Kolm PJ et al. (1997), Xenopus hindbrain patterning requires retinoid ...

XB-ART-15587

Dev Biol 1997 Dec 01;1921:1-16. doi: 10.1006/dbio.1997.8754.

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Xenopus hindbrain patterning requires retinoid signaling.

Kolm PJ , Apekin V , Sive H .

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We have asked how posterior neural tissue is patterned in Xenopus by assaying the involvement of endogenous retinoic acid (RA) in this process and by using the labial Hox gene, HoxD1, as a posterior marker. Although RA is able to inhibit anterior gene expression and activate expression of more posterior genes, the normal role of retinoids in anteroposterior (A/P) patterning is unclear. HoxD1 is an early posterior neurectodermal marker, expressed from midgastrula with a later anterior expression limit in the future hindbrain. We previously showed that HoxD1 was induced as an immediate early response to retinoic acid in naive ectoderm (animal caps). Here, we use a truncated RARalpha2.2 receptor (RARDelta) to dominantly interfere with retinoid signaling. In embryos injected with RARDelta expression of HoxD1 is eliminated. Conjugates of ectoderm and dorsolateral mesoderm show that retinoid receptors are required in the ectoderm for HoxD1 induction. Further, expression of Krox-20 in r3 and r5 of the presumptive hindbrain is compressed into a single stripe that suggests elimination of r5. RARalpha2.2 expression almost precisely overlaps that of HoxD1, suggesting that this receptor may normally activate HoxD1. Expression of neither more anterior genes including cement gland, forebrain, and midbrain markers nor a more posterior spinal cord marker is affected by RARDelta. These data suggest that the posterior hindbrain is the region of the nervous system most sensitive to retinoid loss. Finally, we compare the ability of RA and fibroblast growth factor (FGF) to posteriorize isolated anterior neurectoderm and show that both factors can act directly on this substrate. RA acts in a more anterior domain than does FGF; however, neither factor is equivalent to the natural posteriorizing capacity of the posterior mesoderm. We propose that endogenous retinoid and FGF signals pattern largely nonoverlapping regions along the A/P axis and that posterior neural patterning requires multiple inducers.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a ag1 cdx4 eef1a2 egr2 en2 fgf2 gbx2.2 hoxa1 hoxb9 hoxd1 muc2 otx2 rab40b

