Wettstein DA et al. (1997), The Xenopus homolog of Drosophila Suppressor of...

XB-ART-16970

Development 1997 Feb 01;1243:693-702. doi: 10.1242/dev.124.3.693.

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The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis.

Wettstein DA , Turner DL , Kintner C .

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The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.

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Species referenced: Xenopus
Genes referenced: ank1 dll1 hes5 neurog2 nog notch1 rbpj

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	Fig. 1. (A) Deletion analysis of ICD. Diagram of the X-Notch-1 intracellular domain (ICD) and of the deletions used for mapping ESR-1 induction. NLS-Ank contains the SV40 NLS at the Nterminus (Rupp et al., 1994). The amino acid residues (Coffman et al., 1990) encompassed by each truncation are indicated. The right column summarizes the data from RNase protection assays, such as those shown in Fig. 3, that measure the ability of each construct to induce ESR-1 expression in neuralized animal caps (+++, 100%; ++, 20-40%; +, 10-20%; +/-, 1-5%; or -, not detectable). RAM23 (Tamura et al., 1995) is the subtransmembrane region. Addition of the SV40 NLS (Rupp et al., 1994) to the N terminus of ICD22, 23, 24, and 25 did not alter ESR-1 induction. (B) Different forms of XSu( H)1. Schematic representation of wild-type X-Su(H)1, a form in which the ankyrin repeats from Notch are fused to the carboxy terminus (X-Su(H)1/Ank), a form in which four residues required for DNA binding, between amino acids 193-203, are altered (XSu( H)1DBM), a truncated form in which the carboxy terminal 117 amino acids are deleted X-Su(H)1-Tr, and a form of X-Su(H)1/Ank fused to the hormone binding domain of the human glucocorticoid receptor (hGR).
	Fig. 2. Regions of the X-Notch-1 intracellular domain required for X-Su(H)1 binding. Lane labels indicate the GST fusion protein (see Fig. 1A) used to precipitate in vitro-translated [35S]Met-labeled XSu( H)1. The asterisk marks full-length in vitro-translated X-Su(H)1. The lane labeled ‘Pre-clear’ (see Methods) shows that GST binds no X-Su(H)1. The ‘Input’ lane contains 25% of the [35S]Met-labeled XSu( H)1 used in each binding reaction. Approximate migration of protein markers is indicated. Longer exposure of the gel shows weak binding of X-Su(H)1 to GST-Ank, GST-ICD11, and GST-ICD14, while GST alone still shows no binding.
	Fig. 3. Induction of ESR-1 in neuralized animal caps by ICD and by different forms of XSu( H)1. Test RNAs (1.0 ng/embryo) along with Noggin RNA (0.3 ng/embryo) were injected into two-cell stage embryos. Ectoderm was excised from blastula embryos (stage 10), cultured for approximately 6 hours (equivalent to the neural plate stage), and then assayed by RNase protection assay for expression of ESR-1 RNA and EF-1a RNA (loading control). Each assay includes the probes without RNase treatment (Probes), animals caps injected with just Noggin RNA (Noggin) and stage-matched embryo RNA (Embryos). Position of probe fragments are marked with an arrowhead, while those of protected fragments are marked with arrows. (A) ESR-1 RNA levels in neuralized animal caps expressing different regions of ICD as shown in Fig. 1A. Note that those lacking any portion of the ankyrin repeats fail to or barely induce detectable ESR-1 expression (lanes 5, 11, 14, 15, and 16). Removing the RAM23 region (ICD11, lane 13) markedly reduces the induction of ESR-1 expression relative to ICD (lane 10). Deletion of sequences downstream of the ankyrin repeats (compare lane 4 to 3) also reduces the induction of ESR-1 expression. (B) ESR-1 RNA levels in neuralized animal caps expressing X-Su(H)1 and X-Su(H)1/Ank (see Fig. 1B). Note that X-Su(H)1 has undetectable ESR-1-inducing activity (lane 5) and the ankyrin repeats from ICD (NLS-Ank) have very little inducing activity (lane 4), while XSu( H)1/Ank (lane 7) induces ESR-1 expression to levels found in embryos (lane 8).
