Dufour S et al. (1994), Differential perturbations in the morphogenesis...

XB-ART-20718

J Cell Biol 1994 Oct 01;1272:521-35.

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Differential perturbations in the morphogenesis of anterior structures induced by overexpression of truncated XB- and N-cadherins in Xenopus embryos.

Dufour S , Saint-Jeannet JP , Broders F , Wedlich D , Thiery JP .

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Cadherins, a family of Ca-dependent adhesion molecules, have been proposed to act as regulators of morphogenetic processes and to be major effectors in the maintenance of tissue integrity. In this study, we have compared the effects of the expression of two truncated cadherins during early neurogenesis in Xenopus laevis. mRNA encoding deleted forms of XB- and N-cadherin lacking most of the extracellular domain were injected into the four animal dorsal blastomeres of 32-cell stage Xenopus embryos. These truncated cadherins altered the cohesion of cells derived from the injected blastomeres and induced morphogenetic defects in the anterior neural tissue to which they chiefly contributed. Truncated XB-cadherin was more efficient than N-cadherin in inducing these perturbations. Moreover, the coexpression of both truncated cadherins had additive perturbation effects on neural development. The two truncated cadherins can interact with the three known catenins, but with distinct affinities. These results suggest that the adhesive signal mediated by cadherins can be perturbed by overexpressing their cytoplasmic domains by competing with different affinity with catenins and/or a common anchor structure. Therefore, the correct regulation of cadherin function through the cytoplasmic domain appears to be a crucial step in the formation of the neural tissue.

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Species referenced: Xenopus laevis
Genes referenced: arhgef5 kcnt1

