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Keywords:

  • Delta;
  • Hairy;
  • lateral inhibition;
  • myogenesis;
  • neurogenesis;
  • Notch;
  • signaling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In Drosophila, the Notch signaling pathway is mediated by Delta and Serrate, two sets of transmembrane ligands, which recognize the Notch transmembrane receptor. Binding of the ligand to Notch triggers a set of proteolytic cleavages in Notch, which ultimately results in liberation of its intracellular domain (ICD). ICD, in association with Suppressor of Hairless (Su(H)), translocates into the nucleus and further activates Enhancer of split (E(spl)), which encodes a set of basic helix-loop-helix (bHLH) proteins. These proteins are then thought to repress expression of other proneural genes, leading to repression of neurogenesis (Artavanis-Tsakonas et al. 1999).

Notch signaling is an evolutionarily conserved mechanism that is used throughout the animal kingdom to control cell fates. In Xenopus, for example, Notch signaling components have been identified and proposed to play similar roles in neurogenesis as in Drosophila. X-Delta and Xotch, the Xenopus homolog of Delta and Notch, respectively, are expressed where the primary neurons form (Chitnis et al. 1995), and upon manipulation of X-Delta activity, the number of primary neurons can be modulated (Chitnis et al. 1995; Deblandre et al. 1999). Additional components in this pathway have also been identified in frogs, including XSu(H) (Wettstein et al. 1997), Neuralized (Deblandre et al. 2001), Nrarp (Lamar et al. 2001), ESR 1 and ESR 6e (Deblandre et al. 1999). ESR 1 encodes a member of the bHLH-WRPW family in vertebrates that is similar to E(spl)-C. Transcripts of ESR 1 are present in the neural plate and ESR 1 is thought to be a direct effector of Notch signaling (Wettstein et al. 1997; Schneider et al. 2001).

In addition to neurogenesis, Notch signaling is also involved in regulating myogenesis. In Drosophila, Notch is thought to regulate myogenesis by selecting individual precursors from a cluster of equivalent cells through lateral inhibition (Baylies et al. 1998). Consistent with this, overexpression of pathway components in mouse myogenic cell lines, such as Notch (Kopan et al. 1994; Nofziger et al. 1999), Dl−1 (Delta-like 1; Luo et al. 1997; Jarriault et al. 1998), Jagged (Lindsell et al. 1995), or Su(H) (Kato et al. 1997; Nofziger et al. 1999), leads to blockade of myogenesis. This is also true in chick embryos where overexpression of Delta l reduces expression of MyoD in infected somites (Hirsinger et al. 2001). However, mouse embryos mutant for Notch 1 (Conlon et al. 1995), Dll1 (Hrabe de Angelis et al. 1997) or RBPlk (Barrantes et al. 1999) exhibit essentially normal myogenesis and not essential for myogenic differentiation. These genes are instead considered important for coordinating the process of somite segmentation (Conlon et al. 1995; Barrantes et al. 1999). In addition to Notch signaling, Hairy is also involved in regulating segmentation under control of a segmentation clock. The dynamic expression of Hairy (McGrew et al. 1998; Davis et al. 2001; Jouve et al. 2002), as well as genetic evidence from zebrafish (Henry et al. 2002; Oates & Ho 2002) and mouse (Jouve et al. 2000) suggests an intimate link between Hairy and Notch signaling. Indeed, Delta 1, or a constitutively active form of Notch, could activate transcription of the endogenous Hes 1, the orthologue of Xenopus Hairy 1 (Davis & Turner 2001), or expression of a reporter gene driven by the Hes 1 promoter (Jarriault et al. 1995; Jarriault et al. 1998). A recent report also suggests that the promoter of Xenopus Hairy 2 contains binding sites for Su(H), which are required for its expression in the pre-somitic mesoderm (PSM) (Davis et al. 2001). These findings position Hairy as a potential effector of Notch signaling. However, more detailed studies suggest that this may be the case only in some cells such as PSM and neural precursor cells (Ohtsuka et al. 1999; Jouve et al. 2000), but not in other cells in the developing nervous system (de la Pompa et al. 1997; Davis et al. 2001).

There is another puzzle around Notch signaling in addition to the relationship between Notch and Hairy. Why does overexpression of Notch signaling pathway components inhibit the process of myogenesis in cultured cells while this signaling does not appear to be required in vivo? While there may be redundant signals, the other possible explanation is that Notch signaling has cell context-dependent effects; that is, while in the in vitro cell culture system, Notch signaling is inhibitory for myogenesis, in embryos, it may be a permissive, or neutral, signal.

Cell context-dependent use of Notch activity has been well documented recently in glial versus neural determination in Drosophila. In the bristle lineage of adult flies, Notch signaling inhibits glial fate by negatively regulating expression of gcm (glial cells missing), which is essential for glial differentiation (Van De Bor & Giangrande 2001). In contrast, in the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), Notch is specifically required for gcm expression and the glial fate (Umesono et al. 2002). Consequently, dependent on the cell context, Notch can specify opposite cell fates in different cells.

Notch can also have different effects in the same cell at different developmental stages. This has been best illustrated in the initiation of neural development in the Drosophila eye. In the eye, Notch is required for determination of R8, one of the photoreceptors, in the regulation of a proneural gene Atonal (Baker & Yu 1997; Baonza & Freeman 2001; Li & Baker 2001). When the function of Notch is completely removed at early stages, neural differentiation fails to take place. In contrast, later loss of Notch function, after Atonal expression has been established, results in formation of an excess of R8 photoreceptors (Cagan & Ready 1989; Baker et al. 1996; Baker & Yu 1997). While this indicates that Notch has an either pro- or antineural effect, depending on the cell context (which in this case, is determined by the developmental stages), the mechanism(s) that mediates the cell context, and controls lateral inhibition, has remained elusive.

