Int J Dev Biol
January 1, 2010;
Retinoid signalling is required for information transfer from mesoderm to neuroectoderm during gastrulation.
region of the vertebrate central nervous system
) presents a complex regionalisation. It consists of 7-8 distinct morphological segments called rhombomeres
, each with a unique identity provided by combinations of transcription factors. One class of signalling molecules, retinoids, have been shown to be crucial for hindbrain
patterning through direct trans-activation of Hox genes in the neuroectoderm
. However, how this morphogen acts is not yet fully understood. Here, we show that the retinoid receptor antagonist AGN193109 causes a posterior hindbrain
defect in Xenopus, comparable to that seen in other vertebrates. We show that this defect arises during gastrulation. Blocking endogenous retinoid activity during gastrulation causes downregulation of the most 3'' Hox genes (paralogues 1-5) in gastrula neuroectoderm
, but their initial activation in gastrula
is unaffected. Similar results were obtained in avian embryos: Vitamin A-deficient quail embryos have defective expression of 3 Hox genes (i.e. Hoxb1
) in the neural tube
, but their early expression in the primitive streak and emerging paraxial and lateral mesoderm
is not affected. In Xenopus, depletion of retinoids from mesoderm
by targeted injection of mRNAs for the retinoic acid catabolising enzyme xCYP26
and the cellular retinoic acid binding protein xCRABP
blocks 3 Hox gene expression in the overlying neuroectoderm
. We propose that the gastrula
and its later derivative, the paraxial mesoderm
, is the source of a retinoid, which acts as a transforming (caudalising) signal for the future posterior hindbrain
Int J Dev Biol
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Fig. 1. AGN193109 efficiently depletes retinoid signalling in vivo. (A) The RAR antagonist AGN193109 rescues the RA phenotype. Xenopus laevis embryos incubated in 10-6 M RA show anterior truncations. Embryos incubated in 10-6 M and 10-7 M AGN show a shorter hindbrain area and a large heart oedema with the phenotype being more severe at 10-6 M. When embryos are incubated in equal concentrations (10-6 M) of RA and AGN the resulting phenotype is more like the AGN phenotype. When 10-6 M RA is combined with 10-7 M AGN the phenotype is more like an RA phenotype. NT (control).
Fig. 1. AGN193109 efficiently depletes retin- oid signalling in vivo.(B) AGN treatment caused severe brain malformations. CLSM images of stage 45 tadpole brains labelled with Xen1 and 2G9 antibodies. (B) Control embryo, treated with 0.1% DMSO. (C) 10-6 M AGN treated embryos (fb: forebrain; mb: midbrain; hb: hindbrain, rn refer to rhombomere numbers). In AGN treated embryos, the number of rhombomeres was reduced to 4 or 5.
Fig. 2. Retinoid depletion causes radical molecular truncation of the posterior hindbrain by the end of gastrulation. (Top panel) Whole- mount in situ hybridizations (wISH) on st. 20 Xenopus laevis embryos (A- F). The upper row shows non-treated embryos (indicated by control) and the bottom embryo is treated with 10-6 M AGN (indicated by AGN). All views are dorsal and anterior at the top. (A) Hoxb-1, arrowhead indicates hindbrain expression; (B) Hoxd-3, ar-
rowhead indicates hindbrain expression; (C) En2, Krox-20 and Hoxb-4, top arrowhead indicates En stripe, bottom arrowheads indicate Krox-20 stripes and bar indicates Hoxb-4 stripe; (D) Hoxa-5, arrowhead indicates hindbrain expression and bar indicates spinal cord expression; (E) Krox-20 and Hoxc- 6, arrowhead indicates posterior Krox-20 stripe, bar indicates Hoxc-6 expression; (F) Otx-2 and Xcad3, bar indicates gap between Otx-2 (anterior) and Xcad3 (posterior) expression patterns; (G) XlPOU2, arrowhead indicates hindbrain expression and bar indicates spinal cord expression. (Bottom panel) Whole-mount in situ hybridizations on st. 13 Xenopus laevis embryos (H-M). The upper row shows non-treated embryos (indicated by control) and the right embryo is treated with 10-6 M AGN (indicated by AGN). All views are dorsal and anterior at the top. (H) Hoxd-1; (I) Hoxa-1; (J) Hoxb-1; (K) Hoxd-3; (L) Krox-20 (anterior stripes) and Hoxb-4; (M) Krox-20 (anterior stripes) and Hoxc-6. Arrows in pictures (L,M) localise sparse cells representing the posterior stripe of Krox-20.
Fig. 3. The molecular identity of the hindbrain is determined by retinoid signalling mostly during gastrulation. Whole-mount in situ hybridizations on tadpole (st. 32) Xenopus laevis embryos. The upper row shows non-treated embryos; the middle row embryos treated with 10-6 M AGN from the blastula until the point of fixation; the lowest row embryos treated with 10-6 M AGN from st. 13 until the point of fixation. All views are lateral. (A) Hoxb-1, (B) Hoxd-3, (C) En2, Krox-20 and Hoxb-4, (D) Hoxa-5, (E) Krox-20 and Hoxc- 6. Arrows point to the anterior expression border of each Hox gene.
Fig. 4. AGN incubation affects early expression of Hoxd-1 in the neuro- ectoderm but not in the underly- ing mesoderm. Whole-mount in situ hybridizations on Xenopus laevis em- bryos. Hoxd-1 expression at st. 11 A-C) or st. 12.5 (D-F). Embryos were ncubated with 10-6 M AGN (A,D), 10-6 M RA (C,F) or not treated (0.1% DMSO) (B,E). After photographing the em- bryos were cut along the indicated dashed line and a lateral view of the cut surface is shown next to the right of each embryo. Arrows in (D,E) point to the faint mesodermal expression remaining at that stage (mostly non- involuted mesoderm). Hoxc-6 expres- sion on stage 12 embryos (G-I). Treat- ment with 10-6 M AGN (G), 10-6 M RA (I) or not treated (H).
Fig. 5. Initial mesodermal versus later neural plate expression of Hoxb-1 and Hoxb- 4 expression patterns. Wild-type early (A) and late (C) HH st. 4 embryos are compared to equivalent VAD embryos (B,D); both show expression of Hoxb-1 in the primive streak and later in migrating ingressed cells. Hoxb-4 expression patterns are also shown for HH st. 4 wild-type (E) and VAD (F) embryos, along the primitive streak. At HH st. 8 Hoxb-4 expression pattern includes the neural tube in wild-type (G) but not in VAD (H) embryos. All views are dorsal.
Fig. 6. Effects of targeted retinoid signalling removal in the mesoderm. Whole-mount in situ hybridizations on Xenopus laevis embryos. Hoxa-1 (A) and Hoxb-1 (B) expression at st. 13. NIC: non-injected controls. xCYP26 and xCRABP: Injection of 100 pg xCYP26: 100 pg xCRABP mRNA four times, one time into each macromere at 8-cells stage. Whole embryos (top picture) are shown in a dorsal view with anterior being up. Cut embryos (bottom picture) are shown in a lateral view.
pou3f4 (POU class 3 homeobox 4) gene expression in Xenopus laevis embryos, NF stage 20, assayed by in situ hybridization. dorsal view, anterior up.