XB-ART-38250
Dev Biol
2008 Oct 15;3222:368-80. doi: 10.1016/j.ydbio.2008.07.026.
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Hairy2 functions through both DNA-binding and non DNA-binding mechanisms at the neural plate border in Xenopus.
Nichane M
,
Ren X
,
Souopgui J
,
Bellefroid EJ
.
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The Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest progenitor survival and maintains cells in a mitotic undifferentiated pre-neural crest state. However, its mode of action remains largely unknown. Here we show that a Hairy2 DNA-binding mutant is unable to promote cell survival and to upregulate the expression of early neural border genes but is capable to increase cell proliferation and to expand NC in late embryos. We found that Hairy2 transiently activates in a DNA-binding independent manner the expression of the Notch ligand Delta1 and that Delta1 is required for Hairy2 to promote cell proliferation and to expand NC. Finally, we provide evidence that Hairy2 induces Delta1 through the transcription factor Stat3. Together, these results suggest that Hairy2 has a dual mode of action and may function at the neural plate border through both a DNA-binding and a non-DNA-binding Stat3-Delta1 mediated mechanism.
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Species referenced: Xenopus
Genes referenced: bax bcl2 dlc dll1 hes1 hes2 hes4 hes5.10 hes5.5 hey1 id3 msx1 myc notch1 pax3 six1 snai2 sox10 sox15 sox3 sp6 srrt stat3.2 tbx2 tubb2b
???displayArticle.morpholinos??? dll1 MO1 hes4 MO1 hes4 MO2 stat3.1 MO1
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Fig. 1. Hairy2 has DNA-binding dependent and independent activities. (A) Whole-mount in situ hybridization analysis of embryos injected with Hairy2-GR mRNA (500 pg) or Hairy2DBM-GR mRNA (500 pg) and hybridized with the indicated probes. SoxD is ectopically induced and Msx1 and Pax3 expanded by Hairy2-GR but not by Hairy2DBM-GR. In contrast, both Hairy2-GR and Hairy2DBM-GR increase Sox10. Frequency of embryos with the indicated phenotype: a, 89% ectopic, n = 19; b, 100% normal, n = 26; c, 59% expanded, n = 27; d, 65% expanded, n = 32; e, 93% normal, n = 29; f, 72% normal, n = 28; g, 68% expanded, n = 25; h, 57% expanded, n = 22). Lateral views of tailbud embryos (a,b,g,h) with control and injected sides or dorsal views of neurula embryos (c–f) with the injected side on the right, anterior to bottom, are shown. LacZ was used as lineage tracer. (B) Both Hairy2-GR and Hairy2DBM-GR overexpression (500 pg mRNA each) increases the number of pH3 positive cells (arrows) (a, 75%, n = 24; b, 47% n = 30). Neurula embryos viewed from the dorsal side, with the injected side on the right are shown. Rhodamine dextran was used as a lineage tracer. (C) Injection of Hairy2-GR mRNA (500 pg), but not Hairy2DBM-GR mRNA (500 pg), rescues Bax (50 pg mRNA) mediated embryonic lethality. The external aspect of the embryos at early gastrula stage was monitored for the presence in the injected area of depigmented enlarged dying cells which are expulsed from the embryo and trapped in between the surface of the embryo and the vitelline membrane. Examples of embryos classified as developing normally (a) or showing a low (b) or a high number of dying cells (c) is shown. The percentage of embryos developing normally or showing a mild or severe phenotype observed in each condition is shown in (c) (−, Hairy2-GR mRNA injected minus DEX). The number of embryos examined is indicated. Embryos were injected at the four cell-stages in the animal pole of one blastomere. Rhodamine dextran was used to identify the injected area (not shown). |
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Fig. 2. Hairy2 transiently activates Delta1. (A) Dorsal views of neurula embryos showing Delta1 upregulation upon Hairy2-GR, Hairy2DBM-GR or Hes1-GR injection (500 pg mRNA) (a, 72%, n = 22;b, 83%, n = 36; c, 68%, n = 16, respectively). Q-PCR analysis of Delta1 expression at different time points until tadpole stage in animal caps derived from embryos injected with noggin + Wnt8 overexpressing or not Hairy2-GR mRNA (250 pg/blastomere), with DEX added at the equivalent of stage 12. Note the rapid and transient activation of Delta1 (d). Antero-dorsal view of a late neurula Hairy2 depleted embryo with reduced Delta1 expression in the NC migrating streams (75%, n = 20) (e). Hairy2 MOs (10 