von Dassow G et al. (1993), Induction of the Xenopus organizer: expression ...

XB-ART-22798

Genes Dev 1993 Mar 01;73:355-66. doi: 10.1101/gad.7.3.355.

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Induction of the Xenopus organizer: expression and regulation of Xnot, a novel FGF and activin-regulated homeo box gene.

von Dassow G , Schmidt JE , Kimelman D .

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We have searched for homeo box-containing genes expressed during gastrulation in Xenopus embryos with the expectation that analysis of the spatial and temporal expression of these genes will lead to greater understanding of the regionalization of the mesoderm. We describe the cloning and expression of Xnot, a novel homeo box-containing gene expressed primarily in the gastrula organizing region. We have studied the regulation of Xnot by signaling molecules involved in mesoderm induction and regionalization. Surprisingly, we found that FGF signaling is required for expression of Xnot in the gastrula organizing region, clearly implicating FGF in the induction of dorsal mesoderm. Furthermore, we found that Xnot is initially expressed throughout the embryo and that progressive translation of an unknown protein restricts expression of Xnot to the organizing region. Our results provide experimental evidence supporting the proposed division of Spemann's organizer into independently regulated organizing centers.

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Species referenced: Xenopus
Genes referenced: bmp4 fgf1 inhba nog not tbx2 wnt1 wnt8a

