Xanthos JB et al. (2005), Cloning and developmental expression of Xenopus...

XB-ART-2134

Dev Dyn 2005 Jun 01;2332:631-7. doi: 10.1002/dvdy.20358.

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Cloning and developmental expression of Xenopus Enabled (Xena).

Xanthos JB , Wanner SJ , Miller JR .

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Regulation of actin dynamics, organization, and interaction with cell surface adhesion proteins is critical for tissue morphogenesis during development. The Ena/VASP family of actin-binding proteins function in several cellular processes that involve dynamic regulation of the actin cytoskeleton, including axon guidance, platelet aggregation, cell migration, and cell adhesion. The vertebrate Ena/VASP family is composed of three genes: Ena (Enabled), VASP (Vasodilator Stimulated Phosphoprotein), and Evl (Ena/VASP-Like). To better understand the role of Ena/VASP proteins during vertebrate development, we have cloned and characterized the developmental expression of Ena in Xenopus laevis. Analysis of the temporal expression of Xenopus Ena (Xena) demonstrates that multiple isoforms of Xena are detected throughout embryogenesis and that the presence of different isoforms is developmentally regulated. In situ hybridization analyses reveal that Xena is broadly expressed throughout development. During gastrulation and neurulation, Xena is detected in the neuroepithelium, notochord, and somites. In tadpoles, Xena expression is restricted to dorsal regions of the brain, whereas it is expressed at lower levels throughout the spinal cord. Xena expression is also detected in the notochord, myotome, heart, pronephros, and cranial placodes, including the olfactory and otic placodes. Analysis of the subcellular localization of Xena using a GFP fusion protein revealed that Xena localizes to adherens junctions and focal adhesions in Xenopus animal caps and NIH3T3 fibroblasts, respectively. These results define spatiotemporal windows in which Xena may function during early Xenopus development to modulate actin-dependent processes such as cell adhesion and migration.

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Species referenced: Xenopus laevis
Genes referenced: actl6a enah evl pxn uqcc6 vasp

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	Figure 3. Spatial expression of Xena transcripts during Xenopus development. Whole-mount in situ hybridization was carried out using a digoxigenin-labeled riboprobe. A-F: Xena expression in stage 14 (A), stage 20 (B), stage 25 (C), stage 28 (D), and stage 35 (E,F) embryos. G-I: Transverse sections of stage 35 embryos at the level of the eyes (G), otic vesicle/hindbrain (H), and trunk (I). Nieuwenkoop-Faber stages are indicated in lower right corners. Anterior is to the left in A and B. br, brain; ey, eye; nt, neural tube; ol, olfactory placode; ot, otic vesicle; myo, myotome; no, notochord; pn, pronephros; pnd, pronephric duct; h, heart.
	Fig. 2. Expression of Xena during Xenopus development. A: Western blot analysis of Xena expression using anti–Xena-DELI polyclonal antibodies. B: Western blot analysis of Xena expression using anti-Mena monoclonal antibodies. Nieuwenkoop–Faber stages are indicated at the top of each blot. UE, unfertilized egg
	Fig. 4. Xena localizes to sites of cell– cell and cell–matrix adhesion. A–C: Localization of Xena-GFP (A, green in C) and actin (B, red in C) in Xenopus animal cap cells. Xena-GFP colo- calizes with actin at adherens junctions (colocalization appears yellow in C). D–G: Localization of Xena-GFP (D), Paxillin (E), and actin (F) in NIH-3T3 ﬁbroblasts. Xena-GFP colocalizes with paxillin in focal adhesions at the tips of actin stress ﬁbers (arrowheads). Colocalization of Xena-GFP, paxillin, and actin appears white in G.