Kiyota T and Kinoshita T (2004), The intracellular domain of X-Serrate-1 is clea...

XB-ART-3481

Mech Dev 2004 Jun 01;1216:573-85. doi: 10.1016/j.mod.2004.03.034.

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The intracellular domain of X-Serrate-1 is cleaved and suppresses primary neurogenesis in Xenopus laevis.

Kiyota T , Kinoshita T .

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The Notch ligands, Delta/Serrate/Lag-2 (DSL) proteins, mediate the Notch signaling pathway in a numerous developmental processes in multicellular organisms. Although the ligands induce the activation of the Notch receptor, the intracellular domain-deleted forms of the ligands cause dominant-negative phenotypes, implying that the intracellular domain is necessary for the Notch signal transduction. Here we examined the role of the intracellular domain of Xenopus Serrate (XSICD) in Xenopus embryos. X-Serrate-1 has the putative nuclear localization sequence (NLS) in downstream of the transmembrane domain. Biochemical analysis revealed that XSICD fragments are cleaved from the C-terminus side of X-Serrate-1. Fluorescence microscopic analysis showed that the nuclear localization of XSICD occurs in the neuroectoderm of the embryo injected with the full-length X-Serrate-1/GFP. Overexpression of XSICD showed the inhibitory effect on primary neurogenesis. However, a point mutation in the NLSs of XSICD inhibited the nuclear localization of XSICD, which caused the induction of a neurogenic phenotype. The animal cap assay revealed that X-Serrate-1 suppresses primary neurogenesis in neuralized animal cap, but X-Delta-1 does not. Moreover, XSICD could not activate the expression of the canonical Notch target gene, XESR-1 in contrast to the case of full-length X-Serrate-1. These results suggest that the combination of XSICD-mediated intracellular signaling and the extracellular domain of Notch ligands-mediated activation of Notch receptor is involved in the primary neurogenesis. Moreover, we propose a bi-directional signaling pathway mediated by X-Serrate-1 in Notch signaling.

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Species referenced: Xenopus laevis
Genes referenced: dll1 gal.2 h4c4 hba3 hes5 jag1 maml1 myc myod1 ncam1 notch1 rbpj smpx tubb2b tyro3

