Cui Y (2005), Hairy is a cell context signal controlling Notc...

XB-ART-1027

Dev Growth Differ 2005 Dec 01;479:609-25. doi: 10.1111/j.1440-169X.2005.00823.x.

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Hairy is a cell context signal controlling Notch activity.

Cui Y .

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Notch signaling regulates cell fate determination and many developmental processes. Here we report that lateral inhibition, a major mechanism for Notch activity, is modulated by Hairy, a bHLH-WRPW protein. In Xenopus, Notch can have from inhibitory, permissive to enhancing roles in muscle or neural differentiation. These cell context-dependent effects correlate with Hairy expression levels from high to low, respectively, in the cells. Moreover, Notch effects can be altered upon manipulation of Hairy expression. We propose that Hairy provides a cell context in which a cell can interpret Notch and other extrinsic signals by controlling responsiveness of its target genes; this mode of Hairy-Notch interaction may apply in other systems.

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Species referenced: Xenopus
Genes referenced: acta4 myod1 ncam1 neurod1 neurog1 nog notch1 nrp1 sox2 tbx2 tbxt tubb2b

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	Fig. 1. Different effects of Notch and ESR 1 on myogenesis. (A–C), RNAs as indicated were injected into animal poles of two cell embryos. Animal caps were explanted at late blastula stage and cultured to stage 20. RNA was extracted and processed for RT–PCR assays. Note that while ICD inhibited MyoD function (A), ESR 1, a downstream effector of Notch signaling (B), did not (C). Amounts of RNA injected: (A) MyoD, 1 ng; ICD (lanes 6–8), 0.4 ng, 0.8 ng and 1 ng, respectively; (B) ICD, 1 ng; (C) ESR 1, 0.8 ng; MyoD, 1 ng; ESR 1 (lanes 6–7), 0.8 ng and 1 ng, respectively.
	Fig. 2. Hairy inhibits myogenesis. (A), Embryos were injected into marginal zone of 2-cell embryos with β-gal, alone or with Hairy 2. At gastrula stage the embryos were fixed and stained with Red Gal and MyoD or Xbra, respectively, by in situ hybridization. (B), Embryos (the anterior is up), injected with Tex Red Dextran alone or with Hairy 2, were fixed at stage 25 and stained with the muscle antibody 12/101. (C), RT–PCR analysis shows Hairy inhibits MA expression induced by MyoD. Amounts of RNA injected: (A–B) Hairy 2, 0.1–0.2 ng; β-gal, 0.2 ng; Tex Red Dextran, 10 ng; (C) Hairy 2, 0.2 ng; MyoD, 1 ng; Hairy 2 (lanes 6–8), 0.05 ng, 0.1 ng, and 0.2 ng, respectively.
	Fig. 3. Regulation of Hairy by Notch. (A–C) RNA encoding β- gal alone (A), or together with ICD (B,C), was injected into embryos at the 2-cell stage, and at stage 20 the embryos were stained with Red Gal and processed with a Hairy 2 probe. (C), Close-up of an embryo stained with Red Gal and Hairy 2. Arrows mark cells with both Red Gal and Hairy 2 stain. Arrowhead indicates a cell with Red Gal but less Hairy 2 induction. The inset (C′), shows cells with Red Gal alone. The diffuse blue stain in the upper half of panel (C) was from the endogenous Hairy 2. (D–E), Animal cap assay, performed as in Fig. 1, shows induction of Hairy 2 by ICD (D) and Neurogenin (E). (F–H), Induction of Hairy 2 by Neurogenin. Note the ectopic stain of Hairy 2 in the Neurogenin injected side (arrow, G) as compared with the uninjected side (F). (H), Close-up of an embryo showing induction of Hairy 2 by Neurogenin. The arrow points to a pair of cells with Red Gal and Hairy 2, respectively. (I), Neurogenin activates the neurogenic pathway, which includes among others (such as NCAM and Nrp 1, not shown in the diagram) NeuroD and N-tubulin, in cell 1 (Koyano- Nakagawa et al. 1999), at the same time, together with NeuroD, induces expression of Delta (Chitnis & Kintner 1996; Koyano- Nakagawa et al. 1999) and therefore activates Notch signaling in cell 2. Consequently, in contrast to Notch, Neurogenin has a non-cell autonomous effect in inducing Hairy. Embryos face down in panels A, B, F and G. Amounts of RNA injected: (A–C) ICD, 0.5 ng; β-gal, 0.2 ng; (D, lanes 6–8) ICD, 0.5 ng; (lanes 9– 11), 1 ng; (E) DeltaStu, 0.2 ng; Ngn, 10 pg; DeltaStu (lanes 6–8), 0.1 ng, 0.2 ng, and 0.4 ng, respectively; (F–H) Ngn, 10 pg.
	Fig. 4. Mutual regulation of Hairy and ESR 1. RT–PCR was performed in A, B, D, and E to show that ESR 1 upregulated expression of Hairy 2 (A), that Hairy inhibited expression of ESR 1 in Neurogenin-injected caps (B), and that HΔW, a deletion mutant form of Hairy 2 (C), rescued the repressed expression of ESR 1 by Hairy 2 in VMZ (D) and de-repressed expression of Delta and ESR 1 in normal caps (E). (F), Hairy, induced by Neurogenin through Notch signaling, inhibits ESR 1 in cell 2. At the same time Hairy may determine the fate of cell 1 or other cells surrounding cell 2 by regulating Delta. Amounts of RNA injected: (A) ESR 1 (lanes 4–5), 0.5 ng, 1 ng; (B) Nog, 10 pg; Hairy 2, 0.2 ng; Ngn/nog (lanes 6–8), 10 pg/10 pg; Hairy 2 (lanes 7–8), 0.1 ng, and 0.2 ng, respectively; (D) HΔW, 0.2 ng; ICD/HΔW, 1 ng/0.2 ng; ICD, 1 ng; ICD/Hairy 2 (lanes 7–9), 1 ng/0.4 ng; HΔW (lanes 8–9), 0.2 ng and 0.4 ng, respectively; (E) HΔW, 0.2 ng.
