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Cysteine-rich region of X-Serrate-1 is required for activation of Notch signaling in Xenopus primary neurogenesis.
Kiyota T
,
Kinoshita T
.
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The Notch family genes encode single-pass transmembrane proteins which function in a variety of cell fate specifications in invertebrates and vertebrates. In Xenopus primary neurogenesis, the Notch ligands, X-Delta-1 and X-Serrate-1, mediate Notch signaling and regulate cell differentiation. In the present study, we examined the role of the Serrate-specific cysteine-rich (CR) region in the primary neurogenesis of Xenopus embryos. The ligand constructs containing the DSL (Delta/Serrate/Lag-2) domain in the extracellular region caused a reduction in primary neurons, whereas the DSL-deleted form of X-Delta-1 resulted in the overproduction of primary neurons. However, the DSL-deleted form of X-Serrate-1 or the construct containing only the CR region in the extracellular domain (SerCR) reduced the number of primary neurons. In contrast, the CR-deleted form of X-Serrate-1 (SerACR) lost activity as a Notch ligand, regardless of the presence of the DSL domain within the extracellular domain. Overexpression of X-Delta-1 and X-Serrate-1 strongly induced the expression of Xenopus ESR-1 (XESR-1), a gene related to Drosophila Enhancer of split. SerCR alone also moderately induced the expression of XESR-1, but the SerACR form did not induce this expression. Co-injection of X-Notch-1deltaICD, which deletes the intracellular domain (ICD), with SerCR suppressed a neurogenic phenotype, although co-injection of X-Su(H)1DBM with SerCR did not, indicating that SerCR affects primary neurogenesis through the Notch/Su(H) pathway. These results suggestthatthe CR region of Xenopus Serrate is required for the activation of Notch signaling and cell fate specification in primary neurogenesis.
Fig. 1. (Left) Truncated forms of X-Delta-1 and X-Serrate-1. Various truncation forms lacking portions of the extracellular domain of Notch ligands were
produced. Numbers indicate the amino acid residue numbers. CR, cysteine-rich region; DSL, DSL domain; ELR, epidermal growth factor-like repeats;
SP, signal peptide; TM, transmembrane.
Fig. 2. (Right) Effect of Truncated-Notch ligands on primary neurogenesis. Whole-mount in situ hybridization of N-tubulin (purplish blue) shows
primary neurons (neurula stage, dorsal view, anterior to the left). The injected side labeled by X-gal staining (light-blue) for β-gal faces the top. (A) Injection
of 1.0 ng β-gal RNA alone was used as a control. (B,C) Injection of 1.0 ng X-Delta-1 (B) or X-Serrate-1 (C) RNA. In both cases, a significant reduction of
N-tubulin expression occurred in the injected side. (D,E) Injection of 1.0 ng DlDSL (D) or SerDSL (E) RNA. In both cases, a moderate reduction of N-tubulin
expression occurred in the injected side. (F,G) Injection of 1.0 ng DlδDSL (F) or SerδDSL (G) RNA. DlδDSL caused overexpression of N-tubulin, whereas
SerδDSL showed a suppression of N-tubulin expression. (H) Injection of 1.0 ng SerδCR RNA showed no effect on N-tubulin expression. (I) Injection of
1.0 ng SerCR RNA caused a reduction of N-tubulin expression.
Fig. 3. XESR-1 expression induced by injecting with Notch ligand RNA.
(Upper panel) Indicated RNAs (1ng) along with Green fluorescent protein
(GFP) RNA (0.5ng) were injected into 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 (internal marker). Injection of X-Serrate-1 or
X-Delta-1 led to strong induction of XESR-1 expression. Moderate induction
of XESR-1 was observed in the SerCR-injected sample. However, SerδCR
showed a significant reduction in the inductive capacity of XESR-1 expression,
which was the same as the negative control (no injection or GFP RNAinjection).
(Lower panel) Average levels of XESR-1 expression in triplicate
samples. The level of uninjected sample was taken as 1.
Fig. 4 Notch-dependent effect of SerCR on primary neurogenesis. Whole-mount in situ hybridization of N-tubulin (purplish blue) showing primary neurons (neurula stage, dorsal view, anterior to the left). The injected side is top on each panel, confirmed by X-gal staining (light-blue) for β-gal. (A) Injection of 3.0 ng NδICD RNA led to the overexpression of N-tubulin. (B,C) Co-injection of 3.0 ng
NδICD with 1.0 ng X-Serrate-1 (B) or 1.0 ng SerCR (C) RNA rescued the overexpression of N-tubulin. (D) Injection of 1.0 ng X-Su(H)1DBM RNA
produced a neurogenic phenotype. (E) The neurogenic phenotype caused by 1.0 ng X-Su(H)1DBM was not rescued by co-injection of 1.0 ng SerCR RNA