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	FIG. 1. Expression of the dominant negative RARa2 blocks the teratogenic effects of retinoic acid. Embryos were injected with the dominant negative RAR (RARD), control RNA (RARm), or wild-type RAR (RAR), either alone or in combination. Injected embryos were treated with 1 mM all-trans retinoic acid (RA) from early gastrula (stage 10.5–11) to early neurula (stage 13–14). Embryos were then washed and cultured in RA-free saline until hatching stages (stage 32–35).Uninjected, untreated embryos are shown for comparison. One representative experiment is shown. Comparable data were obtained from five separate experiments. The number of embryos scored in this experiment (n) is indicated beneath each column. (A) Percentage of RA-treated embryos with a given value on the dorsoanterior index scale (DAI). DAI was determined by the following criteria: DAI 2 had no head and severely truncated tail structures; DAI 2 embryos had apparent head structures but no eyes or cement gland; DAI 2 embryos had head structures including cement gland and sometimes eyes. Solid bars, DAI 2; striped bars, DAI 2; cross-hatched bars, DAI 2. (B) Percentage of RA-treated embryos with either eyes or cement gland. These structures were scored as positive even when small or morphologically abnormal. Solid bars, percentage of embryos with eyes; striped bars, percentage of embryos with cement gland. (a), uninjected, untreated; (b), uninjected, RA-treated; (c), RARD injected, RA-treated; (d), RARm-injected, RAtreated; (e), RARD / RARm-injected, RA-treated; (f), RARD / RARa2.2-injected; (g), RARa2.2-injected, RA-treated.
	FIG. 2. Expression of the dominant negative RARa2 blocks induction of HoxD1 and HoxA1 by retinoic acid. (A) Both cells of a two-cell embryo were injected with either the dominant negative RAR (RAR-delta) or control RNA (RARm). Animal caps (AC) were isolated at stage 10 and were treated with 2 1 1007M RA for 7 h. (B) Induction of the RA-inducible genes HoxD1 and HoxA1 was determined by RT-PCR. EF1a expression was used as a control. PCR on RNA samples that had not been reversed transcribed (0RT) was done to assay genomic DNA contamination. Lane 1, uninjected, untreated; lane 2, uninjected, RA-treated; lane 3, RARD-injected, untreated; lane 4, RARD-injected, RA-treated; lane 5, RARm-injected, untreated; lane 6, RARm-injected, RA-treated.
	FIG. 3. Expression of dominant negative RARa2 blocks expression of HoxD1 and alters Krox-20 staining. (A) Albino embryos were microinjected with a mixture of b-galactosidase (b-gal) mRNA (as a tracer) and globin, mutant RAR (RARm), or dominant negative (RARD mRNA in one blastomere of a two-cell embryo as described under Materials and Methods. Embryos were allowed to develop to either late gastrula–early neurula (stage 12.5–13) or midneurula (stage 15–16). All embryos are shown in dorsal view with anterior down. (B) Expression of RARa2.2 and HoxD1 was compared in early neurula embryos. (a), stage 13 uninjected embryo, RARa2.2 probe; (b), stage 13 uninjected embryo, HoxD1 probe; (c), stage 22 uninjected embryo; RARa2.2, Krox-20, En-2, XCG mixed probe; lateral view, anterior left. Bracket, RARa2.2 expression; closed arrows, Krox-20 expression; open arrow, En-2 expression. (C) Expression of ectodermal markers in injected embryos was examined by whole-mount in situ hybridization. All are dorsal views, except g which is a ventroanterior view. Anterior is at the bottom. (a), stage 13 embryo, RARm-injected, HoxD1 probe. Arrow, b-gal staining. (b), stage 13 embryo, RARD-injected, HoxD1 probe. Arrow, b-gal staining. (c), stage 15 embryo, globin injected; En-2, Krox-20, XCG mixed probe. Closed arrows indicate Krox- 20 expression in r3 and r5; open arrow indicates En-2 expression. (d), stage 15 embryo, RARm-injected; En-2, Krox-20, XCG mixed probe. Closed arrows, Krox-20 expression in r3 and r5; open arrow, En-2 expression. (e), stage 16 embryo, dominant negative RARD-injected; En- 2, Krox-20, XCG mixed probe. Closed arrow, single Krox-20 expression band; open arrow, En-2 staining. (f ), stage 15 embryo, dominant negative RARD-injected; En-2, Krox-20, XCG mixed probe. Closed arrow, single Krox-20 expression band; open arrow, En-2 staining. (g), stage 15 embryo, dominant negative RARD-injected; XCG probe. Arrow, XCG staining. (h), stage 15 embryo, RARD-injected; otx2 probe. Arrow, b-gal staining over otx2. (i), stage 15 embryo, RARD-injected; HoxB9 probe. Arrow, b-gal staining over HoxB9.