	Fig. 5. Hairy controls Notch mediated Delta inhibition. (A) Animal cap or VMZ assay was performed by RT–PCR to test the effect of modulated levels of Hairy on Delta or ESR 1 expression. In (B), expression of Hairy 2/1, ESR 1 and Delta in animal caps or VMZ was compared at different stages. (C), A mid-gastrula stage embryo, as viewed from different directions, shows Hairy 2 expression. The embryo, in the panels viewed from the vegetal pole (vegetal) and side (side), was oriented so that the anterior was to the left. (D), An animal cap assay was performed showing that ESR 1 upregulated the expression of Delta. (E), Hairy likely mediates the inhibitory effect of Notch signaling on Delta. However, how Notch regulates Delta, and hence the fate of cell 2, and ESR 1 depends on the expression level of Hairy in cell 1. Question marks indicate possibilities so that the strength of Notch signaling in cell 2 correlates negatively with the endogenous Hairy level in cell 1. Note it may require a higher level of endogenous Hairy for Notch to inhibit Delta than to inhibit ESR 1 and other genes. Amounts of RNA injected: (A) HΔW, 0.2 ng; ICD, 1 ng; HΔW (lanes 6–7, 16–17), 0.2 ng and 0.4 ng, respectively; Hairy 2, 0.2 ng; Hairy 2 (lanes 11–12), 0.2 ng and 0.4 ng, respectively; (E) ESR 1, 0.5 ng.
	Fig. 6. Hairy regulates Notch activity. RT–PCR was performed to compare Notch signaling on MyoD-induced myogenesis in animal caps and VMZ (A), and how a modified level of Hairy in these cells affected Notch signaling (B–C). (D), Embryos injected with ICD (not shown), HΔW or both were stained with a MyoD probe. Arrow indicates enhanced MyoD expression in an ICD/HΔW injected embryo. (E–F), Isolated VMZ were stained with MA (E) and ESR 1 probes (G), or processed with RT–PCR (F). (H), ESR 1 and Hairy, both downstream to Notch signaling, have opposite effects on myogenesis. The relative levels of these two mediators may decide the general effect of Notch. Amounts of RNA injected: (A) ICD, 1 ng; MyoD, 1 ng; ICD (6–8, 12–14), 0.4 ng, 0.8 ng, and 1 ng, respectively; (B) Hairy 2, 0.4 ng; MyoD, 1 ng; MyoD/Hairy 2, 1 ng/0.2 ng; ICD, 0.8 ng; MyoD/ICD, 1 ng/0.8 ng; Hairy 2 (lanes 9–11), 0.1 ng, 0.2 ng, and 0.4 ng, respectively; (C) HΔW, 0.2 ng; MyoD, 1 ng; MyoD/HΔW, 1 ng/0.2 ng; ICD, 0.8 ng; MyoD/ICD, 1 ng/0.8 ng; HΔW (lanes 9–11), 0.1 ng, 0.2 ng, and 0.4 ng, respectively; (D) ICD, 0.5 ng; HΔW, 0.2 ng; (E–G) ICD, 1 ng; HΔW, 0.4 ng; ESR 1, 0.5 ng.
	Fig. 7. Hairy and Notch on Neurogenesis. (A), An RT–PCR assay was used to test the effect of Hairy on expression of neural genes induced by Neurogenin in animal caps. (B), Expressions of Nrp 1, Sox2, N-tubulin, NeuroD and ESR 1 were examined in embryos expressing ICD and Hairy 2 by in situ hybridization. In the NeuroD panel (k), black dots denote the midline of an ICD-expressing embryo. (C–D), Double-staining in situ hybridization was performed to compare the spatial expression patterns of NeuroD/Hairy 2 (C) and ESR 1/Hairy 2 (D). In the right panel of (C), the embryo was cut into two parts, which were probed for NeuroD (Red) and Hairy 2 (purple), respectively. (E), Misexpression of HΔW led to an expansion of the endogenous ESR 1 (arrows; the control was not shown to save the space). Arrows indicate either an ectopic induction or persisted expression of the endogenous gene; arrowheads mark a loss of the endogenous transcripts. All embryos face down or to the reader except those in the right panels of C and D which are to the left. Amounts of RNA injected: (A) Nog, 10 pg; Hairy 2, 0.2 ng; Ngn/nog, 10 pg/10 pg; Hairy 2 (lanes 7–8), 0.1 ng and 0.2 ng, respectively; (B) ICD, 0.3–0.5 ng; Hairy 2, 0.2 ng; (E) HΔW, 0.5 ng.
	Fig. 8. Regulation of Notch activity. For a particular target gene, Notch signaling may be inhibitory, permissive, or enhancing. A cell context signal, Y, which is inhibitory by itself and regulated by Notch and other repressive pathway(s) (RP), plays a critical role in determining what the effect of Notch signaling is. A high level of Y in the cell represses expression of both the target gene and X, an enhancing signal from the Notch pathway and makes Notch inhibitory. X may or may not upregulate Y. A low level of Y makes Notch permissive or enhancing for the target gene. However, if Y has a stronger inhibitory effect than the enhancing effect that X has, Notch signaling, when highly activated, tends to be inhibitory even when the endogenous Y is low. Y may also mediate Notchcontrolled lateral inhibition by regulating Delta.