ng/blastomere) decreases Delta1 expression in animal caps induced to NC at the equivalent of stage 14 (f). (B) Double in situ hybridization showing that Delta1 (purple) and Hairy2 (light blue) expressions overlap in the anterior portion of the lateral neural plate border (arrows) (a). Higher magnification of the lateral neural plate border (a′) and cross section through its anterior portion (a′) are shown. (C) Whole-mount in situ hybridization of Delta1 expression in neurula embryos injected with Id3-GR (1 ng), Hairy2-GR (500 pg) and Id3-GR mRNA (1 ng) or Id3 MO (15 ng). Note that Id3 reduces Delta1 (a, 50% inhibited, n = 18) and counteracts Hairy2 activation of Delta1 (b, 73% unaffected, n = 30) while injection of Id3 MO (15 ng) upregulates its expression (c, 60% upregulated, n = 25). Q-PCR analysis of Delta1 expression in NC induced explants derived from embryos injected with Hairy2 MOs (10 ng/blastomere) or Id3 MO (15 ng/blastomere) as indicated (−, uninjected NC induced caps) (d). Note that Id3 depletion upregulates Delta1 and that Hairy2 knockdown and the double knockdown of Id3 and Hairy2 result in Delta1 inhibition. In all in situ panels, neurula embryos are viewed from the dorsal side. LacZ was used to identify the injected side (right side). |
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Fig. 3. Identification of Hairy2 domains required for Delta1 induction. (A) Schematic representation of Hairy2-GR wild-type and mutants used. The bHLH, Orange domain and WRPW C-terminal motif are indicated. Embryos injected with 500 pg of mRNA of the indicated inducible constructs were tested at neurula stage by in situ hybridization for Delta1 expression. The activity of the different Hairy2 mutants is indicated (+) for active and (−) for inactive. Note that all mutants containing both the N-terminal portion of the protein and the HLH domain are active. All constructs were tested in a minimum of 20 embryos and the activity described was observed in at least 70% of the embryos. (B) Whole-mount in situ hybridization of neurula embryos injected with 500 pg of HRT1-GR or HRT1-NtermHairy2-GR mRNA and analysed for Delta1 expression. Note that in contrast to wild-type HRT1-GR, HRT1-NtermHairy2-GR induces Delta1. Frequency of embryos: a, none induced, n = 24; b, 85% induced, n = 14. Embryos are viewed from the dorsal side, with the injected side to the right. LacZ mRNA was used as lineage tracer. (C) Q-PCR analysis of Delta1 and ESR6e expression in stage 14 NC induced intact or dissociated animal caps derived from Hairy2-GR mRNA (250 pg/blastomere) injected embryos (−, Hairy2-GR mRNA injected minus DEX). Note that Hairy2-GR activates Delta1 in dissociated NC induced caps (a) and that Esr6e induction, which is observed in intact caps as a consequence of Delta1 induction, is lost in dissociated cells (b), demonstrating the effectiveness of cell dissociation. |
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Fig. 4. Hairy2 promotes NC markers and proliferation through Delta1 activation. (A) Q-PCR analysis of Sox10, Snail2 and SoxD expression in NC induced intact or dissociated animal caps derived from Hairy2-GR mRNA (250 pg/blastomere) injected embryos (−, Hairy2-GR mRNA injected minus DEX). Sox10 induction observed upon Hairy2-GR overexpression in intact stage 28 NC induced caps is not observed in dissociated NC induced explants (a). However, repression of Snail2 or induction of SoxD still occurs in dissociated stage 14 NC induced caps (b,c). (B) Whole-mount in situ hybridization of embryos co-injected with Hairy2-GR and Delta1Stu mRNA (1 ng) or Delta1 MO (7.5 ng). Co-injection of Hairy2-GR with Delta1Stu mRNA (1 ng) or Delta1 MO (7.5 ng) affects its ability to induce Sox10 in tailbud embryos, but not its capacity to repress Snail2 or activate SoxD in neurula embryos. Respective repression: (a) 63%, n = 23; (b) 43%, n = 21; (c) 64%, n = 24 and (d) 65%, n = 20. Respective induction: (e) 87%, n = 16 and (f) 60%, n = 28. Lateral views with control and injected sides (a, b, e, f) or dorsal views (c, d) with the injected side on the right are shown. LacZ was used as lineage tracer. (C) Percentage of embryos injected with Hairy2-GR mRNA, Hairy2-GR plus Delta1Stu mRNA (1 ng) or Delta1 MO (7.5 ng) showing increased pH3 staining. Note that co-injection of Delta1Stu mRNA or Delta1 MO blocks Hairy2 ability to promote cell proliferation (−, Hairy2-GR mRNA injected minus DEX). Number of embryos examined is indicated. (D) Percentage of embryos injected as indicated with Bax mRNA (50 pg), Hairy2-GR mRNA (500 pg), Delta1Stu (1 ng) or Delta1 MO (7.5 ng) developing normally or showing a low or high number of dying cells at gastrula stage as shown in Fig. 1C (−, Hairy2-GR mRNA injected minus DEX). Note that Hairy2-GR rescues Bax mRNA mediated embryonic lethality when Notch signaling is blocked. Number of embryos examined is indicated. |
