Schuh TJ et al. (1993), v-erbA and citral reduce the teratogenic effect...

XB-ART-22063

Development 1993 Nov 01;1193:785-98.

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v-erbA and citral reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, in Xenopus embryogenesis.

Schuh TJ , Hall BL , Kraft JC , Privalsky ML , Kimelman D .

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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.

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Species referenced: Xenopus
Genes referenced: lhx1 mt-tr tfap2a thra trna twist1
GO keywords: axis specification [+]

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	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, eye tissue 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.