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Abstract
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.
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.