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	Figure 1. Schematic representation of the truncated NXB-, and N-cadherin, and NcadNCAM hybrid. (,4) The truncated Xenopus N-cadherin/N-CAM hybrid protein is encoded by the pANcadNCAM mRNA and contains N-CAM transmembrane and cytoplasmic domains of the 140-kD mol mass spliced variant form juxtaposed to the 40 aminoterminal aa of the pANcad sequence (B) The truncated Xenopus N-cadherin protein is deleted in its extracellular region from aa 31 to aa 698, and it is encoded by the pANcad mRNA. (C) The truncated Xenopus N-cadherin/XB-cadherin hybrid protein contains N-cadherin aa sequences from aa 1 to aa 30 and aa 699 to aa 769 linked to the XB-cadherin aa sequences between aa 303 and aa 446. The tag VSVG-16 epitope is localized between aa 40 and 41 of the extracellular domain of all the truncated constructs. TM, transmembrane domain; EC, extracellular domain; sig, signal peptide; pre, propeptide sequence; VSVG-16, vesicular stomatitis virus glycoprotein carboxyterminal epitope recognized by a specific P5D4 monoclonal antibody and used as a tag for immunocytoehemistry and whole-mount immunostaining.
	Figure 2. In vivo translation ability of mRNA coding for truncated cadherins or truncated N-cadherin/N-CAM hybrid. The mRNA were coinjected with 35S-trans-label mixture at the two-cell stage of Xenopus embryos. At stage 13-13.5NF, the embryos were homogenized in immunoprecipitation buffer and incubated with anti-VSVG monoclonal antibody P5D4. The radiolabeled proteins immunoprecipitated were loaded on 12% acrylamide gel and revealed by autoradiography. Lane a, truncated NcadNCAM hybrid; lane b, truncated N-cadherin; lane c, truncated NXB-cadherin. Low molecular mass markers electrophoretic mobility are indicated on the left.
	Figure 3. Fate map of the four animal dorsal blastomeres injected at the 32-cell stage. (.4) Lateral view of embryos injected with 0.25 ng of/3-galactosidase mRNA (Ix concentration) at the 32-cell stage and processed for X-gal staining at stage 25NF. X-gal activity is restricted to the anterior neural structures. Staining in the endodermal mass is artefactual. (B) In the inset, a dorsal anterior view of an injected embryo where X-gal staining is localized to the optic vesicles, the brain, the cement gland, and to a lesser extent in the ectoderm and head mesenchyme. (C) Injection sites on a 32-cell stage Xenopus embryo. The four animal dorsal blastomeres (4-AD) A1, A2 (Dale and Slack, 1987) and their A'I, A'2 counterparts across the midline (arrowheads) were the target blastomeres. They are less pigmented and smaller in size than blastomeres of the ventral side. (D) Embryos injected with ~-gaiactosidase mRNAs are processed for X-gal staining before sectioning at stage 25NE Cells expressing the B-galactosidase activity are restricted to the neural lineage and the cement gland. Bar, 260 gm in A, 200 gm in B, 400 #m in C, and 106 gm in D.
	Figure 4. Perturbations produced by injection of pANXBcad mRNAs at 1× concentration. Whole-mount immunostaining of an injected (A and B) and control (D and E) embryo at the stage 32NF with the anti-N-CAM 4D monoclonal antibody and the 12/101 monoclonal antibody directed against skeletal muscle. A and D are the lateral views, and B and E are the dorsal views. Numerous N-CAM positive cell clusters are observed (B, arrowheads), as well as a delayed development of eye and of cranial nerve emergence (A, arrows) in the case of the injected embryos when compared to the control (D, arrows, and E). No defect in somitic development is detectable at this stage. Morphology of an injected (C) and control (F) embryo at stage 4 INE Abnormal development of anterior structure is visible in C, which represents a typical injected embryo. The main defect is observed on the eye development (arrowheads). Bar, 290 #m in A, B, D, and E and 640/zm in C and E
	Figure 5. (A) Quantification of the perturbations obtained for the different truncated cadherins: the results are expressed in percent of embryos with malformations for increased concentrations of mRNA (0.1 x, 1 x, and 5 x concentrations corresponding to 0.025, 0.25, and 1.25 ng of mRNA per blastomere, respectively). Increased concentrations of mRNA induce more severe malformations in anterior structures. (B) Grades of perturbations induced by the injection of the different mRNA. The grades were scored by determining the percent of perturbed embryos showing a perturbed morphogenesis of the retina that remains fused to the central nervous system (lefipanel) or a disorganization in the retinal layers (right panel). pANXBcad mRNA at the 1 x and 5 x concentrations primarily induced severe developmental defects of anterior structures, with the retina remaining attached to the floor of the diencephalon. In contrast, similar concentrations of pANcad mRNA induced mainly abnormalities in the organization of the plexiform layers of the retina. Embryos injected with pANcadNCAM mRNA exhibit only perturbations in the organization of the retinal layers.
	Figure 6. Ramon y C~jtd trichrome histological staining of stage 41NF embryos. Histologically stained section of a control embryo (A) showing, from left to right, the brain with an eye on each side and the pharynx. (D) Typical embryo showing perturbations in retinal cell layers (arrowhead). The mesencephalon and the pharynx have a normal morphology and localization, while the organization of the retinal layers are altered with additive photoreceptor and neuropil clusters clearly observed at higher magnification (E and F, arrowheads and arrows). (G, J, and K) Another type of perturbation observed after injections of mRNA coding for truncated NXB-cadherin at Ix or 5 x or N-cadherin at 5 × concentration. Optic vesicles exhibit strong defects in their morphogenesis with retinas remaining fused to the ventral part of the brain (G, arrowheads). They either exhibit additive photoreceptor and neuropil clusters (J, arrowheads) or more drastic perturbations with delayed and aborted morphogenesis of the eye (K). Morphology of a control eye (B and C, at higher magnifications). From left to right on B and from top to bottom on C, the pigmented retinal epithelium (pe), the outer segment of the photoreceptors (os), the nuclear layer (nl), the plexiform layer (pl), and the ganglion cell layer (gcl) can be identified. Higher magnifications of a representative retina showing perturbation in its cell layer organization (E and F). Note a neuropil clustering near the photoreceptor layer (arrow) and an additional photoreceptor cell clustering in the nuclear layer (arrowheads). Higher magnification of fused retinas (H, I, and L) showing also stronger alterations of the eye structure represented by multiple small retina arrangements composed of all cell layers that were juxtaposed into the same eye cup (H and/, neuropil layer, arrows; double-photoreceptor layer, arrowhead). Bar, 100 #m in A, D, G, J, and K, 23 tim in L, 20 #m in B, E and H, and 10 gm in C, F and L C and F are made with the Nomarski optical system.
	Figure 7. Sections of a stage 23NF embryo injected with pANXBcad mRNA and/3-galactosidase mRNA (A), with pANXBcad mRNA (B and C), or with pANcadNCAM mRNA (D). In A, the embryo was processed for X-gal staining before sectioning. The sections are stained with the anti-N-CAM polyclonal antibody (B), and with the P5D4 monoclonal antibody directed against the epitope "tag" (C and D). (A) Many/3gal-positive cells are seen in the diencephalon, in the optic vesicle, and in the cement gland. Note that these cells do not have a radial arrangement (arrowheads), as compared to their unlabeled or very weakly labeled neighbors. Most strongly labeled/3galpositive cells are found as clusters at the vicinity of the eye cup (arrows), a few are also seen in the lumen of the central nervous system. (B) These cells (arrows), as well as all the neuroepithelial cells, express N-CAM. (C) The truncated cadberin was mostly detected in the intensely labeled/3gal-positive cells outside the optic cup. (D) The truncated N-CAM is expressed in a number of well-organized neuroepithelial cells. Bar, 50 #m in A, B, and C, and 30/~m in D. de, diencephalon; ov, optic vesicle; ce, cement gland.
	Figure 8. Catenins interact with truncated cadherins in injected embryos, mRNA were coinjected with 35S-Trans-label mixture at the two-cell stage of Xenopus embryos. At stage 13-13.5NF, the embryos were homogenized in SDS-free immunoprecipitation buffer and incubated with P5D4 monoclonal antibody. The radiolabeled proteins immunoprecipitated were loaded on 7.5 % acrylarnide gel and revealed by autoradiography. The catenins (arrows) coprecipitating with the truncated N-cadherin and NXB-cadherin (arrowheads) were shown respectively on lanes A and B. Molecular weight markers were indicated on the left.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase