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Treatment of late blastula/early gastrula stage Xenopus embryos with all-trans retinoic acid results in disruption of the primary body axis through effects on both mesoderm and neuroectoderm. This effect of retinoic acid, coupled with the known presence of retinoic acid in Xenopus embryos has led to the proposal that retinoic acid may be an endogenous morphogen providing positional information in early development. To further elucidate the role of retinoic acid in early Xenopus development, we have attempted to interfere with the retinoic acid signalling pathway both at the level of retinoic acid formation, by treatment with citral (3,7-dimethy-2,6-octadienal), and at the level of nuclear retinoic acid receptor function, by microinjection of v-erbA mRNA. The feasibility of this approach was demonstrated by the ability of citral treatment and v-erbA mRNA injection to reduce the teratogenic effects of exogenous retinol and retinoic acid, respectively, in early Xenopus development. Interestingly, v-erbA mRNA injection and citral treatment of gastrula stage embryos resulted in tadpoles with a similar set of developmental defects. The defects were chiefly found in tissues that received a contribution of cells from the neural crest, suggesting that at least a subset of neural crest cells may be sensitive to the endogenous level of retinoic acid. In accord with this proposal, it was found that the expression patterns of two early markers of cranial neural crest cells, Xtwi and XAP-2, were altered in embryos injected with v-erbA mRNA. These results indicate that structures in addition to the primary axis are regulated by retinoic acid signalling during early Xenopus development.
Fig. 1. v-erbA rescue of RA-treated embryos. (A) Uninjected
embryos and embryos microinjected with v-erbA mRNA or the verbA
mutant 82t mRNA were treated with 2 mM RA for 25-40
minutes at stage 9.5-10.5. The number of hatched embryos and the
presence of cement glands, eye tissue or two complete eyes was
scored on day 3 or 4 (n = number of embryos scored). (B) 4-cell
embryos were injected in two dorsal or two ventral blastomeres
prior to RA treatment as described for A above. (C) Example of verbA
rescue of RA-treated embryos; left, 1 untreated embryo;
bottom row, range of phenotypes of RA-treated embryos; top row,
range of phenotypes of RA-treated embryos injected with v-erbA
mRNA.
Fig. 2. Effects of v-erbA on RA- or activininduced
Xlim-1 expression in animal pole
cell explants. Animal pole cell explants
from uninjected, 82t and v-erbA mRNAinjected
embryos were treated with (A) RA
(5 mM), or (B) activin (4 ng/ml) for 6-7
hours. Induction of Xlim-1 expression was
assessed by RNAse protection assays. An
EF-1 probe was included as a control to
assess relative RNA loading. Control
samples contained only tRNA and probes.
The results from one representative
experiment are shown.
Fig. 3. Effects of v-erbA on
Xenopus development.
Uninjected embryos and embryos
injected with 1.6-4 ng of v-erbA
or 82t mRNA were examined at
various stages of development.
(A) Anterior view of late neurula
stage embryos unilaterally
injected with v-erbA mRNA.
Arrowhead indicates reduced
cement gland formation and
arrow indicates reduced head fold
on side of the embryo that
received the v-erbA mRNA
injection. Dorsal is at the top.
(B) Lateral view of uninjected
(left) and v-erbA mRNA-injected
(right) tailbud stage embryos.
Anterior is right, dorsal is at top.
82t mRNA-injected embryos
appeared to be identical to
uninjected embryos. (C) Dorsal
view of one uninjected (left) and
three v-erbA mRNA-injected
tadpole stage embryos. Anterior
is at the top, except for the third
embryo which is in the reverse
orientation. 82t mRNA-injected
embryos appeared to be identical
to the uninjected embryo shown.
(D) Dorsal-lateral view of eight
tadpole stage embryos injected
with v-erbA mRNA (right) and
two 82t mRNA-injected controls (left). Note the sharp dorsal bend in the primary axes of the v-erbA mRNA-injected embryos. (E) Lateral
view of tadpole stage embryos (top four) depicting the range of effects of a high level (4-6 ng) of v-erbA mRNA injection. An uninjected
control embryo is shown at the bottom. Anterior is at the right. Injection of equivalent amounts of 82t mRNA had no apparent effects on
embryonic development.
Fig. 4. Effects of dorsal versus ventral v-erbA mRNA injection on
Xenopus development. (A) Four-cell embryos were injected with
one of the indicated RNAs in two dorsal or two ventral
blastomeres and embryos were scored at the tadpole stage (see
Fig. 3C). Data presented are the combined results from two
experiments. Number of embryos n=55 (uninjected), n=45 (82t),
n=17 (ventral v-erbA), n=30 (dorsal v-erbA). (B) Embryos were
injected with v-erbA mRNA at the two-cell stage and the levels of
v-erbA protein present at the indicated stages was assessed by
immunoblotting. Arrow indicates the v-erbA protein (Mr 75´103)
which was not present in uninjected controls. Similar results were
obtained using an antiserum (gift of R.N. Eisenman) that
recognizes the gag-portion of v-erbA.
