Reynaud-Deonauth S et al. (2002), Notch signaling is involved in the regulation o...

XB-ART-7654

Differentiation 2002 Jan 01;694-5:198-208.

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Notch signaling is involved in the regulation of Id3 gene transcription during Xenopus embryogenesis.

Reynaud-Deonauth S , Zhang H , Afouda A , Taillefert S , Beatus P , Kloc M , Etkin LD , Fischer-Lougheed J , Spohr G .

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During Xenopus embryogenesis, XId3, a member of the Id helix-loop-helix protein family, is expressed in a large variety of differentiating tissues including epidermis, cement gland, brain, neural tube, neural crest cell derivatives, somites, and tailbud. Transcription of XId3 is mediated by several cis-regulatory elements including an enhancer of 440 bp located 870 bp upstream from the transcription initiation site. The enhancer activity in embryos was studied using transgenic methodology. A galactosidase reporter gene, driven by a regulatory element composed of the enhancer and a minimal promoter derived from the XId3 gene, was expressed in transgenic embryos with a profile that faithfully reproduced that of the endogenous XId3 gene. The pattern resulted from a synergistic effect between the enhancer and the promoter, and in vitro transactivation assays showed that transcription can be stimulated by Notch signaling. The presence of potential Su(H) binding sites, in both the enhancer and the promoter, suggests that these represent candidates for in vivo cis-regulatory elements. The data presented here suggest that Notch control of differentiation may involve activation of transcription of Id, a negative regulator of bHLH transcription factors.

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Species referenced: Xenopus
Genes referenced: ggt1 id3 myc notch1 rbpj

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	Fig. 1 Activity of CAT reporter genes driven by enhancer and promoter elements derived from the XId3 gene in Xenopus embryos. A Constructs used to determine location and activity of the GTG enhancer. (a) Map of the upstream part of the Xenopus XId3 gene showing the location of the GTG enhancer and of restriction sites relevant for the construction of the reporter genes. (b) CAT reporter genes with progressively 5ø-deleted upstream sequences from the XId3 promoter. (c) The GTG Enhancer, the tetrameric oligo GTG1 and GTG2 constructs and a minimal c-myc promoter construct used as a control. B CAT activity assay performed with each construct described in b. As control for basic CAT activity, the minimal c-myc reporter was used. C Comparison of the GTG enhancer activity with different 5ø-deleted Id promoter constructs. D Activity assay for the GTG1 and GTG2 constructs in parallel with the GTG enhancer.
	Fig. 2 Expression driven by the XId3 enhancer/promoter tandem in transgenic embryos and expression of the endogenous gene determined by in situ hybridization. A A plasmid containing the galactosidase encoding sequence driven by the XId3 enhancer/ promoter tandem was linearized with NotI and used to create transgenic embryos as described. In this experiment, 11 out of 22 neurulae stained positively for galactosidase, nine of them are shown. The two embryos that are not shown stained very strongly with abnormal development similar to embryo number 3. Brightness and contrast of embryo number 1 has been raised to visualize the asymmetric expression in right and left side somites. B Three selected embryos showing a regular and consistent transgene expression pattern. C Tailbud stage of a transgenic embryo expressing the XId3 driven galactosidase gene. The transgene is strongly expressed in the growing apex of the tailbud, where cells have a maximal proliferation rate, and progressively silenced in the differentiating somites, and finally reduced to a pattern corresponding to the ordered myotome nuclei. D Higher magnification of an embryo showing transgene expression in the skin (Sk), brain (B), spinal cord (Sp), eye vesicle (Ey), ear vesicle (Ea), cement gland (Ce), neural crest derivatives (Nc), notochord (No), myotomes (My) and tailbud (Tb). E Whole-mount in situ hybridization of tailbud stage embryos with a XId3 RNA probe. The major positive signals, represented in brown color, are indicated. Abbreviations are as in 2B.
	Fig. 3 Expression driven by the XId3 enhancer/promoter tandem visualized in sections of transgenic embryos. In this embryo, the left side stained more intensively than the right one. The sections are oblique with respect to the dorsal axis. As a consequence the notochord and the neural tube appear elongated. A Oblique section of the head area of a tailbud stage embryo showing a scattered staining pattern. B Oblique section of the central body part of the embryo shown in A. Dorsal fin (Df), spinal cord (Sp), myotomes (My) and notochord (No) are indicated.
	Fig. 4 Immunocytochemical detection of Id protein in developing Xenopus embryos. A A stage 22 embryo. B Head of a stage 35 embryo. Staining is observed in brain (B), eye (Ey), neural crest derivatives (Nc), cement gland (Ce) and skin (Sk). C Tail of a stage 35 embryo. D Magnified view of somites from C.
	Fig. 5 GTG enhancer and promoter of the XId3 gene. Nucleotide sequence of the GTG enhancer (A) and of the proximal promoter (B) of the XId3 gene. The potential Su(H) binding sites, the DraIII, SspI and EcoRI restriction sites are boxed, the TATA box is underlined. The areas from which the synthetic oligonucleotides GTG1 and GTG2 were derived are indicated with arrowed lines.
	Fig. 6 Transactivation by Notch, in an oocyte assay system, of a CAT reporter gene driven by the GTG enhancer, GTG1 and GTG2 constructs. A Cartoon illustrating the transactivation assay. Oocytes are microinjected with a mRNA encoding the cytoplasmic portion of Notch or, as a control, the mutated form of Notch described in the text. Eight hours later, the CAT reporter gene is injected into the germinal vesicle. 20–24 hours later the oocytes are lysed in at least triplicate batches of 10–20 oocytes each, an extract is prepared, and CAT activity determined. B Transactivation by Xenopus Notch mRNA of CAT reporter genes driven by GTG-Enhancer-, GTG1- and GTG2-constructs. Approximately 150 oocytes were injected with each Notch mRNA and 8 hours later with the indicated reporter genes (50 oocytes/reporter gene) as indicated under Methods. Extracts were prepared for CAT activity determination another 24 hours later. C Comparison of the abilities of mouse and Xenopus Notch to transactivate the GTG1-CAT constructs. Injections were performed and oocytes processed as described under B.