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First published online August 11, 2005
doi: 10.1242/10.1242/dev.01961

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ß-Catenin-dependent Wnt signalling controls the epithelial organisation of somites through the activation of paraxis

Claudia Linker^1,*,,, Cynthia Lesbros^1,*, Jérôme Gros¹, Laura W. Burrus², Alan Rawls³ and Christophe Marcelle^1,

¹ Laboratoire de Génétique et de Physiologie du Développement (LGPD). Developmental Biology Institute of Marseille (IBDM), CNRS UMR 6545. Université de la Méditerranée, Campus de Luminy, case 907, 13288 Marseille, Cedex 09, France
² Department of Biology, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA
³ School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA

View larger version (115K): [in a new window]   Fig. 1. Axial or lateral structures do not affect the epithelial structure of the dermomyotome. (A) Schematic of the experiment: separation of the anterior PSM from axial structures (neural tube and notochord) by making a slit through the three germ layers of the embryo. Embryos were then cultured for 18-20 hours. (B) Scheme of the separation of PSM from lateral structures. (C,D) Transverse sections of embryos in E and G. (E,F) Expression of Fz7 and paraxis is maintained after separation of the PSM from axial structures. The residual expression of paraxis and Fz7 along the neural tube is due to remnants of somitic tissue and ectoderm that have not been completely separated from the neural tube (and confirmed on sections, not shown). (G) Expression of Fz7 is not affected after separation of the PSM from lateral structures.

View larger version (103K): [in a new window]   Fig. 2. Ectoderm maintains the epithelial structure of somites. (A) Schematic of the procedure: separation of the anterior PSM from overlying ectoderm by the introduction of an impermeable membrane (blue), followed by 3-24 hours of culture. The position of the membrane at the end of the experiment matches perfectly the region where no staining is observed (see C-H). (B-D) Expression of Fz7 after 3 (B), 12 (C) or 24 (D) hours of culture. The position of the membrane is indicated in B. (E,F) Expression of Pax3 after 12 (E) or 24 (F) hours of culture. (G,H) Expression of paraxis after 12 (G) or 24 (H) hours of culture. (I,J) Transverse sections of C (I) and D (J) at the positions indicated.

View larger version (62K): [in a new window]   Fig. 3. Specific functions of Wnt6 and Wnt1 in the epithelialisation and patterning of somites. (A) Schematic of the procedure: separation of the PSM from overlying ectoderm with a membrane (blue) after the injection of Wnt-expressing cells (in red) and followed by 24 hours in culture. (B) Transverse section through F. (C) Transverse section through M. (D-I) Expression of markers after the injection of Wnt6-(D-I) and Wnt1-expressing (J-O) cells: (D) Fz1; (E) Fz2; (F) Fz7; (G) pax3; (H) paraxis; (I) Wnt11. Wnt-expressing cells were stained with fixable CM Tracker DiI; their exact position, defined under UV examination, was recorded and is indicated in each panel by broken lines. The corners of the impermeable membranes are indicated in each embryo.

View larger version (101K): [in a new window]   Fig. 4. Wnt6 activates the ß-catenin pathway. Projections of confocal dorsal views (A,B,E,F,I,J) and confocal transverse sections (C,D,G,H,K,L) of somites electroporated with GFP (A-D), DN ß-catenin (E), DN Lef1 (F-H), DN Lef1 and GFP (1:1, J), DN Lef1 and paraxis (1:1, J-L). Sections are stained with a combination of phalloidin red, recognising F-actin (in red) and an antibody directed against N-cadherin (in blue), together with the GFP (in green). Sections in D,H,L show the N-cadherin staining only. (A-D) Control, GFP electroporated somites with typical long bottle-shaped cells (arrowheads in A) that display the adherens junction-specific markers F-actin and N-cadherin at their apical end (C,D). (B) Enlargement of cells in A. (E-H) Dermomyotome cells electroporated with DN ß-catenin (E) or DN Lef1 (F-H) display a round morphology with a clear redistribution of the adherens junction markers at the plasma membrane (G,H). (J-L) Dermomyotome cells expressing DN Lef1 together with paraxis display a normal epithelial-like morphology (J) with adherens-junction marker at the apical end (K,L). (M) Quantification of experiments shown in A-L, where coloured bars represent the percentage of epithelial and round cells in somites electroporated with GFP only (yellow bar), DN Lef1 and GFP (blue bar), or a combination of DN Lef1 and paraxis (purple bar). After GFP electroporation, only 1.5% of the cells displayed a round morphology. The electroporation of DN Lef1+GFP increases the number of round cells to 62.5%, while the co-electroporation of DN Lef1 and paraxis significantly rescues the epithelial morphology, as only 24.8% of the cells display a round morphology. Standard deviations are indicated.

View larger version (79K): [in a new window]   Fig. 5. ß-Catenin regulates paraxis expression. (A) Schematic of the experiment: embryos were halved with one half processed for Fz7 expression and the other half processed for paraxis expression, as seen in D. (B) Schematic of the procedure: impermeable membranes were positioned at each side of the embryo and the embryos were allowed to grow for 10 hours. Embryos were then cut in half and processed for Fz7 and paraxis as before (E). (C) Somites (dorsal view) electroporated with a DN ß-catenin construct lose paraxis expression in the electroporated region (black arrowheads), whereas control somites express paraxis in the entire dermomyotome (white arrowheads). (F,G) Dorsal electroporation of DN Lef1 construct. Electroporated cells, which express GFP (G), do not express paraxis (F).

View larger version (93K): [in a new window]   Fig. 6. Maintaining a continuous source of Wnt6 or paraxis counters the de-epithelialisation of the dermomyotome. (A) Expression of Paraxis in an embryo injected with Wnt6-expressing cells at E2.5 and incubated for 48 hours. (B) Enlargement of the boxed area in A, showing the maintenance of a high paraxis expression around the injected cells. (C) Fluorescence image of B, showing the position of the DiI-labelled cells. (D) Transverse section of the same embryo. (E) Enlargement of D. Arrow indicates a dermomyotome-like epithelial structure located in the vicinity of the injected cells (outlined). (F-I) Confocal pictures of sections of embryos electroporated with paraxis CLGFPA in the dorsal region of the dermomyotome, stained with antibodies against GFP (in green), ß-catenin (in blue) and phalloidin red, recognising F-actin (in red), (F,G) Twenty-four hours after electroporation, the morphology (shown with GFP staining) and polarity (recognised by F-actin and ß-catenin staining at the adherens junctions) of cells overexpressing paraxis is normal. (G) Enlargement of F. (H,I) Forty-eight hours after electroporation, cells over-expressing paraxis organise in clusters of cells that maintain contacts through their apical ends, expressing F-actin and ß-catenin (arrowheads). (I) An enlargement of H.

View larger version (26K): [in a new window]   Fig. 7. A model for the sequential action of Wnts on somite epithelialisation and compartimentalisation. (A) The Wnt6 signal, secreted by the ectoderm, binds to Fz7 receptor expressed by the cells of the segmental plate. (B) This interaction activates the canonical, ß-catenin pathway that controls the expression of paraxis and maintains the epithelial structure of the dorsal part of the somite, while the ventral region disaggregates to form the sclerotome. (C) Cells in the medial-most region of the dermomyotome receive a Wnt1 signal from the dorsal neural tube, which activates Fz2 receptor and induces Wnt11 expression in this region, thus defining the dorsomedial compartment of the dermomyotome.