Fig. 5. Sections of control and v-erbA mRNA-injected embryos. Uninjected embryos and embryos injected with v-erbA or 82t mRNA at
the two-cell stage were fixed, sectioned and stained. In all sections examined, 82t mRNA-injected controls appeared identical to
uninjected controls and showed no obvious developmental defects. (Magnification = 62.5´). (A,B) Lateral sagittal sections of control
(uninjected) (A), and v-erbA mRNA-injected (B) embryos at the tailbud stage. Arrowheads indicate the visceral pouches that partition the
intervening branchial arches in the control embryo, which are absent or present in a rudimentary form in the v-erbA mRNA-injected
embryo. Dorsal is at the top, anterior is to the right. (C,D) Ventral coronal sections of control (82t mRNA-injected; C) and v-erbA mRNAinjected
(D) embryos at the tailbud stage. Arrowheads indicate branchial arches. Anterior is to the right. (E,F) Mid-sagittal sections of
control (uninjected; E) and v-erbA mRNA-injected (F) embryos at the tailbud stage. The foregut is designated by f and the arrow indicates
the region ventral to the notochord discussed in the text. Dorsal is at top, anterior is to the left. (G,H) Transverse sections of control
(uninjected; G) and v-erbA mRNA-injected (H) embryos at the tailbud stage. Arrows indicate developing heart. Dorsal is at the top.
(I,J) Coronal sections of control (82t mRNA-injected) (I) and v-erbA mRNA-injected (J) embryos at the tadpole stage. The ventral portion
of the eye is designated by e; arrow indicates gut structures. Anterior is to the left.
Fig. 6. Effects of v-erbA on Xtwi and XAP-2 expression. Wholemount in situ hybridization analysis of Xtwi (A,B) and XAP-2 (C,D) expression in control (82t mRNAinjected; A,C) and verbA mRNA-injected (B,D) embryos (stage 23). Arrows indicate staining of hyoid crest cells, which are found just posterior to mandibular crest cells (surrounding the future eye) and anterior to the anterior and posterior components of the branchial arch crest. Dorsal is at the top in all panels.
Fig. 7. Rescue of retinol-treated embryos by citral. Stage 9.5-10.5
embryos were treated with 25 mM retinol +/- 30 mM citral or
related compounds overnight (approx. 16 hours). (A) Lateral view
of three retinol-treated embryos (right), three embryos treated with
retinol and citral (left), and one untreated embryo (bottom) at the
tadpole stage. (B) Embryos treated with retinol +/- citral or
related compounds were scored on day 4 for the presence of
cement gland, eyetissue or two complete eyes. The results from
one of two representative experiments is presented. n = number of
embryos scored. citral: 3,7-dimethyl-2,6-octadienal; nerol: 3,7-
dimethyl-2,6-octadien-1-ol; nitrile: 3,7-dimethyl-2,6-
octadienenitrile.
Fig. 8. HPLC analysis of the effects of citral on endogenous RA levels in Xenopus embryos. (Left) Chromatogram of reversed phase
HPLC separation of a mixture of authentic retinoid standards. Peaks: a, 4-oxo-all-trans RA (810 ng/ml); b, 4-oxo-13-cis RA (1050
ng/ml); c, all-trans-RAG (300 ng/ml); d, 13-cis-RA (265 ng/ml); e, 9-cis-RA (283 ng/ml); f, all-trans RA (216 ng/ml); h, retinol (700
ng/ml). Sensitivity: 0.04 AUFS. (Middle) Sample chromatogram of HPLC analysis of retinoids extracted from 100 stage 12 Xenopus
embryos. Peak c coeluted with the authentic all-trans-RAG (242 ng/g); peak d coeluted with the authentic 13-cis-RA (102 ng/g); peak e
coeluted with the authentic 9-cis-RA (160 ng/g); peak f coeluted with the authetic all-trans-RA (100 ng/g); peak g coeluted with the
authentic didehydro-retinol (3104 ng/g); peak h coeluted with the authentic retinol (2748 ng/g) and peak i coeluted with the authentic
retinal (240 ng/g). Sensitivity: 0.005 AUFS. (Right) Sample chromatogram of HPLC analysis of retinoids extracted from 100 stage 12
Xenopus embryos treated with 60 mM citral for 2.5 hours. Peak a coeluted with the authentic 4-oxo-all-trans-RA (120 ng/g); peak b
coeluted with the authentic 4-oxo-13-cis-RA (240 ng/g); peak c coeluted with the authentic all-trans-RAG (238 ng/g); peak d coeluted
with the authentic 13-cis-RA (44 ng/g); peak f coeluted with the authentic all-trans-RA (38 ng/g); peak g coeluted with the authentic
didehydro-retinol (3442 ng/g); peak h coeluted with the authentic retinol (3200 ng/g); peak i coeluted with the authentic retinal (840
ng/g). Sensitivity: 0.005 AUFS.
Fig. 9. Effects of citral
on early Xenopus
development.
(A) Lateral view of
tailbud stage embryos
untreated or treated for
16 hours with
increasing
concentrations of
citral, beginning at
stage 10. Top two
embryos are controls;
bottom, left to right,
embryos treated with
20 mM, 40 mM, and
60 mM citral,
respectively. Anterior
is at right. (B) Dorsal
view of control (left)
and citral-treated
(60 mM, 16 hours
beginning at stage 10)
embryos at the tadpole
stage. Anterior is at
right for control
embryos and left for
citral-treated embryos. (C) Embryos treated with 60 mM citral for 16 hours beginning at the indicated stages were scored for abnormal
head morphology at the tadpole stage. Numbers above bars indicate the total number of embryos scored in two independent experiments.