In this report, we attempt to address how a cell context regulates Notch-mediated lateral inhibition and Notch activity. We show that Notch signaling in the frog embryo regulates Hairy and with a negative feedback loop, Hairy controls Notch-derived lateral inhibition by regulating expression of Delta. We further find that Notch has different effects on myogenesis. While in the ectodermal cells, Notch inhibits myogenesis induced by a myogenic protein, MyoD, it has a different effect in ventral marginal zone (VMZ) cells. The different interpretation of Notch function correlates with the expression levels of Hairy in these cells. Modification of cellular Hairy activities drives the effect of Notch from being inhibitory to permissive or enhancing. These observations have led us to propose Hairy as a cell context signal, which, by controlling responsiveness of its target genes, sets up an intrinsic bias with which the cell interprets Notch and other extrinsic signals. We provide evidence that this model applies to neurogenesis as well and speculate that Hairy-like molecules may have a universal role in metazoans in regulating Notch and other signaling pathways.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plasmids and molecular cloning

ESR 1 was cloned out using PCR based on the published sequence. Hairy 2B, which was obtained from screening a library (Grammer et al. 2000), was used in this study. The mutant form of Hairy 2, HΔW, was created by deleting the part of the cDNA encoding the last four amino acids WRPW and subcloning it into CS2 +107. For microinjection into frog embryos, RNAs were transcribed with an RNA synthesis kit (mMESSAGE mMACHINE, Ambion, Austin, TX, USA) from the linearized cDNA.

RNA injection, animal cap assay and RT–PCR

RNAs as indicated in the text were injected into animal poles of 2-cell stage embryos. Animal caps were explanted at stage 9 (Nieukwoop & Faber 1967) and cultured to various stages. These caps were either fixed and processed for in situ hybridization or subjected to RT–PCR analysis.

RNAs from various tissues (embryos, animal caps, or VMZ) were isolated using a Proteinase K based lysis buffer, treated with RNase-free DNase to remove traces of genomic DNA, and were reverse transcribed in a 20 µL reaction. From each tissue, 1 µL of the cDNA was amplified in a PCR reaction (25 µL) with [32P]-dCTP included to compare expression levels of different markers. Samples were resolved on a 5% polyacrylamide gene. Data were analyzed with PhosphorImager Storm 820 (Molecular Dynamics, Sunnyvale, CA, USA). Primers used in this study are all listed here: EF1α upstream (U), 5′-CAGATTGGTGCTGGATATGC-3′; EF1α downstream (D), 5′-ACTGCCTTGATGACTCCTAG-3′; MA (U), 5′-GCTGACAGAATGCAGAAG-3′; MA (D), 5′-TTGCTTGGAGGAGTGTGT-3′; ESR1 (U), 5′-GCTGTTCAGTTCCTGTGCTATTACC-3′; ESR1 (D), 5′-TTTGTTGGTGTTGCTTGCCAG-3′. Hairy1 (U), 5′-AACTCTTCATCCCCAGTGGCTG-3′; Hairy1 (D), 5′-GCAGGTTCCTCAGGTGTTTCAC-3′; Hairy2 (U), 5′-CGAGCACAGAAAGTCGTCCA-3′; Hairy2 (D), 5′-CGTTAAATCCGGCTCTGTACTT-3′; Delta (U), 5′-TCTGGCTTCAACTGTGAG-3′; Delta (D), 5′-AACCTCGTGCACATTGAC-3′; N-tubulin (U), 5′-CTTCCGTGGAAGAATGTC-3′; N-tubulin (D), 5′-GAGCCTTTGTCATCAAGC-3′; N-CAM (U), 5′-CACAGTTCCACCAAATGC-3′; N-CAM (D), 5′-GGAATCAAGCGGTACAGA-3′; NeuroD (U), 5′-CCCATGTATTCCACGTCA-3′; NeuroD (D), 5′-GCAGGATAGTGCATAGTG-3′; Nrp 1 (U), 5′-GGGTTTCTTGGAACAAGC-3′; Nrp 1 (D), 5′-ACTGTGCAGGAACACAAG-3′.

In situ hybridization

In situ hybridization of Xenopus embryos or tissues was performed as described previously (Sive et al. 2000). For double-staining, two different probes are labeled with digoxigenin and fluorescein, and detected with antidigoxigenin and antifluorescein antibodies (Boehringer Mannheim, Indianapolis, IN, USA), respectively. Fast Red (Boehringer Mannheim) was used for the first stain. The reaction was stopped with several washes of Ptw (PBS with 0.1% Tween 20) and samples were heated at 65°C for 10 min in 5 mm EDTA to deactivate the alkaline phosphate. The second stain was performed in BM Purple (Boehringer Mannheim) as in the regular protocol (Sive et al. 2000).

Immunocytochemistry

RNAs were injected into one blastomere of 2-cell embryos. Tex Red Dextran (10 ng; Molecular Probes, Eugene, OR, USA) was co-injected as a lineage tracer. After being fixed in MEMFA (Moon & Christian 1989) for 1 h at room temperature, the embryos were incubated with 12/101 antibody (1:100 dilution) in PBS with 20% serum overnight in the cold room, followed by incubation with the secondary antibody (1:200; peroxidase-conjugated goat antimouse IgG (H + L), Jackson ImmunoResearch laboratories, Bar Harbor, ME, USA). Diaminobenzidine (DAB)/H2O2 was used for staining.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Different effects of Notch intracellular domain and ESR 1 on MyoD activity in ectodermal cells

Notch signaling is generally thought to affect cellular differentiation and cell fate determination indirectly by biasing a choice between two fates, although it can also be instructive in some tissues (Artavanis-Tsakonas et al. 1999; Gaiano & Fishell 2002; Sato et al. 2002). In order to address the role of Notch signaling on myogenesis in frogs, we started by asking how Notch modifies MyoD function in ectodermal cells. RNA encoding MyoD (1 ng), or MyoD with ICD (0.4 ng, 0.8 ng, 1 ng) was injected into animal poles, and animal caps were collected and subjected to RT–PCR analysis using primers for MA (skeletal muscle actin). As shown in Figure 1A, co-injection of ICD inhibited the expression of MA induced by MyoD (Fig. 1A), suggesting that Notch signaling inhibits myogenesis in ectodermal cells.

image

Figure 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.