	Fig. 4. Inducible forms of X-Su(H)1/Ank and ICD. hGR/ICD22 or hGR/X-Su(H)1/Ank RNA (1.0 ng/embryo) were injected along with Noggin RNA (0.3 ng/embryo) into two-cell stage embryos and the induction of ESR-1 expression was measured in animal cap assays as described in Fig. 3. (A) Neuralized animal caps expressing the indicated constructs were either left untreated (-) or exposed (+) to dexamethasone (DEX) for 3 hours. Note that ESR-1 expression increases following dexamethasone induction. Both forms also induced ESR-1 expression in the absence of dexamethasone, probably as a result of expressing them at levels high enough to overwhelm mechanisms for cytoplasmic retention. (B) Neuralized animal caps expressing the indicated constructs were either left untreated (0) or exposed to dexamethasone for three (3), two (2), or one (1) hour before harvesting. As a control, neuralized animal caps were treated for 3 hours with dexamethasone (Noggin+DEX). (C) Animal caps from embryos injected with hGR/ICD22 RNA were (+) or were not (-) neuralized (Noggin) and had (+) or had not (-) been treated with either cycloheximide (CHX) and/or dexamethasone (DEX), as indicated. This experiment was carried out both in the presence and absence of Noggin, because we knew that ESR-1 expression can be induced to higher levels in neuralized versus nonneuralized animal caps, and we were concerned that the ability of noggin to neuralize animal caps would be blocked by cycloheximide. Consistent with this notion, the induction of ESR-1 in neuralized animal caps appeared to be affected to a small degree by the addition of cycloheximide (lane 9 versus 7), while no such effect was observed in nonneuralized caps (lane 5 versus 3). ESR-1 RNA expression levels in lanes 2-9 after normalizing to EF-1a are: 59, 174, 24, 96, 73, 180, 89, and 300.
	Fig. 5. ESR-1 expression is blocked by X-Su(H)1DBM. Test RNAs (1.0 ng/embryo), as indicated above each lane, along with Noggin RNA (0.3 ng/embryo) were injected into two-cell stage embryos, and the induction of ESR-1 expression was measured in animal cap assays as described in Fig. 3. (A) Expression of X-Delta-1 in neuralized animal caps induces the expression of ESR-1 (compare lanes 2 and 6). This induction is reduced by the inclusion of XSu( H)1DBM (DBM) or X-Su(H)1 but not by X-Su(H)1-Tr, a carboxyterminally truncated form of X-Su(H)1 (see Fig. 1B). Quantitation of the ESR-1 signal in lanes 3-6 is: 91, 24, 24, and 60, respectively. (B) Compilation of inhibition of X-Delta-1-mediated ESR-1 induction by X-Su(H)1 and X-Su(H)1DBM (DBM). The number of data points is indicated (n=) and error bars indicate the true population standard deviation.
	Fig. 6. The effects of X-Su(H)1, X-Su(H)1/Ank and X-Su(H)1DBM on primary neurogenesis. Albino Xenopus embryos were injected once at the two-cell stage with test RNAs (1.0 ng/embryo) along with lacZ RNA (0.2 ng/embryo). At neural plate stages, the embryos were processed both for b-galactosidase expression (light blue) to mark the injected side, and for the expression of N-tubulin (dark purple) to mark the formation of primary neurons. Primary neurons arise in three stripes on either side of the midline of the neural plate: a long medial stripe, a short more anterior intermediate stripe, and a wide lateral stripe. Shown are dorsal views, with anterior to the left and the injected side oriented up in each panel. Embryos shown in each row are from the same experiment. Expression of: (A) wild-type X-Su(H)1 has little or no effect on the formation of primary neurons; (B) X-Su(H)1/Ank abolishes primary neurons; (C,F) X-Su(H)1DBM produces a neurogenic phenotype; (D) X-Su(H)1 along with X-Su(H)1DBM ameliorates the neurogenic phenotype; (E) X-Su(H)1/Ank along with X-Su(H)1DBM completely reverses the neurogenic phenotype; (G)X-Delta-1 reduces the number of primary neurons; (H) X-Su(H)1 along with X-Delta-1 partially reverses the effects of X-Delta-1; and (I) X-Su(H)1DBM along with X-Delta-1 not only reverses the effects of X-Delta-1 but also results in a neurogenic phenotype.
	Fig. 7. Forms of X-Su(H)1 affect the pattern of expression of X-Delta-1 and XNGNR1. The effects of test RNAs on the formation of primary neurons was assayed as described in Fig. 6. Embryos were injected with lacZ alone (A,D), X-Su(H)1 (B,E), or X-Su(H)1DBM (C,F) and analyzed for the expression of X-Delta-1 (A-C) or XNGNR1 (D-F). Note that the X-Su(H)1DBM increases the expression of both XNGNR1 and X-Delta-1.
	rbpj (recombination signal binding protein for immunoglobulin kappa J region) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15/16, dorsal view, anterior left.