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	Fig. 1. Effect of the Notch ligands on primary neurogenesis of the Xenopus laevis embryo. Whole-mount in situ hybridization of N-tubulin ( purplish blue) shows primary neurons (neurula stage, dorsal view with anterior to the left). The injected side is the upper side of each panel, which confirmed by b-galactosidase (b-gal) staining (sky-blue). (A) The effect of wild-type ligands (X-Delta-1 (A), X-Serrate-1 (B)) or dominant-negative ligands (X-Delta-1Stu (C), X-Serrate-1Eco (D)) on primary neurogenesis. Although the former decreases the number of primary neurons, the latter increases those on the injected side.
	Fig. 3. The intracellular domain of X-Serrate-1 is localized in the nuclei. (A) Several GFP-fused Notch ligands were expressed in the ectoderm of the Xenopus embryo. The tissue was dissected at the gastrula (A,B) or neurula (C) stage, and then subjected to fluorescence microscopic analysis. Nuclear DNA was stained with DAPI. (A) XDICDGFP and XSICDGFP are strongly localized in the nucleus, but not MycGFP and XSICDmtGFP mutated in the putative NLS. (B) XDlGFP is found on the plasma membrane and in the cytoplasm as cis-endocytosed granules. XSerGFP and XSermtGFP mutated in the putative NLS of X-Serrate-1 are detected only on the plasma membrane at the gastrula stage. (C) XDlGFP or XSermtGFP at the neurula stage is the same as that in the gastrula stage. Note that nuclear localization of GFP is found in the neuroectoderm injected with XSerGFP. Arrow indicates the accumulation of XSerGFP fluorescence in the nucleus. Bar is 20 mm and common to A.
	Fig. 4. The intracellular domain of X-Serrate-1 is cleaved. (A) Lysates of Xenopus embryos uninjected or injected with synthetic RNA of the construct were immunoprecipitated (IP) with anti-myc (A,B,E) or anti-GFP (C,D) antibody, and then the precipitates were analysed by immunoblot (IB) with anti-myc (B,C,E) or anti-GFP (A,D) antibody. Gray arrow indicates heavy chain of IgG used for immunoprecipitation. Black asterisk indicates the band of MycGFP. Black and opened arrowheads indicate bands of full-length XDlMycGFP and XSerMycGFP, respectively. (A) MycGFP, XDlMycGFP and XSerMycGFP are detected at 45, 135 and 185 kDa, respectively. (B) Immunoblot with the anti-myc antibody after immunoprecipitation with the anti-myc antibody. Note that 88 and 80 kDa proteins of the N-terminus of XDlMycGFP (gray arrowhead and asterisk) are detected in addition to the 135 kDa protein, and that a 120 kDa protein of the N-terminus of XSerMycGFP (black arrow) is detected in addition to the 185 kDa protein. (C) As the same as Fig. 4A, MycGFP, XDlMycGFP and XSerMycGFP are detected at 45, 135 and 185 kDa, respectively. (D) Immunoblot with the anti-GFP antibody after immunoprecipitation with the anti-GFP antibody. Note that the XSICDGFP band is detected from the embryo injected with XSerMycGFP construct, and that XDICDGFP band is barely detected in the case of XDlMycGFP. (E) Immunoblot with the anti-myc antibody after immunoprecipitation with the anti-myc antibody. Note that 40, 42 and 45 kDa proteins of the C-terminus of XDlcMyc (gray arrowhead and asterisk) are detected in addition to the 115 kDa protein, and that a 40 kDa protein of the C-terminus of XSercMyc (black arrow) is detected in addition to the 180 kDa protein.
	Fig. 5. The intracellular domain of X-Serrate-1 suppresses the primary neurogenesis. The upper side of each panel is the injected side, which is indicated by b-gal staining (light blue). (A) XDICD-injected embryo. Effect of XDICD is not detected in the injected side. (B) XSICD-injected embryo. Primary neurons are suppressed in the injected side. XSICD can rescue the neurogenic phenotype caused by the dominant-negative forms of the Notch ligands. (C) XSICDmtinjected embryo. Point mutation induced in NLS causes the neurogenic phenotype in the injected side. (D) XSermt-injected embryo. Suppressive effect on primary neurogenesis is not observed in the injected side. XSICDmt and XSermt cannot rescue the neurogenic phenotype by the dominant-negative forms of the Notch ligands.
	Fig. 6. X-Serrate-1 inhibits the induction of N-tubulin expression in neuralized animal cap. (A) Truncated BMP receptor (tBR) alone induces neural marker genes. tBRRNA (2 ng) along with MycGFP RNA (0.5 ng) were injected into the animal pole of both blastomeres of two-cell stage embryos. Animal cap was excised from the blastula stage (stage 8), cultured until the normal embryo reached stage 14 or 25, then assayed by quantitative RT-PCR for expression of NCAM, N-tubulin, MyoD, alpha-globin and Histone H4 (loading control). WE, whole embryo (positive control); 2RT, negative control. (B) Schematic diagram illustrating the animal cap assay. Indicated RNAs or tBR RNA (2 ng) along with FITC (5 ng) was injected into the animal pole of each blastomere of two-cell stage embryos. Animal cap was excised from the blastula stage (stage 8), cultured until the normal embryo reached stage 25. (C) Expression of N-tubulin was assayed by in situ hybridization. X, uninjected side. (C) Injection of FITC alone as negative control. (D) Injection of tBR RNA causes induction of N-tubulin-expression (asterisk). (E) Injection of tBR and X-Delta-1 (Dl) RNA (1 ng). Expression of N-tubulin occurs in the X-Delta-1-injected side as same as tBR alone (asterisk). (F) Injection of tBR and X-Serrate-1 (Ser) RNA (1 ng). Expression of N-tubulin is inhibited in the X-Serrate-1-injected side (asterisk).
	Fig. 7. The intracellular domain of X-Serrate-1 does not activate the expression of XESR-1. (upper) Indicated RNAs (1 ng) along with MycGFP RNA (0.5 ng) were injected into the animal pole of both blastomeres of two-cell stage embryos. Animal cap was excised from the blastula stage (stage 8), cultured until the normal embryo reached stage 9.5, then assayed by quantitative RT-PCR for expression of XESR-1 and Histone H4 (loading control). Injection of X-Serrate-1 or NICD led to strong induction of XESR- 1 expression. However, injection of XSICD showed an inductive capacity as low as the background level (uninjected or MycGFP RNA-injection). -RT, negative control. (lower) Average levels of XESR-1 expression in triplicate samples. The level of uninjected sample was taken as 1.
	Fig. 8. Putative action of X-Serrate-1. See text for details. XSer, X-Serrate- 1; XSICD, X-Serrate-1 intracellular domain; NICD, X-Notch-1 intracellular domain; Ps, Presenilin; CSL, CBF1/Suppressor of Hairless/Lag-2; Mam, Mastermind; X, unknown proteolytic factor.