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While Notch signaling inhibits myogenesis in mouse cell lines (Kopan et al. 1994; Kato et al. 1997; Luo et al. 1997; Jarriault et al. 1998; Nofziger et al. 1999) as well as in chicken embryos (Hirsinger et al. 2001), its role in frog myogenesis is rather confusing. For example, expression of an activated mutant Xotch causes an increase in muscle tissue (Coffman et al. 1993), but the same manipulation with a mouse mutant Notch (mNotchIC) leads to repression of myogenesis (Kopan et al. 1994), and yet in embryos where Notch signaling is blocked with a Delta mutant (DeltaStu) the muscle differentiates normally (Jen et al. 1997). In agreement with these reports, as shown below, activation of Notch signaling in the frog embryo, as well as in explanted tissues, often displays different, sometimes opposite phenotypes, which appear to be dependent upon cell types as well as how strongly Notch signaling is activated.

To investigate the mechanism underlying the Notch activity, we expressed ESR 1, a downstream effector of Notch signaling, in an animal cap assay to ask how ESR 1 would affect MyoD function (Fig. 1B; see also Wettstein et al. 1997; Schneider et al. 2001). RNA encoding MyoD, or MyoD with ESR 1 (0.8 ng or 1 ng) was injected into animal poles of 2-cell embryos, and RT–PCR was performed. As shown in Figure 1C, in contrast with the observation that ICD strongly inhibits the function of MyoD, co-expression of ESR 1 did not significantly affect MyoD activity (Fig. 1C).

The ESR 1 effect on myogenesis was investigated by expressing its RNA (0.5 ng) in embryos. In such embryos, expansion of MyoD expression was often seen (62% (55), not shown). This suggests that while considered as a transcription repressor, ESR 1 can enhance expression of some genes, such as MyoD (and Delta; see below), although the mechanism is currently not known. This finding further implies that ESR 1 is not the effector that mediates the Notch-triggered inhibitory effect on myogenesis and that there must be another downstream component in the Notch signaling pathway.

Hairy inhibits myogenesis

We considered Hairy to be an excellent candidate for being downstream of Notch and negatively regulating MyoD activity. This is because the promoter of the Hairy 2 gene contains potential binding sites for Su(H) (Davis et al. 2001), and Hairy 1 severely inhibits myogenesis (Umbhauer et al. 2001). Like Hairy 1, Hairy 2 also shows strong antimyogenic activity, as injection of its RNA (0.1–0.2 ng) downregulated expression of endogenous MyoD (55% (31)), the general mesoderm marker Xbra (83% (23); arrows in Fig. 2A), as well as Xwnt8 (74% (23); data not shown). Interestingly, like Hairy 1, Hairy 2 did not inhibit other dorsal markers such as goosecoid or chordin (data not shown; Umbhauer et al. 2001).

image

Figure 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.

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Consistent with the finding that the early mesoderm markers were blocked by expression of Hairy 2, differentiated muscle, as stained with a monoclonal antibody 12/101, was also lost on the injection side of the embryo (81% (38)), suggesting that muscle failed to form in the affected area (arrow, Fig. 2B). In agreement with this, co-expression of Hairy 2 RNA inhibited MA induction by MyoD in ectodermal cells (Fig. 2C). Therefore Hairy is a candidate for being in the Notch signaling pathway and mediating Notch-induced inhibition on myogenesis.

Since Hairy 2 and Hairy 1 share functional redundancy, and Hairy 2 is more abundantly expressed than Hairy 1 in frog embryos (see below), we have used Hairy 2 rather than Hairy 1 in our experiments.

Hairy is under the regulation of Notch signaling

We then tested whether Hairy 2 is under the regulation of the Notch pathway in frog embryos. While previous studies have suggested that Hes 1 may be an effector for Notch signaling in some cells, it functions independently of the Notch cascade in other cells. For example, Hes 1 expression is not disturbed in Notch 1 mutant embryos (de la Pompa et al. 1997).

In situ hybridization with an antisense probe for Hairy 2 was performed in embryos injected unilaterally with either β-galactosidase (β-gal; 0.2 ng) alone (Fig. 3A) or with ICD (0.5 ng; Fig. 3B,C). β-gal was used to trace the injected cells. While expression of β-gal never induced expression of Hairy 2 (48), ICD not only strongly expanded its endogenous expression domain, but also induced a significant amount ectopically (79% (86); Fig. 3B).

image

Figure 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.

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An RT–PCR analysis was performed to test this further in ectodermal cells. As shown in Figure 3D, while there was some level of Hairy 2 RNA expressed in uninjected animal caps, injection of ICD increased the RT–PCR signal by an average of 2.5-fold (for 0.5 ng of ICD RNA) and 4-fold (for 1 ng of ICD RNA), as determined by densitometry analysis of the bands (Fig. 3D). This suggests that, as in the PSM (Jouve et al. 2000) and the neural progenitor cells (Ohtsuka et al. 1999), Hairy (both Hairy 2 and Hairy 1; data not shown) may act as an effector for Notch signaling.

If Notch lies upstream of Hairy 2 and upregulates its expression, Neurogenin should also do so. Neurogenin is a bHLH protein and plays a critical role in promoting neuronal differentiation in Xenopus embryos (Ma et al. 1996). Like other bHLH proteins such as XASH-3, NeuroD, and ATH-3, Neurogenin can induce neuronal differentiation in the neural epithelium of the neural plate as well as in the non-neural ectoderm (Lee et al. 1995; Chitnis & Kintner 1996; Ma et al. 1996; Takebayashi et al. 1997). At the same time, Neurogenin induces Delta expression to activate Notch signaling in a negative feedback loop that inhibits neuronal differentiation (Ma et al. 1996; Koyano-Nakagawa et al. 1999). An elevated level of Delta may also activate Notch signaling by upregulating Notch expression (Huppert et al. 1997).

Consistent with these observations, Neurogenin (10 pg) upregulated expression of Hairy 2 transcripts in animal caps (Fig. 3E), and this induction appears to be dependent on Notch signaling, since coexpression of DeltaStu (0.1 ng, 0.2 ng, 0.4 ng), which blocks the Notch pathway (Jen et al. 1997), reduced its expression to the background level (Fig. 3E).

Induction of Hairy 2 transcripts by Neurogenin was confirmed by in situ hybridization. Like Notch, Neurogenin (10 pg) injection not only expanded the endogenous Hairy 2 domain, but also induced Hairy 2 ectopically (88% (44); arrow, Fig. 3G). Since Hairy 2 is induced by Neurogenin through Notch signaling, its transcripts should be present in the cell (cell 2) that neighbors Neurogenin-expressing cell (cell 1, Fig. 3I).

