López SL et al. (1995), Differential effects of retinoic acid and a ret...

XB-ART-18951

Dev Dyn 1995 Dec 01;2044:457-71. doi: 10.1002/aja.1002040411.

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Differential effects of retinoic acid and a retinoid antagonist on the spatial distribution of the homeoprotein Hoxb-7 in vertebrate embryos.

López SL , Dono R , Zeller R , Carrasco AE .

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An antibody raised against the recombinant Xenopus laevis Hoxb-7 protein (López and Carrasco [1992] Mech. Dev. 36:153-164) recognizes the 30 kDa translation product of the Hoxb-7 gene in X. laevis and the cognate nuclear protein in chicken embryos. The X. laevis Hoxb-7 protein was expressed maternally and zygotically. Treatment of X. laevis and chicken embryos with either all-trans retinoic acid (RA) or the retinoid antagonist Ro 41-5253 (Ro; Apfel et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:7129-7133) during early development induced malformations of the neural tube and complementary changes in the expression domain of the homeoprotein Hoxb-7. Treatment of X. laevis embryos with retinoic acid during gastrulation induced an anterior shift of the Hoxb-7 expression domain and was correlated with an enlargement of rhombomere r7. In addition to a reduction in rhombomere numbers and of forebrain size, various malformations involving all three germ layers were observed. Treatment of X. laevis embryos with the antagonist Ro before or during gastrulation caused a progressive reduction of the Hoxb-7 domain and also dose-dependent malformations of all three germ layers. RA or Ro treatment of chicken embryos from the beginning of gastrulation caused changes of the Hoxb-7 expression domain very similar to those observed in X. laevis. In particular, either a dose-dependent loss of the Hoxb-7 protein in the neural tube or an ectopic expression in the forebrain region was observed. The results of this study indicate that endogenous retinoids regulate the spatial expression of homeobox-containing genes in vertebrates.

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Species referenced: Xenopus laevis
Genes referenced: hoxb7 tbx2

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	Fig. 2. Distribution of the Hoxb-7 protein in stage 47 X. laevis tadpoles following RA treatment during gastrulation. a and b: Whole mount immunostained embryos. Arrows mark the rostral (isthmus) and the caudal (obex) limits of the hindbrain a, left: Different focal plane through the same embryo to show the Hoxb-7 protein distribution. Note that its anterior boundary coincides with the junction of r6 and r7. a, right: Untreated control embryo showing the rhombomeres ot the hindbrain. Arrowheads mark the boundaries of rhombomeres r l (rostral) to r7 (caudal). b: An embryo treated with 1 pM RA during stage 10.25. Note the alteration of its rhombomere pattern and of the Hoxb-7 domain. Arrowheads mark the rhombomeric boundaries. c and d: Coronal sections to reveal the antero-posterior extension of the Hoxb-7 domain in the neural tube. The sections are counterstained with Hoechst 33258. Arrowheads mark the somite boundaries. The arrow indicates the caudal hindbrain boundary. c: Control embryo. d: Embryo treated with 0.1 FM RA during stage 10. e and f: Transverse sections at the level of the IX-X ganglionic complex. Asterisks mark the cellular population that most likely derives from the epibranchial placode. Note that these do not express the HOXb-7 protein. These photographs were taken using Nomarski interference contrast. e: Control embryo. The arrow indicates the root of the IX-X cranial ganglia. f: Embryo treated with 1 pM RA during stage 10. g and h: Transverse sections at the forelimb bud level. Photographs were taken using Nomarski interference contrast. g: Control embryo. h: Embryo treated with 1 pM RA during stage 10. Note the hypertrophic pronephric duct and tubules. b: pharyngeal pouch; e: ectodermal cells; i: isthmus; m: mesenchymal cells associated with the ectodermal rod; 0: obex; ov: otic vesicle; p: pronephros; s: somite: sl, s2: first and second somite; t: neural tube.
	Fig. 3. Alterations observed after treatment of gastrulating X. laevis embryos with Ro 41-5253. All embryos were immunostained with Hoxb-7 antibodies. a: Untreated control embryo, stage 37. The arrow points to the Hoxb-7domain within the CNS. b: An embryo treated with 7.5 pM Ro from stage 9 to 13 was fixed when control siblings reached stage 37. Note the reduced otic vesicle and eye, the latter being shifted rostrally. The branchial arches were absent and the Hoxb-7protein was not detected in the neural tube as revealed by transverse histological sections (data notshown). Note the hypertrophic coelomic cavity on the ventral side of the embryo. c: Untreated control embryo, stage 46. The arrow points to the Hoxb-7 domain in the CNS. The small arrowheads point to the rhombomeric boundaries within the hindbrain. d: An embryo treated with 1.5 pM Ro from stage 9 to 13 was fixed when control siblings reached stage 46. Note the decreased Hoxb-7protein levels in the neural tube (open arrow) and the alterations of rhombomere morphology (compare with c). b: branchial arches; c: coelomic cavity; e: eye: ov: otic vesicle.
	Fig. 4. Treatment of X. laevis embryos with Ro during morula to late blastula stages. a and b: Embryos immunostained for the Huxb-7 protein. a: Untreated control embryo, stage 33. The arrow points to the Hoxb-7 domain in the CNS. c and e indicate the planes of sections shown in panels c and e. b: Ro treated embryo (7.5 pM), fixed when control siblings reached stage 33. The head of this embryo is located to the left of the panel. Note that the eyes and otic vesicles are absent. No Hoxb-7 protein is present in the neural tube of this embryo. The hypertrophic melomic cavity is wrapped in epidermal outgrowths, as confirmed by histology. d and f indicate the planes of sections shown in panels d and f. c to f: Coronal sections of the control (c, e) and Ro treated embryo (d, f) as indicated in a and b. c: Section to show the normal distribution of the HOXb-7 protein in the neural tube. The arrowhead indicates the anterior boundary of the ffoxb-7expression domain in the neural tube (hindbrain). e: Section to show the normal HOXb-7 expression in somites (arrowheads) and the pronephros. d,f: Coronal sections of the treated embryo shown in b. Note the complete lack of Hoxb-7 expression in the neural tube and the paraxial mesoderm. The pharyngeal arches and cavity are completely absent. The coelomic cavity is hypertrophic and associated with epidermal outgrowths (see b and data not shown). ov: otic vesicle; c: coelomic cavity; n: neural tube; p: pronephros.