López SL and Carrasco AE (1992), Retinoic acid induces changes in the localizati...

XB-ART-24053

Mech Dev 1992 Feb 01;363:153-64. doi: 10.1016/0925-4773(92)90066-s.

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Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

López SL , Carrasco AE .

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We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.

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Species referenced: Xenopus laevis
Genes referenced: hoxa6 hoxb7 hoxc6 rara rarb

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	Fig. 1. Inmunolocalization of Xebl and Xeb2 protein in untreated Xenopus laevis embryos. (A) Stage 30 embryo, showing Xebl protein distribution in the neural tube and the metamerization points of the pronephros (arrowhead). Notice labelled cells migrating between the neural tube and the lateral mesoderm. (B) The same as shown in panel A, but at higher magnification. See nuclear staining of cells spreading through the pronephros and lateral mesoderm (arrowhead). ov, otic vesicle. (C) Dorsal view of a whole mount stage 45 embryo stained with Xeb2 antibody. Notice strong labelling in the IX-X ganglia (arrow) and in the caudal hindbrain. The branchial arches are also stained (E, F, G) and indicate planes of sections shown in panels E-G. (D) The same embryo as in panel C, but slightly shifted laterally to show Xeb2 expression in the lung bud (arrow). t indicates plane section shown in panel I. (E) Transverse section of stage 45 embryo at the level of the basibranchial region, showing nuclear staining with Xeb2 antibody (arrowhead). (F) Transverse section of the same embryo as in 1E, but at the level of the nucleus of the IX-X ganglia (ga), (arrow). (G) A more posterior section of the same embryo, showing labelled cells spreading into the fore-limb rudiment. lb, limb bud. (H) Transverse section of the same embryo at the level of the caudal hindbrain. Notice cells expressing Xeb2 at high levels located dorso-laterally in the neurectoderm. (I) A more posterior section at the level of the caudal hindbrain, showing labelled cells in the neuroepitelium and a very strong staining in the lung buds (arrow). lu, lung bud.
	Fig. 2. Stage 37 embryos treated with RA at stage 11 and stained with Xebl (left figures) and Xeb2 (right figures) antibodies. (A, B) control embryos. (C, D) Embryos treated with 10 -6 M RA. (E, F) Embryos treated with 10 -5 M RA. Notice the ectopic site of Xebl expression in Fig. 2C and the enlargement of the Xeb2 expression domain that reaches the anterior end of the CNS (Fig. 2F).
	Fig. 3. Stage 37 embryos treated with 10 -7 M RA at different stages of gastrulation and stained with Xeb2 antibody. (A, B) Control embryos at different magnification. (C, D) Embryo treated at stage 10.25. (E, F, H) Embryo treated at stage 11. (F, H) two different optic planes. (G) treatment at stage 12. Notice the anteriorization of the domain of Xeb2 expression in the hindbrain and the branchial arches upon treatment with RA. See also the disorganization of the pronephros (arrow, panel E), the labelled cells in the migratory postotic stream (arrow, panel H), and the displacement of the otocyst and the malformed eyes to more anterior positions (F). ov, otic vesicle.
	Fig. 4. Stage 37 embryos treated during gastrulation with increasing concentrations of RA, and stained with Xebl (upper embryo) or Xeb2 (lower embryo) antibodies. Con, control embryos. (A, B, C) 10 -7, 10 -6, 10 -5 M RA treated embryos at stage 10.25.
	Fig. 5. Scheme illustrating the current hypothesis leading to displacement of a putative morphogen gradient. The indicated changes in the gradient moves the Xeb2 expression boundary to a more anterior position whereas the Xebl domain is not affected by the exogenously applied concentration of RA.