The cell autonomous induction of Hairy 2 by Notch was confirmed by in situ hybridization as shown in Figure 3C (arrows). Similarly, in Neurogenin RNA injected embryos, we were also able to identify some Hairy 2 positive cells at an ectopic site, which were in close proximity to Neurogenin expressing cells (arrow, Fig. 3H), suggesting that the Hairy 2 transcripts in these cells might have been induced by Neurogenin non-cell autonomously.

Taken together, our data are consistent with the previous report in that Hairy is regulated, at least in part, by Notch signaling in frog embryos (Davis et al. 2001).

Mutual regulation of Hairy and ESR 1

Since Hairy is a downstream component in Notch signaling, we wished to know how it relates to ESR 1. As assayed by RT–PCR, overexpression of ESR 1 (0.5 ng, 1 ng) in animal caps led to an upregulation of Hairy 2 (Fig. 4A), suggesting that Hairy 2 may be downstream of ESR 1. However, it is also possible that ESR 1 and Hairy 2 are both downstream to Notch in parallel pathways. Moreover, co-injection of an RNA for a dominant negative ESR 1 with ICD never downregulated expression of Hairy 2 (data not shown), suggesting that Hairy 2 is under regulation of both Notch and ESR 1.

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Figure 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.

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To test how Hairy regulates ESR 1, we overexpressed Neurogenin in neuralized caps (co-injected with 10 pg of noggin) to strongly induce ESR 1 expression (Koyano-Nakagawa et al. 1999), since ICD in these cells often showed a weak induction. As shown in Figure 4B, co-injection of Hairy 2 (0.1 ng, 0.2 ng) with Neurogenin (10 pg) led to a strong reduction in ESR 1 expression (Fig. 4B). This suggests that Hairy inhibits Neurogenin-induced ESR 1 expression. This inhibition is presumably not due to a direct inactivation of the Neurogenin protein since if that were true, Hairy should have inhibited all genes that are induced, including Nrp 1 (see Fig. 7A).

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Figure 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.

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We engineered a mutation in the Hairy 2 construct, and used it to inhibit activity of the wild-type protein. Hairy, like other members in the bHLH-WRPW family, has three domains: a bHLH domain, which is at the N-terminal, an Orange domain in the center and a WRPW domain at the C-terminal (Davis & Turner 2001; Fig. 4C). The WRPW domain is required for mediating the inhibitory effect of the protein by interacting with the Groucho family of general transcription repressors (Paroush et al. 1994). Therefore, by deleting this domain, we created a Hairy mutant called HΔW, in the hope that it would interfere with activity of the wild-type protein when they heterodimerize (Davis & Turner 2001). As expected, while Hairy 2 (0.4 ng) inhibited induction of ESR 1 by ICD in VMZ tissues (Fig. 4D), addition of HΔW (0.2 ng, 0.4 ng) to ICD/Hairy 2 (1 ng/0.4 ng) injected tissues overcame the inhibitory effect of Hairy 2 and led to a complete rescue of ESR 1 expression (Fig. 4D). The stronger induction of ESR 1 in ICD/Hairy2/HΔW groups than in the ICD sample suggests that ESR 1 is repressed by the endogenous Hairy, therefore upon alleviating repression, the cells are more responsive to the ICD signal (see below). Similarly in animal caps, HΔW rescued, although partially, the inhibitory effect of Hairy on MyoD-induced MA expression (data not shown). Therefore HΔW acts as a Hairy interfering mutant. To further test the specificity of this mutant, we coexpressed RNAs encoding HΔW (1 ng) and ESR 1 (0.5 ng) in VMZ tissues, to ask if HΔW interferes with the ability of ESR 1 to activate MA (Fig. 6E). Our data confirmed that while HΔW strongly inhibited the activity of Hairy 2, it failed to do so with ESR 1 (Fig. 4D; data not shown).

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Figure 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 (68, 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.

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We then asked how modified Hairy 2 activity in the cell would affect the expression of ESR 1. As shown in Figure 4E, consistent with the observation that addition of Hairy 2 to Neurogenin-injected cells inhibited ESR 1 expression (Fig. 4B), injection of HΔW, which interferes with activity of the wild-type Hairy, increased the ESR 1 signal by 5.7-fold in uninjected animal caps (Fig. 4E), suggesting that the endogenous Hairy negatively controls expression of ESR 1 (see also Fig. 4D).

Significantly, Hairy 2 not only controls the expression of ESR 1, but also regulates Delta. As shown in Figure 4B,E, addition of Hairy 2 led to a reduction of Delta expression in Neurogenin-expressing caps (Fig. 4B), and inhibition of the endogenous Hairy activity by HΔW (0.2 ng) led to a de-repression of Delta in normal animal cap cells (2.6-fold increase; Fig. 4E). Significantly, since the Delta level controls Notch-mediated lateral inhibition (Kunisch et al. 1994), we speculated that Hairy may regulate this process through modifying Delta (Fig. 4F).

Endogenous Hairy controls Notch-mediated Delta inhibition

An animal cap assay was performed in which we tested Delta expression in the cells overexpressing Notch ICD (1 ng) alone, with Hairy 2 (0.2 ng, 0.4 ng) or HΔW (0.2 ng, 0.4 ng). These cells were considered as a single cell in this assay since they all inherited the injected RNAs. Therefore, the Delta level in this cell should indicate if it has strong or weak lateral inhibition were it in contact with another cell(s).

As shown in Figure 5A, expression of HΔW in animal caps led to a robust expression of Delta (and ESR 1); addition of HΔW to ICD also resulted in Delta and ESR 1 upregulation (4–10-fold increase; Fig. 5A). However, when the similar assay was performed in VMZ, we noted that overexpression of Hairy 2, or co-injection of Hairy 2 with ICD did not lead to a significant repression on Delta expression, while the same manipulation led to a strong inhibitory effect on ESR 1. Similarly, coexpression of HΔW with ICD did not lead to an upregulation of Delta, while there was a significant increase in the ESR 1 induction (Fig. 5A).

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Figure 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.

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Notably, while overexpression of ICD in animal caps led to a nearly complete repression of Delta expression (90% reduction; lanes 3 and 5, Fig. 5A), the same injection did not have a profound effect in VMZ (5% reduction; lanes 8 and 10, Fig. 5A). This suggests that Notch could mediate either a strong (in animal caps) or weak (in VMZ) lateral inhibition in different cells.