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	Figure 1. (A) The nucleotide sequence of Xnot and its predicted amino acid sequence. The homeo box sequence is indicated by a solid underline, and the acidic region is indicated by a broken underline. The predicted initiation codon and 5' nucleotides that match the consensus translational initiation sequence (Kozak 1987) are indicated in boldface type. (B) Comparison of the Xnot homeo box sequence to other homeo box sequences (Laughon 1991). The four predicted helices within the borneo box region are indicated (Laughon 1991). (C) Schematic illustration of the Xnot clone, with the boxed region indicating the open reading frame. The positions of the homeo box and acidic regions are indicated. The portion of the cDNA used to produce an RNase protection probe is also shown.
	Figure 2. Developmental expression of Xnot. Poly(A) + RNA was isolated from 50 unfertilized eggs and 50 embryos each at different stages, separated on a gel, blotted, and hybridized with a full-length probe made from the Xnot cDNA. (Lane 1) Unfertilized egg; (lane 2) stage 10 (early gastrula); (lane 3) stage 12 (late gastrula); (lane 4) stage 15 (neural plate stage); (lane 5) stage 19 (neural tube stage); (lane 6) stage 24 (tailbud). The blot was rehybridized with an EF1-~ probe (Krieg et al. 1989) to verify the loading of RNA in each lane.
	Figure 3. Analysis of Xnot expression by RNase protection. (A) RNA was isolated from five unfertilized eggs and five embryos each at different stages of development. RNA was analyzed by RNase protection using a mixture of Xnot and EFI-a probes, with the EFI-a probe at a reduced specific activity. Note that the MBT occurs at 7 hr and that 10 hr corresponds to a stage 10 (early gastrula) embryo. Stage 12 is late gastrula stage, stage 15 is neural plate stage, stage 19 is neural tube stage, and stage 24 is tailbud stage. (B) Distribution of Xnot in a stage 9 (late blastula) embryo. Stage 9 embryos were dissected into animal (An), middle {Mid), and vegetal (Veg) thirds and analyzed as in A.
	Figure 4 Spatial distribution of Xnot transcripts during early development. Xnot expression was analyzed by the whole-mount in situ procedure using albino embryos. (A) Vegetal view of stage 10.5 embryos. The dorsal lip is at the top. Note the intense staining just above the dorsal lip and the equatorial staining well above the blastopore lip (arrow). (B) Close-up of stage 10.5 embryo. The sharply delimited staining of Xnot is apparent above the blastopore lip in the prenotochordal territory. The staining does not extend all the way to the lip (arrow) as with other organizer-specific genes (Cho et al. 1991; Dirksen and Jamrich 1992; Ruiz i Altaba and Jessell 1992; Taira et al. 1992). (C) Dorsal view of stage 12 embryos. The equatorial expression is no longer present. Xnot is found only along the dorsal midline in the region that will give rise to the notochord. Anterior is at the top. (D) Close-up of a stage 12 embryo demonstrating the gradient of expression at the anterior end and the sharp termination of expression at the blastopore. Anterior is at the top. (E) Stage 16 embryo. Xnot expression extends from the anterior (top) to the posterior along the dorsal midline, with the strongest expression in the posterior. Two new spots of Xnot expression appear on either side of the neural plate. (F) Stage 23 embryos. Xnot is expressed only in the posterior part of the notochord and floor plate along the body axis. In the anterior region, the two spots of Xnot expression have almost converged as the neural plate folds up. Anterior is at the top. (Left embryo) Dorsal is at left; (middle embryo) dorsal view; (right embryo) dorsal is at right. (G) Stage 30 embryos. Only the most posterior tip of the body axis (the tailbud, t) contains Xnot transcripts. In the anterior region, the two spots of Xnot expression have converged in the epiphysis (e), the precursor of the pineal body. Faint expression in other regions of the brain is also apparent.
	Figure 5. Expression of Xnot along the dorsal midline. Embryos were processed for whole-mount in situ hybridization and embedded in paraffin, and transverse sections were cut. (A) Section through the middle of a stage 12.5 (late gastrula) embryo. Staining is observed only at the dorsal midline in all three germ layers. The archenteron was slightly deformed during sectioning. (B) Posterior section of a stage 16 (neural plate stage) embryo. Xnot expression is observed along the dorsal midline in the notochord (n), archenteron roof (a), and in the floor plate (f) of the neural tube. (C) Posterior section of a stage 18 (neural fold stage) embryo. The same regions expressed Xnot as in B. Dorsal is at the top of each section.
	Figure 6. Activation of Xnot expression by mesoderm-inducing agents. Animal caps were removed at stage 8 and cultured with various treatments. RNA was isolated at the equivalent of stage 12 for RNase protection analysis using a mixture of Xnot and EFI-a probes, with the EFI-a probe at a reduced specific activity. (A) Both FGF and activin induce Xnot expression. (Lane 1) No factors added; (lane 2) FGF added; (lane 3) activin added; (lane 4) activin and cycloheximide (CHX) added. (B) Addition of cycloheximide alters the levels of Xnot. (Lane 1) No factors added; (lane 2) cycloheximide added; (lane 3) activin added; (lane 4) activin and cycloheximide added. (C) Retinoic acid (RA) does not significantly affect the expression of Xnot. (Lane 1) No factors added; (lane 2) FGF added; (lane 3) FGF and retinoic acid added; (lane 4) activin added; (lane 5) activin and retinoic acid added.
	Figure 7. Xnot expression is altered by cycloheximide and injected Xwnt-8, DVR- 4, and FGF dominant-negative receptor RNAs. Xnot expression was analyzed by the whole-mount in situ procedure using pigmented embryos. (AI Cycloheximide was added to stage 9 embryos that were then left to develop until control embryos reached stage 12. Lateral view with the animal pole at top. The lack of staining in the vegetal region is an artifact of the whole-mount procedure. (B} Cycloheximide was added to stage 10 embryos that were then left to develop until control embryos reached stage 12. Vegetal view with dorsal side at top. (C) Embryos were injected with Xwnt-8 RNA in one of the ventral vegetal cells of an 8- to 16-cell embryo. Embryos were fixed at stage 11. Vegetal view. Note the two sites of Xnot expression at the equator (arrows). (D) Embryos were injected with DVR-4 RNA at the equator in the two dorsal cells of four-cell embryos. Embryos were fixed at the equivalent of stage 11. Vegetal view with dorsal side at top. Note the lack of Xnot expression. (E) Embryos were injected with RNA encoding the dominant-negative mutant of the Xenopus FGF receptor in the two dorsal cells of four-cell embryos. Embryos were fixed at the equivalent of stage 12. Vegetal view with dorsal side at top. No Xnot expression was detected.
	Figure 8. Summary of the regulation of Xnot expression. Both FGF and activin are able to induce the expression of Xnot, which is suggested to occur by the inhibition of a negative regulator of Xnot, which could either be a transcriptional repressor or a protein that leads to the degradation of Xnot RNA. DVR-4 can inhibit the expression of Xnot and could work directly or by the activation of the inhibitor of Xnot. Retinoic acid does not regulate the levels of Xnot transcription and, rather, is suggested to work at a later time in development or on a parallel pathway. Because Xnot is a likely transcription factor, its expression in the prenotochordal region is suggested to regulate other notochord- and floor plate-specific genes.