Cibois M et al. (2013), A gene regulation network controlled by Celf1 p...

XB-ART-47830

Biol Open 2013 Aug 21;210:1078-83. doi: 10.1242/bio.20135629.

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A gene regulation network controlled by Celf1 protein-rbpj mRNA interaction in Xenopus somite segmentation.

Cibois M , Gautier-Courteille C , Kodjabachian L , Paillard L .

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Somite segmentation is impaired in Xenopus celf1 morphant embryos. The Celf1 RNA-binding protein targets bound mRNAs for rapid degradation, and antisense approaches demonstrated that segmentation defects in celf1 morphants were due to a derepression of rbpj mRNA. Rbpj protein is a key player of Notch signalling. Because segmentation involves complex cross-talk between several signalling pathways, we analysed how rbpj derepression impacted these pathways. We found that rbpj derepression stimulated the Notch pathway. Notch positively controlled the expression of cyp26a, which encodes a retinoic acid (RA)-degrading enzyme. Thus, rbpj derepression led to cyp26a overexpression and RA attenuation. It also repressed fgf8, consistent with an inhibition of FGF signalling. Pharmacological inhibition of the FGF pathway repressed cyp26a, but rbpj derepression was sufficient to restore cyp26a expression. Hence, while it was known that the FGF pathway antagonized RA signalling through expression of cyp26a, our results suggest that Rbpj mediates this antagonism. Furthermore, they show that the post-transcriptional repression exerted by Celf1 on rbpj mRNA is required to keep cyp26a expression under the control of FGF signalling. We conclude that rbpj repression by Celf1 is important to couple the FGF and RA pathways in Xenopus segmentation.

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Species referenced: Xenopus
Genes referenced: celf1 cyp26a1 dlc dusp6 fgf8 gal.2 mespa msgn1 notch1 rbpj
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	Fig. 1. Impact of rbpj overexpression on dlc expression. We injected nLacZ mRNA and control (left panel) or the target-protector (middle panel) morpholinos into one blastomere of two-cell Xenopus embryos and allowed the embryos to develop to the tailbud stage. We then stained them for β-galactosidase activity (red dots) and dlc by in situ hybridisation (ISH). The right panel shows the percentage of embryos with a staining intensity in the injected side above, equal to, and below (respectively green, yellow and red) that in the control side. We compared the distributions between these three classes by a chi-square test and we show the p-value. The photographs are lateral views of the injected sides, anterior left. The positions of the anterior PSM (Ant) and the posterior PSM (Post) are indicated.
	Fig. 2. Impact of rbpj overexpression on the RA signalling pathways. (A) We injected the pRARE-luciferase plasmid and dnRAR mRNA or control or TP morpholinos together into one blastomere of two-cell embryos, which we allowed to develop to the early tailbud stage. We treated the embryos with retinoic acid where indicated, and we extracted proteins and measured luciferase activities (relative light unit). We show here the mean luciferase activities, ± SEM, of 8–16 embryos per condition. We compared the mean values between the controls and the other conditions by a Student's t-test and we show the p-values. (B) We injected nLacZ mRNA and dnRAR or control or the target-protector morpholinos, we allowed the embryos to develop to the tailbud stage and we stained them for β-galactosidase activity and mespa mRNA as in Fig. 1. We compared the distances between the posterior tip of the embryo and the posterior limit of mespa expression, and the right panel shows the percentage of embryos with a distance in the injected side above, equal to, and below (respectively green, yellow and red) that in the control side. We compared the distributions, between these three classes, of the control and the other conditions by a chi-square test and we show the p-values. The photographs are dorsal views, anterior left, injected-side up. Panel C is the same as panel B, but the ISH revealed cyp26a expression. The right panels show the percentage of embryos with a staining intensity in the injected side above, equal to, and below (respectively green, yellow and red) that in the control side. The photographs are dorsal views, anterior left, injected-side up, except the posterior views.
	Fig. 3. Impact of rbpj overexpression on the FGF signalling pathways.We injected nLacZ and control or TP MO, or dnRAR mRNA, as indicated, into one blastomere of two-cell embryos, which we allowed to develop until the tailbud stage. Where indicated, we treated the embryos for 2 hours with SU5402 (embryos injected with control MO were treated with DMSO) and we stained them for β-galactosidase activity (red dots) and fgf8 (A), msgn1 (B), or dusp6 (C) mRNA by ISH. We sorted DMSO and SU5402-treated embryos into 5 classes depending on their staining intensities. The right panels show the percentages of embryos within each of these classes, and, for injected embryos, the percentage of embryos with a staining intensity in the injected side above, equal to, and below (respectively green, yellow and red) that in the control side. We compared the distributions, between the classes, of the control and the other conditions by a chi-square test and we show the p-values. All the photographs are dorsal views, anterior left, injected-side up.
	Fig. 4. Rbpj mediates the repression exerted by FGF signalling on the RA pathway.We injected nLacZ mRNA and control (left panel) or the TP (middle panel) morpholinos into one blastomere of two-cell embryos and allowed them to develop to the tailbud stage. We treated embryos for 2 hours with SU5402. We detected β-galactosidase activity and mespa (A), cyp26a (B) or dlc (C) mRNAs by Red-gal staining and ISH. We also treated control morpholino-injected embryos with DMSO, stained them with the same probes, and we sorted DMSO and SU5402-treated embryos into 5 classes depending on their staining intensities (dlc, cyp26a) or the distance between the posterior tip of the embryo and the posterior limit of expression (mespa), and the right panels show the percentages of embryos within each of these classes. For injected embryos treated with SU5402, the right panels also shows the percentage of embryos with a staining intensity or a distance to posterior tip in the injected side above, equal to, and below (respectively green, yellow and red) that in the control side. We compared the distributions, between the classes, of the control and the other conditions by a chi-square test and we show the p-values. (A,B) dorsal views, anterior left, injected-side up, (C) lateral views of the injected side, anterior left.
	Fig. 5. Model for the relationships between the RA, FGF and Notch pathway in Xenopus PSM.Three intra-pathway feedback loops attenuate each pathway (RA stimulate cyp26a and FGF stimulates dusp6 while Notch represses dlc). In addition, the RA pathway either stimulates (through fgf8) or represses (through dusp6) the FGF pathway, the FGF pathway stimulates the Notch pathway (through dlc), and the Notch pathway represses the RA pathway (through cyp26a). (A) The Celf1 RNA-binding protein attenuates the Notch pathway by limiting the abundance of Rbpj protein through a post-transcriptional control. Notch signalling depends on FGF signalling through Dlc. (B) When the TP MO impairs the repressive interaction between Celf1 and rbpj mRNA, rbpj is overexpressed, which stimulates the Notch pathway. Consequently, the RA pathway is repressed (due to cyp26a overexpression) and the FGF pathway is repressed (due to fgf8 repression). FGF repression does not lead to Notch repression owing to rbpj overexpression, and relieving the repressive interaction between Celf1 protein and rbpj mRNA uncouples the Notch and RA pathways from the FGF pathway.

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