Since manipulation of Hairy activities could affect Delta expression, this cell context-dependent Delta inhibition mediated by Notch signaling may be controlled by levels of Hairy in these cells. Another possibility is that Notch signaling can generate different amounts of Hairy in ectoderm and VMZ, which has been ruled out by our comparison analysis (data not shown). We then assayed for Hairy expression in these two tissues by RT–PCR. Tissues were prepared at stage 10 and either processed for the assay immediately or left to age to stage 13 or 20. As shown in Figure 5B, in all stages examined, there was a marked difference in Hairy 2 expression in these tissues (1.5–3.6-fold difference). Hairy 2 was expressed consistently at higher levels in ectodermal cells than in VMZ. Hairy 1 expression followed the same pattern although at the early neurula stage, there was no significant difference in the two cells. Therefore, the different levels of Hairy in ectodermal versus VMZ likely accounts for the different effect from Notch signaling in these cells. Consistent with the observation that Hairy inhibits expression of Delta (Fig. 4B), in ectodermal cells where there was high Hairy expression, Delta is expressed at low levels. Conversely, in VMZ where there was a low level of Hairy, Delta is high. The difference for Delta expression between these two cells was much larger at early stages (3.2-fold difference at stage 10, and 2.6-fold at stage 13), and then the difference reduced to minimal at later stages (Fig. 5B). Interestingly, ESR 1, which represents the readout of Notch signaling, was expressed at higher levels in ectodermal cells at early gastrula/neurula stages, but this pattern was reversed at tadpole stages. This suggests that in addition to Notch, other signaling cascades or mechanisms may be involved in regulating the expression of Hairy 2 (Davis et al. 2001).

Hairy 2 expression at midgastrula stage was examined by in situ hybridization. As shown in Figure 5C, the transcripts were abundantly present in the ectodermal and dorsal mesodermal cells, but expressed less in the ventral side, confirming the results from our PCR analysis.

Importantly, activation of the Notch pathway in the embryo leads to upregulation of Hairy (Fig. 3); however, this induced Hairy does not compromise the Notch cell context-dependent effect. For example, ICD injection results in much stronger induction of ESR 1 in VMZ than in ectodermal cells (lanes 10 and 5, Fig. 5A), suggesting that the endogenous Hairy is an important regulator.

Furthermore, whether Notch can inhibit Delta is also dependent on the endogenous Hairy activity. Only when it is high, such as in ectodermal cells (cell 1, Fig. 5E), can Delta be downregulated by Notch activation. In contrast, in VMZ cells where the endogenous Hairy activity is low, Delta expression is resistant to Notch regulation. Interestingly, while ESR 1 upregulates Delta (Fig. 5D), its own expression is subjected to regulation by Hairy (Fig. 4B,E). This suggests that while ESR 1 plays a part in regulating Delta, Hairy, by inhibiting both Delta and ESR 1, is more important in this regard. Nevertheless, conceivably, ESR 1 may exhibit its effect in some developmental processes such as myogenesis when the endogenous Hairy level is low (Fig. 5E).

Hairy determines Notch activity on myogenesis

We compared the effects of Notch signaling on MyoD-induced myogenesis in ectodermal cells and VMZ. Consistently, while ICD (0.4 ng, 0.8 ng,1 ng) displayed a strong inhibition on MA expression induced by MyoD (1 ng), it did not seem to inhibit MA in VMZ (Fig. 6A). This suggests that for myogenesis, Notch is an inhibitory signal in the ectodermal cells for this dose range, while it is permissive or neutral in isolated VMZ. Since Hairy is expressed at different levels, that may account for the different effect of Notch signaling in these two cells.

To test this, we manipulated the levels of endogenous Hairy in these cells, asking how that would affect Notch signaling. In a VMZ assay, we expressed RNA for MyoD (1 ng), either alone or with ICD (0.8 ng), in the presence of increased Hairy 2 RNA (0.1 ng, 0.2 ng, 0.4 ng). MA expression was assayed as shown in Figure 6B. Consistent with its effect on myogenesis, the addition of Hairy 2 switched Notch from being permissive to inhibitory, as evidenced by the downregulation of MA expression (lanes 10, 11 over lane 8, Fig. 6B).

The activity of endogenous Hairy in ectodermal cells was brought down by expression of HΔW and the effect of Notch signaling on myogenesis was examined in this compromised background. As shown in Figure 6C, while ICD (0.8 ng) strongly inhibited MyoD-induced MA induction in normal ectodermal cells (lane 8 over lane 5, Fig. 6C), MA induction recovered significantly in cells when Hairy 2 activity was decreased with HΔW (0.1 ng, 0.2 ng, 0.4 ng; 2-fold increase in lane 11 over lane 8, Fig. 6C). While comparison of MA levels in these samples indicated that a lower level of Hairy in animal cap cells still could not change Notch from being inhibitory to enhancing, HΔW RNA injection clearly made Notch embark on this track. The incomplete rescue of the ICD inhibitory effect on MyoD, echoed by the similarly incomplete rescue of ICD inhibition on Delta expression in ectodermal cells (Fig. 5A), suggests that the cellular microenvironment established by the high level of Hairy defies exogenous efforts to compromise it. VMZ cells are even more refractory, that is, addition of Hairy could not in any way restore the ability of ICD to inhibit Delta (Fig. 5A). These findings highlight an important role for the endogenous Hairy in establishing a cell context.

We examined the effect of Notch signaling on myogenesis in frog embryos by in situ hybridization. While expression of ICD (0.5 ng; data not shown) or HΔW (0.2 ng) did not have any effect on MyoD expression, co-injection of these RNAs led to a significant increase in MyoD transcripts in a majority of the injected embryos (55% (38); arrow, Fig. 6D). This was further confirmed by RT–PCR, where co-injection of ICD with HΔW enhanced MyoD expression by 23% over the control, and more than 70% increase over the ICD group (10 individual embryos were analyzed; data not shown). This suggests that when Hairy level is low enough, Notch signaling enhances the myogenic pathway.

