May 1, 2003;
Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus.
We analysed the role of Notch
signalling during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway we found that Notch
expands the floor plate domain of sonic hedgehog
and represses the notochordal markers chordin
, with a concomitant reduction of the notochord
size. We propose that within a population of the early organiser with equivalent potential to develop either as notochord
or floor plate, Notch
activation favours floor plate development at the expense of the notochord
, preferentially before mid gastrula
. We present evidence that sonic hedgehog
, suggesting that secreted Sonic hedgehog
may be involved or reinforcing the cell-fate switch executed by Notch
. We also show that Notch
signalling requires Presenilin to modulate this switch.
[+] show captions
Fig. 3. Shh signalling down-regulates chd, resembling the effect of Notch. (A-E) In situ hybridisation of chd in early gastrulae, vegetal views, dorsal up, injected side on the right. (F-G′) In situ hybridisation of shh in neurulae, dorsal views, anterior up. (H) In situ hybridisation of N-tubulin and shh in neurulae. (I) In situ hybridisation of gli3 in neurulae. (J) External morphology at tadpole stages. (A-E) Embryo injected with (A) 2 ng of nuc-lacZ, (B) 1 ng of notchICD, (C) 2 ng of X-su(H)DBM, (D) 1 ng of X-shh mRNA, (E) 2 ng of X-shh-ds RNA. (F) Control embryo. (F′) Higher magnification of the embryo shown in F. (G) Embryo injected with 2 ng of X-shh-ds RNA to show the degradation of endogenous shh transcripts (77% of injected embryos, n=22). (G′) Higher magnification of the embryo shown in G. (H,I) Embryos injected with 2 ng of X-shh-ds RNA on the right side. (J) Control embryo (left) and two embryos injected with 1 ng of X-shh-ds RNA per blastomere at the 2-cell stage showing different grades of cyclopia (right).
Fig. 4. Comparison of the expression patterns of Xotch, chd and shh. In situ hybridisation of (A,D,G,J) Xotch, (B,E,H,K) chd and (C,F,I,L) shh. (A-C) Vegetal view of an early gastrula, stage 10.25, dorsal side up, showing (A) the distribution of Xotch mRNA in the dorsal marginal zone, (B) the expression of chd in the organiser and (C) shh transcripts, first seen at this stage in the organiser and found in a subdomain of the chd territory. (D-F) Vibratome section in the sagittal plane of an early gastrula showing (D) the expression of Xotch in the epithelial and subepithelial layers of the organiser, (E) the expression of chd in the epithelial and subepithelial layers and in the deep zone of the organiser and (F) a patch of cells expressing shh in the subepithelial layer of the organiser, with some faint staining in the epithelial layer. The arrowheads in D,E,F point to the dorsal blastoporal groove. Distinctions in the cellular composition of the gastrula organiser followed the criteria of Hausen and Riebesell (Hausen and Riebesell, 1991). (G) Late gastrula/early neurula embryo, dorsal view, anterior up, showing the distribution of Xotch transcripts. Interestingly, we observed asymmetries in several embryos, with higher levels of Xotch mRNA on the right side. The yellow line indicates the plane of section shown in J. (H) Dorsal view of a late gastrula, anterior up, showing the expression of chd in the involuted cells. (I) Dorsal view of a late gastrula, anterior up, showing the expression of shh in the dorsal midline. (J) Transverse vibratome section at the level of the posterior trunk of the same embryo as in G. Xotch transcripts are found in proneural domains (p) of the neural plate, presomitic mesoderm (s) and in the developing floor plate (f) but are not found in the notochord (n). Notice higher levels of Xotch mRNA in the proneural and presomitic domains on the right side. More anterior sections also showed the presence of Xotch transcripts in prospective floor plate cells, although at lower levels, and their absence from the notochord (not shown). (K) Sagittal section of the same embryo shown in H, anterior to the left; the blastopore is at the right margin. Chd transcripts are distributed along the notochord. (L) Sagittal section of the same embryo shown in I, anterior to the left; the blastopore is at the right margin. Shh transcripts are found in the three dorsal midline layers, in a dorsal to ventral gradient.
Fig. 6. Presenilin up-regulates shh and down-regulates chd in a Notch-dependent way. In situ hybridisation of (A-F) shh and (G-P) chd. Whole embryos in A-C,G-I,M,N are shown in dorsal views, anterior up. (A) Stage 14 embryo injected with 4.5 ng of nuc-lacZ mRNA (nucβgal). (B) Stage 14 embryo injected with 2 ng of X-ps-α . (C) Stage 14 embryo injected with 2 ng of X-ps-α plus 2 ng of X-su(H)DBM. (D) Transverse section of the same embryo shown in A. (E) Transverse section of the same embryo shown in B. Notice the expansion of the floor plate domain of shh on the injected side (arrow). (F) Transverse cut of a stage 16 embryo injected with 2 ng of X-ps-α plus 2 ng of X-su(H)DBM. Owing to the strong inhibition, the visualisation of shh expression in 50μ m vibratome sections from these embryos was difficult. In order to appreciate differences between the injected and non-injected side, we show here a transverse cut obtained with a scalpel. The asterisk indicates the repression of shh expression in the floor plate domain on the right side, where X-gal stained cells are preferentially located. Notice that the notochord is complementarily augmented on this side. Overall, the notochord is larger than in embryos of the same stage injected with equal amounts of nuc-lacZ mRNA (not shown). The broken line in D-F outlines the notochord. (G) Stage 13 embryo injected with 4 ng of nuc-lacZ mRNA. (H) Stage 13 embryo injected with 2 ng of X-ps-α. (I) Stage 13 embryo injected with 2 ng of X-ps-α plus 2 ng of X-su(H)DBM. (J,K,L) Transverse sections of the embryos shown in G, H and I respectively. (M) Stage 12.5 embryo injected with 5 ng of a control morpholino. (N) Stage 12.5 embryo injected with 5 ng of an antisense morpholino oligonucleotide against X-ps-α. (M,P) Transverse sections of the embryos shown in M and N respectively. All sections, except F, are Nomarski interference contrast views.
chrd.1 (chordin, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.25, vegetal view, dorsal up.