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Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis

Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA

View larger version (43K): [in a new window]   Fig. 1. (A) Convergent extension occurs in both dorsal mesodermal and posterior neural tissues. Axial mesoderm (red) involutes and undergoes convergent extension in parallel to convergent extension of overlying posterior neural ectoderm (blue). AR, archenteron; BC, blastocoel. (B-D) Cell behaviors during convergent extension. (B) Dorsal mesoderm (Shih and Keller, 1992a); (C) neural-over-mesoderm (Elul and Keller, 2000); (D) isolated posterior neural ectoderm (Elul et al., 1997). Red, mesoderm; Blue, posterior neural. In C, light blue indicates notoplate.

View larger version (60K): [in a new window]   Fig. 2. Targeted injections of mutant Xdsh elicits distinctly different phenotypes. (A) Injection of Xdsh-D2 into the dorsal animal blastomeres at the eight-cell stage targets expression predominately to the neural ectoderm (blue); injection into the dorsal vegetal blastomeres targets expression to the dorsal axial mesoderm (red). (B) Control embryos at stage 20. (C) Dorsal-animal injection of Xdsh-D2 inhibits neural tube closure, and neural plates remain partially open at stage 20 (arrow). In other cases, neural plates close but fail to fuse normally (arrowhead). (D) Embryos injected dorsovegetally close their neural tubes normally; however, a few do display improper fusion of the neural folds (arrowhead). (E) Control embryos at stage 30. (F) At stage 30, embryos expressing Xdsh-D2 in the neural ectoderm exhibit a severe dorsal flexure and failure to straighten the A/P axis. (G) At stage 30, embryos expressing Xdsh-D2 in the mesoderm do not display dorsal flexure, but instead fail to elongate and are shorter and wider than controls. (H) Control embryo at stage 30. (I) Embryos injected with Xdd1 animally at the eight-cell stage. (J) Embryos injected vegetally at the eight-cell stage with Xdd1.

View larger version (72K): [in a new window]   Fig. 3. Lineage tracing of targeted injections. (A) Control embryo. (B) Embryo injected with Xdsh-D2 into the dorsal animal two blastomeres at the 16-cell stage. Embryo displays severe dorsal flexure. (C) Fluorescence view of embryo in B; GFP fluorescence is present in the dorsalmost tissues and absent from ventral tissues. (D) Embryo injected with Xdsh-D2 into the dorsal vegetal two blastomeres at the 16-cell stage, showing short stout morphology. (E) Fluorescence view of embryo in D; GFP fluorescence is present in the both dorsal and ventral tissues and is excluded from the neural tube.

View larger version (47K): [in a new window]   Fig. 4. Mutant Xdsh inhibits neural convergent extension in Keller sandwich explants. (A) Keller sandwiches were prepared by removing the entire dorsal marginal zone, which consists of dorsal axial mesoderm (AM, red), posterior neural ectoderm (PNE, blue), and some anterior ectoderm (yellow), from two different embryos. These two explants are cultured together with the deep cells facing, and superficial epithelia (black) surrounding the recombinate. Convergent extension of neural and mesodermal portions transforms the initially rectangular explant into a stereotyped morphology with two distinct domains of elongation and a collar region at the interface. (B) The stereotypical morphology of elongated Keller sandwiches in control explants (n=15). (C) Explants expressing Xdsh-D2 fail to elongate and no distinction can be made between mesodermal and ectodermal regions (n=11/11 display reduced elongation). (D) Explants expressing Xdd1 also fail to elongate (n=5/5 display reduced elongation). (E) Observation of GFP localization (green) demonstrates targeting of injected Xdsh-D2 to the neural ectoderm. Upper panels show explant morphology, lower panels show distribution of Xdsh-D2 GFP fluorescence (green). Note that the mesoderm does not express Xdsh-D2 (arrowheads) and has elongated; the adjacent neural tissue is expressing Xdsh-D2 (green) and has failed to elongate or narrow.

View larger version (85K): [in a new window]   Fig. 5. Xdsh-D2 expression does not inhibit posterior neural cell fates. (A) Control embryo stained for HoxB9. HoxB9 staining is normal, extending from behind the head (arrow) to the tip of the tail (arrowhead). (B) Embryos injected animally at the eight-cell stage with 1 ng of Xdsh-D2; HoxB9 staining is present from the back of the head to the tip of the tail. (C) In embryos in which the posterior neural folds have completely failed to meet, two separate domains of HoxB9 expression can be seen. (c') Anterior view of the embryo in C shows the two domains of HoxB9 expression, one on each side, extending from behind the head (arrows) to the posterior limit of each side of the embryo (arrowheads). (D) Control embryo stained for Krox20. (E) Krox-20 staining pattern is normal in embryos injected animally with Xdsh-D2 mRNA at the eight-cell stage. (F) In some severely affected embryos, Krox20 staining pattern is mildly distorted, but both stripes are discernible. (d'-f') Detail of staining patterns for embryos in D-F; arrowhead marks stripe in rhombomere 3; arrows mark rhombomere 5. (G-H) Xdsh-D2 expression in the mesoderm does not inhibit posterior neural cell fates. (G) Control embryos stained for HoxB9 and Krox20. (H) Embryos injected with 1ng of Xdsh-D2 mRNA into the dorsal vegetal blastomeres at the eight-cell stage and stained for HoxB9 and Krox20.