In order to test this in a more defined context, VMZ were isolated from embryos which had been injected with RNA for HΔW (0.4 ng), ICD (1 ng), or both, and assayed for MA expression by in situ hybridization and RT–PCR analysis. As shown in Figure 6E, while ICD RNA injection induced expression of MA in some VMZ, coexpression of ICD with HΔW led to its induction at a much higher level, which was confirmed by RT–PCR (Fig. 6F).

ESR 1 was able to induce MA in VMZ by itself (Fig. 6E,F), therefore a higher level of MA induction by ICD/HΔW injection might be due to the enhanced expression of ESR 1, which was repressed by the endogenous Hairy (Fig. 4E). Consistent with this hypothesis, co-injection of ICD and HΔW led to a higher expression of ESR 1 over the ICD group in VMZ (Fig. 6G; also Fig. 5A).

Collectively, these data suggest that the level of Hairy in a cell determines whether Notch signaling is inhibitory, permissive, or enhancing for myogenesis in the frog embryo. It may do so, at least partially, by regulating the level of ESR 1 (Fig. 6H).

More importantly, since the endogenous Hairy appears to selectively regulate a large number of genes, we were prompted to consider Hairy as a cell context signal, which controls how a cell responds to an external signal, such as Notch. As a cell context signal, its own expression is regulated by different mechanisms. In support of this, while the Hairy 2 promoter contains binding sites for Su(H) (Davis et al. 2001) and its expression can be upregulated by Notch, misexpression of DeltaStu does not significantly decrease its endogenous transcripts (Fig. 3E), and the spatial expression pattern in the embryo does not always correlate with that of ESR 1 (Fig. 5B; see also Davis et al. 2001). Together, our data suggest that Hairy, regulated by Notch and other mechanisms, provides a cell (or tissue) specific setting which may bias the cell towards a particular decision in response to Notch, or other external stimuli. This may explain why Notch often displays opposite effects in different contexts.

Hairy and Notch in neurogenesis

To test the Hairy–Notch relationship in other systems, we chose to examine neurogenesis, since Notch is known to regulate neural development. In Xenopus, an early landmark for neurogenesis is the expression of N-tubulin, whose expression marks neurons (Oschwald et al. 1991; Chitnis et al. 1995). Previous studies have shown that the generation of neurons from neural progenitors is promoted by the activity of a variety of neural bHLH proteins and a balanced inhibition by Delta–Notch signaling. Neurogenin, and other bHLH proteins, not only promotes expression of genes involved in neuronal differentiation such as NCAM (a general neural marker; Kintner & Melton 1987), NeuroD and N-tubulin (markers of differentiated neurons; Lee et al. 1995; Ma et al. 1996), it activates the lateral inhibition machinery at the same time (Chitnis 1995; Chitnis & Kintner 1996; Koyano-Nakagawa et al. 1999). We therefore tested if Hairy could affect Neurogenin-induced neurogenesis in ectodermal cells.

Consistent with our observation that a high level of Hairy makes Notch signaling inhibitory, co-injection of RNA for Hairy (0.1 ng, 0.2 ng) with Neurogenin (10 pg) in ectodermal cells led to inhibition of some neural genes such as N-tubulin, NCAM, and NeuroD, although Nrp 1 was essentially not affected (Fig. 7A). An alternative interpretation is that a high level of Hairy itself is inhibitory, thus inhibiting the Neurogenin-induced neurogenesis.

We compared the spatial expression patterns of these neural markers in embryos expressing either Notch ICD or Hairy 2 in order to further study the relationship between them. Expression of Nrp 1 as well as Sox2 and Sox3 in either ICD or Hairy 2-injected embryos (not shown) was not dramatically reduced; occasionally there was some enhancement instead (Nrp 1, 14% (21); Sox2, 12% (32) for ICD-injected embryos; arrows, Fig. 7Bb and 7Bd). Since Nrp 1 is a pan-neural marker, and Sox2 and Sox3 are important for neural competence (Kishi et al. 2000), these findings suggest that there is an intrinsic link between neural competence and Notch/Hairy signaling.

We further examined the expression of N-tubulin, NeuroD and the Notch downstream gene ESR 1 in ICD- and Hairy 2-injected embryos. When ICD (0.3 ng–0.5 ng) was injected unilaterally into embryos, N-tubulin was inhibited uniformly throughout the injected side (94% (31); arrowhead, Fig. 7Bf). However, NeuroD was more severely inhibited in the head area of the embryo than in the trunk (52% (29); arrows/arrowhead, Fig. 7Bj and 7Bk); in the injected embryos, the highly expressed anterior domain of NeuroD was often completely lost while the weaker, posterior strips often persisted, although at a significantly reduced level. This suggests that for NeuroD expression, injection of the same amount of Notch can elicit different responses, which are cell context-dependent. Consistent with our observation, injection of NeuroD causes formation of ectopic neurons of high density in the trunk of Xenopus embryos (Chitnis & Kintner 1996), which was interpreted as NeuroD being relatively refractory to Notch inhibition.

ESR 1 expression offers a more striking example for this cell context-dependent effect of Notch signaling. In ICD-expressing embryos, ectopic ESR 1 was induced only in cells outside of the head region (arrows, Fig. 7Bn,o); within the head area, ESR 1 was never induced and the endogenous ESR 1 was even inhibited, sometimes completely by the ICD injection (67% (36); arrowheads, Fig. 7Bn,o). This suggests that Notch signaling, dependent on different cell contexts, regulates these neural genes differently. Since in myogenesis Hairy controls Notch activity, and indeed Hairy 2 is expressed at a much higher level in the head than in the trunk (Fig. 3A; Fig. 7C,D), it is likely that Hairy also regulates Notch signaling in neural development.

There are some significant differences between Hairy 2 and ICD effects on these genes. While injection of Hairy, like ICD, inhibited expression of NeuroD (53% (30)) and ESR 1 (65% (34)) more strongly in the head than in the trunk (arrowheads, Fig. 7Bl,p), ectopic N-tubulin expression was sometimes seen in the trunk (34% (35); arrow, Fig. 7Bh), although in other embryos its expression was lost throughout the injection side (60% (37); arrowhead, Fig. 7Bg). The ectopic N-tubulin expression was never seen in ICD-injected embryos. This is likely because in the trunk, low levels of the endogenous Hairy makes Hairy a permissive, or even weakly enhancing signal for some genes such as NeuroD, Nrp 1, and the neural competence factors Sox2 and Sox3; alternatively, this may be due to possible interactions between Hairy and other signaling cascades, such as BMP (data not shown). In contrast, Notch signaling is more likely to be permissive than enhancing, since another downstream gene, ESR 1, also inhibits neurogenesis (data not shown; see also Schneider et al. 2001).

