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The vertebrate central nervous system (CNS) is induced by signals emanating from the dorsal mesoderm, or organizer, that divert the ectoderm away from an epidermal and towards a neural fate. Additional signals from the organizer pattern the neural ectoderm along the anteroposterior axis. We devised highly specific methods utilizing constitutively active or dominant negative receptors to evaluate the role of retinoids in neural patterning. Microinjection of these reagents either augments or reduces retinoid signaling in specific regions of the embryo. We show that increased receptor activity suppresses anterior neural structures while dominant negative receptors lead to anterior enhancement. Similarly, microinjection of the dominant negative receptor leads to the loss of posterior marker genes. We demonstrate that retinoid receptors comprise a critical component in neural posteriorization and are required for proper neuronal differentiation. These results support a quantitative role for retinoid signaling in regionalization of the CNS.
Fig. 1. Activity of mutant xRARs in vitro and in vivo. (A) Schematic diagram of xRARα expression constructs utilized in this study. (B) Effect of wild-type and mutant xRARαs on reporter gene response to all-trans RA in transfected CV-1 cells. (C) Effect of wild-type and mutant xRARs on reporter gene response to all-trans RA in microinjected embryos. (D) Wild-type and mutant xRARs alter the morphological response to exogenous all-trans RA in microinjected Xenopus embryos. (E) Wild-type (xRARα1) and dominant negative RARs (xRARα1405*) bind a retinoic acid response element whereas the control construct (xRARα2386*) does not.
Fig. 2. Constitutively active and dominant negative xRARα have apparently opposite effects on embryonic pattern formation. Wild- type and mutant xRAR mRNAs were evaluated for their ability to alter morphology along the A/P axis. Injections or treatments were as follows: (A) uninjected, (B) xRARα1405*, (C) VP16-xRARα1, (D) 10−7 M tRA. mRNAs were injected bilaterally at the two-cell stage and the embryos allowed to develop until fixation at stage 38-40. Microinjections were performed with both α1 and α2 dominant negative receptors. The strongest and most consistent phenotypes were elicited by xRARα1405* and this receptor was utilized for subsequent studies.
Fig. 3. Mutant xRARα mRNAs alter patterning along the anteroposterior axis. Position specific molecular markers were employed to evaluate the effects of xRARα mRNA injection on A/P neural specification. In situ hybridization analysis of stage 16-18 embryos. In each panel the staining pattern on the injected side should be compared with that on the control, uninjected side. (A-D) The injected side, marked by X-gal staining, is oriented to the right, in all other panels the injected side is on the bottom. (A-H) Xotx-2 (Blitz and Cho, 1995), (I-L) Xen-2 (Hemmati-Brivanlou and Harland, 1989), (M-P) Xen-2 and xKrox-20 (Bradley et al., 1993), (Q-T) Hox-B9 (Wright et al., 1990). (C,D,G,H) The line is oriented at the posterior boundary of Otx-2 staining, the arrow indicates regions of increased (C,G) or decreased (D,H) Otx-2 staining. (K,L) The line is parallel with the anterior edge of the neural folds, arrows indicate the location of En-2 staining. (O,P) The line is parallel with the anterior edge of the neural folds, arrows indicate En-2 and Krox-20 staining on the injected side. (Q-T) Arrows indicate the anterior border of Hox-B9 staining on the injected side, the lack of an arrow in S indicates no staining. Note that the β-galactosidase injected embryos in A,E,J,M,Q are younger than the others resulting in a broader neural plate, however, there is no difference in marker gene expression between the injected and uninjected sides of the embryo.
Fig. 4. Retinoid signaling is required for neuronal differentiation. Markers of both neural position and neuronal differentiation were employed to evaluate requirements for xRAR signaling. In situ hybridization analysis of stage 16 embryos (A-H) Xlim-1 (Taira et al., 1992), (I-L) N- tubulin (Chitnis et al., 1995).