View larger version (98K): [in a new window]   Fig. 6. Xdsh-D2 expression does not inhibit notochord differentiation. (A) Control embryos stained for notochord-specific antigen Tor70 (dorsal view, anterior towards the right). (B) Embryos injected dorsoanimally with Xdsh-D2. (C) Embryos injected dorsovegetally with Xdsh-D2. Side views of embryos are shown in a'-c'.

View larger version (57K): [in a new window]   Fig. 7. Xdsh-D2 inhibits convergent extension in XBF-2-expressing animal caps. (A) Control uninjected animal caps do not elongate (n=0/40). (B) Animal caps injected with 0.5 ng of XBF-2 elongate as a result of neural convergent extension. Some caps elongate significantly (left); others elongate slightly (middle); and some caps fail to elongate (right). 45% of caps elongated to some degree; n=55. (C) Co-expression of 1 ng of Xdsh-D2 with 0.5 ng of XBF-2 strongly suppresses elongation in all caps. Very weak elongation (see caps at far left) was observed in only 7% of caps (n=45).

View larger version (65K): [in a new window]   Fig. 8. Xdsh-D2 inhibits convergent extension in heterochronic neural plate grafts. (A) Neural plate grafts were prepared by removing the neural ectoderm of stage 11.5 embryos and grafting in animal caps from injected embryos. (B) Grafted GFP expressing animal caps are induced to become neural tissue (Mariani et al., 2001; Ribisi et al., 2000), are integrated into the host embryo, and undergo normal neural morphogenesis as shown by the convergent extension of the posterior portion of a GFP-expressing graft (b'). (C) Dorsal view of embryos with GFP-expressing grafts develop normally and close their neural tubes; GFP expression (c') demonstrates the extent of convergent extension of the graft (n=9 recombinates from three experiments). White arrows indicate the anterior and posterior limits of the GFP-expressing graft. (D,E) Dorsal view of embryos with Xdsh-D2-expressing grafts that fail to close their neural tubes (red arrowheads); GFP fluorescence of the Xdsh-D2 construct (d',e') indicates that the grafts fail to converge and extend (n=14 recombinates from three experiments). (F) Unmanipulated embryos at stage 30 are straight and elongated. (G) Embryos with GFP-expressing grafts are also elongated and straight, although they do display a very subtle dorsal flexure as a result of grafting. (H) Embryos with Xdsh-D2 grafts are elongated but develop with a pronounced dorsal flexure.

View larger version (81K): [in a new window]   Fig. 9. Neural convergent extension is required for elongation of the A/P axis. (A-D) Tor70 notochord staining of manipulated embryos. GFP grafted (A) and mock-operated (B; see Materials and Methods) embryos develop with normal A/P axes. Embryos with Xdsh-D2-expressing grafts into the neural plate (C) or with ablated posterior neural plates (D) develop with severe dorsal flexure. (E) Quantitation of notochord length in manipulated embryos, expressed relative to length of unmanipulated control embryos. Control, n=12; GFP-grafted, n=6; Xdsh-D2 grafted, n=10; mock, n=4; ablated, n=4. Data shown are mean±s.e.m.

View larger version (90K): [in a new window]   Fig. 10. Targeted injection of other Wnt antagonists has differential effects. (A) Control embryos. (B) Embryos injected animally with Xwnt5a mRNA display dorsal flexure. (C) Embryos injected vegetally with Xwnt5a are not ‘kinked’ but are foreshortened. (D) Embryos injected animally with Nxfz-8 mRNA display severe dorsal flexure. (E) Embryos injected vegetally with Nxfz-8 display two phenotypes. Many embryos do not display dorsal flexure (top), similar to other vegetally injected embryos in this study. Other embryos do display dorsal curvature (bottom). This phenotype may be due to higher diffusibility of the small, secreted Nxfz-8 protein.

View larger version (31K): [in a new window]   Fig. 11. Parallel forces in axial elongation. (A,B) In normal embryos, elongation of the posterior neural ectoderm (blue), dorsal mesoderm (red), and ventral tissues (green) all contribute to the generation of a straightened and elongate A/P axis. (C) In embryos injected dorsoanimally with Xdsh-D2, strong inhibition of neural convergent extension and mild effects on mesodermal convergent extension conspire to generate a dorsally kinked and foreshortened A/P axis. (D) In dorsovegetally injected embryos, strong inhibition of mesodermal convergent extension results in a shortened axis, while neural and ventral elongation produce a straight axis. (E) In grafted Xdsh-D2 embryos, the embryo is dorsally curved, owing to the failure of neural convergent extension, and mesodermal convergent extension elongates the axis, though not completely.