The cell context-dependent effect of Hairy on expression of these genes suggests that Hairy is inhibitory in the head, and permissive or enhancing in the trunk for the genes studied. These different effects must have an impact on the expression of these endogenous genes. To explore this possibility, double-staining in situ hybridization was performed to examine the expression patterns of NeuroD and ESR 1, respectively, with Hairy 2. Consistent with our observation that Hairy has different effects in different regions of the embryo, in the head, neither NeuroD nor ESR 1 was expressed in the same domain as Hairy 2, while in the trunk, both were coexpressed with Hairy 2 (Fig. 7C,D).

The complementary expression of NeuroD and ESR 1 with Hairy 2 in the head of Xenopus embryos suggests that in that region, Hairy may have a role in restricting the expression domain of these genes. To test this, the endogenous Hairy 2 was inhibited by HΔW and expression of NeuroD and ESR 1 was examined in these embryos. As shown in Figure 7E, misexpression of HΔW led to a weak expansion of the endogenous ESR 1, although no NeuroD expansion was noticeable (data not shown). This suggests that Hairy 2 levels must be higher in individual cells in the head than in the trunk, so that cells only in this region can repress the endognous ESR 1; furthermore, as a cell context signal, Hairy may have a preset hierarchy in controlling expression of its target genes to ensure a well defined response to external stimuli.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lateral inhibition and Hairy

Notch signaling has been proposed to regulate cell fates through lateral inhibition. In this model, neighboring cells, whether they are equivalent, intrinsically biased, or extrinsically biased, communicate through Delta–Notch signaling, which, with a feedback mechanism, amplifies and consolidates the differences between Notch and Delta levels. Eventually this difference causes one cell to enter the receiving mode and the other signaling mode with two different cell fates (Chitnis 1995; Artavanis-Tsakonas et al. 1999).

A key feature for this model is that Notch signaling must be inhibitory in the receiving cell; in addition, Delta must also be downregulated. However, our data, as well as those from others, suggest that Notch signaling can have different effects, either inhibitory, permissive or enhancing; and on the neighboring cell, Notch signaling can be either permissive or inhibitory. Our studies indicate that Hairy levels in the receiving cell play a critical role in determining the effect of Notch on both cells. When the level of Hairy is high, it makes Notch inhibitory, and consequently Delta expression is downregulated, which transmits a strong lateral inhibition on the next cell. As a result, these two cells, with one being in the receiving mode and the neighbor in the signaling mode, end up with two different fates. Conversely, when the level of Hairy is low in the receiving cell, Notch signaling is permissive or even enhancing, such that it does not interrupt Delta expression. Therefore, with a weak lateral inhibition, both cells have the same strength of Notch signaling and adopt the same fate (see the model in Figure 5E). This happens when a group of cells maintain the same cell fate as shown in Figure 7C,D for NeuroD and ESR 1 anterior expression domains. Indeed, in these domains Hairy expression is completely off.

Genetic studies from Drosophila have indicated that E(spl)-C is involved in regulating the Notch–Delta loop. Mutation of this gene, like Notch or Delta mutants, causes neural hyperplasia at the expense of epidermis. In addition, a mutant cell inhibits its neighboring wild-type cell and causes them to adopt an epidermal fate (Heitzler et al. 1996). These data have been interpreted as E(spl)-C downregulating Delta, although there could be another plausible explanation, that is, it is another gene, controlled by E(spl)-C, that causes the mutant phenotype.

In the frog system, ESR 1, the frog homolog of E(spl)-C, while inhibiting expression of some neural genes (data not shown and Schneider et al. 2001), upregulates Delta. In addition, when the endogenous Hairy activity is inhibited, there is a concomitant increase in Delta and ESR 1 expression. This suggests that in the frog embryo Delta is under dual regulation of Hairy and ESR 1. However, it is Hairy, but not ESR 1, that is involved in downregulating Delta. The discrepancy between frogs and flies may suggest a selected use of Notch signaling components in different systems.

Notch signaling and the dose-dependent effect

We noticed that Notch has different effects, depending on the doses we employed. In ectodermal cells, for example, 0.2 ng of ICD RNA showed little, if any, inhibitory effect on MyoD-induced myogenesis (data not shown), while 1 ng almost completely abolished MA induction by MyoD. In contrast, 1 ng of ICD RNA injection usually induces MA expression in isolated VMZ. On the other hand, in embryos, while injection of 0.2–0.5 ng of ICD RNA does not have much effect on MyoD expression, 4 ng decreases it by 30% (data not shown). These data argue that different cells (ectodermal vs. VMZ) have different ‘thresholds’ for interpreting Notch signaling as inhibitory or permissive, but the recurring theme is that the more Notch, the more likely it is inhibitory. Presumably this is due to the inhibitory effect of Hairy, which is produced in proportion to the Notch expressed (data not shown).

Notch, Hairy and gene-specific regulation

Our data also suggest that for the same input of Hairy or Notch signaling, there is a significant difference in the response from different genes. Genes that are involved in myogenesis such as MyoD, Xwnt8, and the pan-mesoderm marker Xbra, are strongly inhibited by Hairy, while those involved in the dorsal development such as goosecoid and chordin are not. In the neurogenic pathway, N-tubulin, NeuroD, ESR 1, Sox2/3, and Nrp 1 all have unique response to Notch signaling. For some of them such as Sox2/3 and Nrp 1, Notch signaling is permissive; it is strongly inhibitory for N-tubulin, and is cell context-dependent, either enhancing or inhibitory, for NeuroD and ESR 1.

The effect of Notch signaling on selecting primary neurons in Xenopus

Studies overexpressing Notch ICD or Delta, and blocking Notch signaling in frog embryos have suggested that Notch signaling is involved in selecting primary neurons through lateral inhibition (Chitnis et al. 1995). Consistent with this proposal, Delta is expressed in the neural plate where the primary neurons will differentiate. Widespread expression of Delta or ICD reduces the number of neurons; further, neurons with higher density form within the proneural domains when Notch signaling is blocked, which is achieved by expressing DeltaStu.

