly affected by treatments that upregulate (dnGSK3, Fz3-MO) or downregulate (Fz1-MO) the canonical Wnt pathway.

View larger version (58K): [in this window] [in a new window]   Fig. 5. CheWnt3 drives the development of oral fate. (A) Confocal images of early gastrula stage Clytia embryos stained with phalloidin (green) and To-Pro3 (red), showing characteristic phenotypes obtained following Wnt3 RNA injection into eggs before fertilisation. Presumptive endoderm cell ingression took place over a broad area of the embryo, with a graded distribution of CheBra apparent. (B) Equivalent observation at the 1-day planula stage, showing the lack of a distinguishable morphological oral-aboral axis. (C,D) Representative in situ hybridisation images of CheBra and FoxQ2A expression at early blastula stage (15 hpf). In embryos injected with CheWnt3 RNA, CheBra expression has expanded to the entire body with a residual graded distribution, and FoxQ2A expression has been lost. Scale bars: 40 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Restoration of oral poles in Wnt3-MO embryos by ectopic CheWnt3 expression. (A-C) Confocal images of early gastrula Clytia embryos stained with phalloidin (green) and ToPro3 (red). (B) Uninjected; (A,C) Injected with Wnt3-MO prior to fertilisation; for C, Wnt3 RNA was further injected into one blastomere at the 8-cell stage (n=15). In C, higher-magnification images of the region derived from the RNA-injected cell oral end, labelled by co-injected fluorescent dextran (blue), are shown beneath. Asterisks indicate the involution of cells neighbouring those containing Wnt3 RNA. (D) Representative in situ hybridisation image of CheBra (oral) and CheFoxQ2a (aboral) expression in early gastrulae (15 hpf) derived from Wnt3-MO-injected eggs (left). Wnt3 RNA was injected into a blastomere at the 4-cell stage after Wnt3-MO injection (right). Scale bars: 40 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 7. A current model for axis determination in Clytia. (A) In early stages (from egg to early blastula), the ligand CheWnt3 and its receptor CheFz1, produced from animally concentrated RNAs, are required for canonical Wnt signalling in the the animal hemisphere. CheFz1 RNA is distributed as an animal-vegetal gradient in the cytoplasm, whereas CheWnt3 RNA is tightly localised to the animal cortex. CheWnt3 protein probably adopts a wider distribution upon translation (see text). The negatively acting receptor CheFz3, produced from a vegetal cortical RNA, is likely to be the major factor restricting canonical Wnt signalling activation to the future oral end. (B) In later stages, tightly localised zygotic CheWnt3 expression at the oral pole becomes a major factor restricting canonical Wnt pathway activation. Wnt3 is also involved in the reciprocal negative regulation between the receptors CheFz1 and CheFz3 by unknown mechansims. Arrows and dotted lines represent signalling pathways and transcriptional regulation pathways, respectively. The coloured boxes represent RNA distribution, the area with a red dashed outline the predicted Wnt3 protein distribution, and green circles β-catenin stabilisation and nuclear localisation.

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