	FIG. 4. Expression of dominant negative RARa2 blocks induction of HoxD1 by mesoderm. (A) Both cells of a two-cell embryo were injected with either the dominant negative RAR (RARD) or control RNA (RARm). Animal caps (AC) were isolated at late blastula (stage 9) and conjugated with stage 12 dorsolateral involuted mesoderm (DLM). Conjugates were cultured until siblings of the animal caps had reached early neurula (stage 14). (B) HoxD1 expression was visualized by whole-mount in situ hybridization (purple stain). DLMs were isolated from FLDX-injected embryos, which was visualized using an anti-fluoroscein antibody (blue staining). bp, blastopore; D, dorsal; V, ventral. a, RARm-injected animal caps; b, RARD-injected animal caps; c, RARm-injected animal caps conjugated with DLM. Arrows indicate HoxD1 expression. Note that in the one conjugate without HoxD1 expression, the mesoderm and ectoderm do not form as complete contact as the other conjugates. d, RARD-injected animal caps conjugated with DLM.
	FIG. 5. Retinoic acid can posteriorize gene expression in isolated anterior dorsal ectoderm. (A) Anterior dorsal ectoderm (aDE) was isolated from midgastrula (stage 11.5) embryos, as indicated on the diagram. Explants were incubated in either 1006 M retinoic acid (RA) or 0.25 mg/ml bFGF until control embryos reached stage 28 when RNA was prepared from explants as described under Material and Methods. bl, blastoceol. (B) Analysis of gene expression by RT-PCR. The diagram on the right indicates the relative A/P position of expression of the tested markers, including XAG (cement gland), otx2 (cement gland, forebrain, midbrain), En-2 (midbrain–hind-brain boundary), Krox-20 (rhombomeres 3 and 5 of hindbrain),Xgbx-2 (anterior boundary in r1), HoxD1 (anterior boundary at r4/5 boundary in hindbrain), RARa2.2 (anterior boundary posterior to r5 in hindbrain), HoxB9 (spinal cord), and Xcad3 (anterior boundary in spinal cord). Muscle actin (MSA) was assayed to determine the extent of mesoderm induction. EF1a was used as a control. PCR on RNA samples that had not been reversed transcribed (0RT) was done to assay genomic DNA contamination. Xgbx-2, Xcad3, and MSA were assayed in a separate experiment than the other markers, with lane 3, aDE plus bFGF; lane 4, whole embryo.
	FIG. 6. Midgastrula posterior mesendoderm posteriorizes anterior dorsal ectoderm. (A) Anterior dorsal ectoderm (aDE) was isolated from midgastrula (stage 11.5) embryos, as indicated on the diagram. Explants were either conjugated to posterior dorsal mesoderm (pDM) as indicated or incubated in 1006 M retinoic acid (RA) until embryos reached stage 28. RNA was prepared from explants as described under Materials and Methods. bl, blastoceol. (B) Analysis of gene expression by RT-PCR. The diagram on the right indicates the relative A/P position of expression of the tested markers, including XAG (cement gland), Xash3a (forebrain, midbrain, hindbrain, spinal cord), En-2 (midbrain–hindbrain boundary), Krox-20 (rhombomeres 3 and 5 of hindbrain), Xgbx-2 (anterior boundary in r1), HoxA1 (anterior boundary in hindbrain), HoxD1 (anterior boundary at r4/5 boundary in hindbrain), RARa2.2 (anterior boundary posterior to r5 in hindbrain), and HoxB9 (spinal cord). EF1a was as a control. PCR on RNA samples that had not been reversed (0RT) was done to assay genomic DNA contamination. Lane 1, pDM; lane 2, aDE; lane 3, aDE plus pDM; lane 4, aDE plus RA; lane 5, whole embryo.
	FIG. 7. Model of RA and FGF function in the embryo. RA and FGF pattern overlapping domains of the dorsal ectoderm. Dorsal views of late gastrula embryos are shown. Fates of different regions of the neural plate are indicated on the left. Domains of the embryo affected by RA are hatched. Domains of the embryo affected by FGF are stippled. A, anterior; P, posterior; bp, blastopore. (A) The domain of the embryo dependent on RA signaling for proper gene expression includes the hindbrain caudal to rhombomere 4 and potentially the anterior spinal cord. The domain of the embryo where FGF signaling is required spans from the anterior spinal cord to the posterior of the embryo. Extreme anterior regions of the embryo (unshaded; presumptive forebrain and midbrain) do not require either retinoid or FGF signaling for proper pattern formation. (B) A broad region of the embryo is sensitive to RA treatment. Genes expressed from the presumptive cement gland to the middle of the presumptive hindbrain are repressed by RA, while more posterior genes are induced by RA. The most posterior domain of the embryo is not sensitive to RA treatment. While the extreme anterior of the embryo (cement gland, forebrain, midbrain) is refractory to FGF treatment, some genes expressed from the midbrain–hindbrain boundary posterior to the anterior spinal cord are repressed by exogenous FGF. In contrast, genes expressed in the presumptive spinal cord to the posterior end of the embryo are induced by FGF treatment.