In this report, we have presented data that expression levels of Hairy determine if Notch signaling is inhibitory. However, as discussed above, Notch shows different effects, depending on the amount of ICD injected. Therefore, it is easy to understand that overexpression of either Delta or ICD in the embryo inhibits formation of neurons since above a certain threshold, Notch signaling can be inhibitory, although the endogenous Hairy level is low in the proneural domains.

Delta expression is more stringently dependent on endogenous Hairy. When it is low, Notch signaling does not downregulate Delta; in a cell such as one in the VMZ tissue, exogenous Hairy does not even help. This is in contrast to other genes, such as ESR 1 which is regulated by Notch in a more sensitive way (Fig. 5A). However, while in ectodermal cells, Notch regulates Delta (Fig. 5A, lanes 5–7); similarly manipulation of the endogenous Hairy activity in VMZ cells did not modify Delta expression in the dose range we employed (Fig. 5A, lanes 10–12 and 15–17). We consider Delta is relatively resistant to Notch regulation, that is, it requires more stringent regulation (i.e. higher level of the endogenous Hairy activity) such that it is subject to regulation by Notch activation. Notably, ESR 1 regulation by Notch is also Hairy activity-dependent. For example, in ectodermal cells, a mild inhibition of endogenous Hairy activity did not lead to significant upregualtion of ESR 1 (Fig. 5A, lanes 5 vs. 6). Similarly, in VMZ cells, a mild increase of Hairy activity did not significantly inhibit ESR 1 expression (Fig. 5A, lanes 10 vs. 11). Therefore, we predict that Delta expression must also be regulated in the same way as ESR 1 by Notch, although it requires a cell context established by much higher endogenous Hairy activity, as in ectodermal cells. Had we more dramatically modified the endogenous Hairy activity in VMZ cells, which often compromised the cells’ ability to survive and therefore prevented us from doing so, we would have seen that Delta expression is subject to Notch regulation in the same way as ESR 1. This context, that is, low endogenous Hairy activity, makes a cell like those in the VMZ tissue interpret Notch as a permissive signal; at the same time, since Delta expression tends not to be repressed, the neighboring cells would have similar strength of Notch activation. As a consequence, cells in this zone would reveal Notch as a permissive or even an enhancing signal (i.e. MA induction by Notch alone; Fig. 6).

Collectively, our data suggest that the endogenous Hairy may play a critical role in establishing an environment for Notch signaling to play out its effect. Therefore, since Hairy 2 is expressed at relatively low levels in the neural plate (Fig. 3A), our data do not support a role for Notch mediated Delta downregulation in selecting primary neurons. Nevertheless, the DeltaStu misexpression phenotype suggests that in the normal embryo, Notch signaling is inhibitory. As a result, in cell 2 (Fig. 4F) that is next to a Neurogenin-expressing cell (cell 1), the high level of Notch signaling, created by high levels of Delta in cell 1 as activated by Neurogenin (Kunisch et al. 1994), is sufficient for inhibiting N-tubulin in this cell, but not sufficient for inhibiting Delta. Under this condition Notch signaling in cell 2 does not actively modulate the fate of cell 1 or other cells that surround it through lateral inhibition mechanism. We consider this an example of ‘weak selection’. As a result, while these two cells may still adopt different fates, the basal level of Notch signaling in cell 1 is not perturbed and acts as an inhibitory signal. This makes the threshold higher for induction of the complete set of genes that are involved in neurogenesis, which could lead to partial expression of the whole set of neural genes in this cell. On the other hand, if the Hairy level is low enough in cell 2, or the gene of interest is refractory to Hairy/Notch inhibition, both cells within this territory could adopt the same fate or express the gene uniformly (‘zero’ or ‘minus’ selection), since in this scenario, Notch signaling acts as a permissive, or even enhancing signal. Examples of this category include Nrp 1, and Sox 2/3, which lack the salt and pepper expression pattern since they are not inhibited by Hairy and Notch signaling, and the anterior domains of NeuroD/N-tubulin. Conceivably, a high level of Hairy in cell 2 will strongly inhibit the neurogenic cascade in this cell and at the same time, due to the inhibition of Delta in cell 2, it decreases the strength of Notch signaling in cell 1. This dual inhibition, accomplished by the activation and inhibition of Delta in cell 1 (as induced by Neurogenin) and cell 2 (as repressed by Notch signaling in a high Hairy activity context) respectively, makes a ‘strong selection’ possible, which may lead to a reinforcement of the neural fate decision in cell 1. This likely occurs in those cells that reside across the border of Hairy 2 expression and anterior domains of NeuroD/N-tubulin. We anticipate that the same mode of Notch/Hairy interaction applies to the procedure of myoblast selection. The dynamic expression pattern of Hairy at early stages, however, prevents us from performing a more detailed analysis, which involves direct measurement of lateral inhibition in vivo by targeted delivery of ICD into a subset of cells in the embryo.

A universal model for Notch action

Collectively, our data suggest that in both Xenopus myogenesis and neurogenesis, and possibly other systems, Notch signaling can have different effects on the receiving cell as well as on the neighboring cell. These effects are dependent on the level of a Hairy, or Hairy-like inhibitory protein ‘Y’ (Fig. 8) in the cell. This cell context signal, which may be regulated by other mechanism(s) (repressive pathway, RP), controls the responsiveness of its target genes and ‘X’, an enhancing signal. A high level of Y sets a repressive tone for the genes in response to Notch or other external signals. Y also regulates the expression level of Delta, hence indirectly participating in the cell fate determination (Fig. 8). Cell context change may have an important impact on cell fate determination and other developmental processes (Li & Baker 2001; Van De Bor & Giangrande 2001; Umesono et al. 2002). We think this may be a universal model for describing Notch activities in different contexts and species and will provide a framework for the future research on Notch signaling.

image

Figure 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 Notch-controlled lateral inhibition by regulating Delta.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was performed at the University of California, Berkeley, where Y. C. was a postdoctoral fellow. Y. C. would like to thank Dr J. Gurdon and C. Kintner for kindly providing plasmids and Dr J. Christian and Dr A. H. Monsoro-Burq for reading this manuscript. This study was supported by an NIH grant.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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