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Fig. 5. Rescue of Hairy2 depletion by Delta1. (A) Whole-mount in situ hybridization of embryos injected with Hairy2 MOs (10 ng) alone or with Delta1 mRNA (1 ng) as indicated. Delta1 restores Snail2 (a, b) and Sox10 (c, d) expression in Hairy2 MOs injected embryos. Respective inhibition: (a) 76%, n = 17; (b) 30%, n = 24; (c) 50%, n = 16; (d) 15%, n = 28. Dorsal views of neurula embryos (a, b) with the injected side on the right or lateral views of tailbud embryos (c, d) with control and injected sides revealed by LacZ staining are shown. (B) Percentage of embryos injected with Hairy2 MOs (10 ng) alone or together with Delta1 mRNA (500 pg) showing reduced pH3 staining. Delta1 injection partially rescues the inhibition of proliferation observed in Hairy2 depleted embryos (−, rhodamine dextran injected alone). Number of embryos examined is indicated. (C) Whole-mount in situ hybridization of tailbud embryos injected with Hairy2 MOs (10 ng) with Su(H)Ank-GR mRNA (500 pg). Su(H)Ank-GR efficiently restores Sox10 expression in Hairy2 MOs injected embryos. Rescue of Sox10 is not observed in − DEX control embryos. Respective inhibition: a, 77% n = 18; b, 42% n = 21. Lateral views with control and injected sides identified by LacZ staining are shown. |
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Fig. 6. Id3 is required for NC development downstream of Delta1. (A) Whole-mount in situ analysis of embryos injected with Delta1 mRNA (1 ng), DeltaStu mRNA (1 ng) or Delta1 MO (7.5 ng) as indicated. Note that Delta1 overexpression increases Id3 (a, 50% induced, n = 16). Conversely, Id3 is reduced in Delta1Stu mRNA or Delta1 MO injected embryos (b, 55%, n = 24; c, 67%, n = 18). (B) Activation of Notch signaling by injection of Delta1 mRNA (1 ng) rescues Id3 and Snail2 inhibition by Hairy2-GR in neurula embryos (a, 60% reduced, n = 23; b, 93% rescued, n = 15; c, 78% inhibited n = 18; d, 67% rescued, n = 30). Q-PCR analysis of Id3 (e) and Snail2 (f) expression in NC induced explants derived from embryos injected with Hairy2-GR mRNA (250 pg/blastomere) with or without Delta1 mRNA (500 pg/blastomere) as indicated (−, uninjected NC induced caps). Note that co-injection of Delta1 mRNA rescues Id3 and Snail2 repression by Hairy2-GR. (C) Id3 MO (15 ng) blocks Delta1 (1 ng mRNA, a–d; 500 pg mRNA, e) induction of Snail2, Sox10 and cell proliferation in embryos. Percentage of embryos with the indicated phenotype: a, 55% increased, n = 20; b, 70% inhibited, n = 26; c, 68%, increased n = 19; d, 62% reduced, n = 33. Percentage of embryos injected with Delta1 mRNA (500 pg) with or without Id3 MO (15 ng) showing increased pH3 staining (e). Note that co-injection of Id3 MO reduced Delta1 ability to stimulate cell proliferation (−, rhodamine dextran injected alone). Number of embryos examined is indicated. (D) Id3-GR mRNA (1 ng) injection partially rescues Snail2 in Delta1 (7.5 ng) depleted neurula embryos and Sox10 at tailbud stage but not in − DEX control embryos. Respective inhibition and rescue: a, 87% inhibited, n = 30; b, 70% unaffected, n = 37; c, 62% inhibited, n = 16; d, 67% unaffected, n = 21. Percentage of embryos injected with Delta1 MO (7.5 ng) with Id3-GR mRNA (1 ng) showing decreased pH3 staining (e). Note that Id3 partially rescues the inhibition of cell proliferation observed in Delta1 MO injected embryos. Number of embryos examined is indicated. In all panels, neurula embryos are viewed from the dorsal side, with the injected side revealed by LacZ staining oriented to the right. Tailbud embryos are viewed from the lateral side. Control and injected sides are shown. |
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Fig. 7. Stat3 is necessary for Hairy2 non-cell-autonomous function. (A) Whole-mount in situ hybridization of embryos injected with Hairy2-GR mRNA (500 pg), Stat3 MOmis (15 ng) or Stat3 MO (15 ng) as indicated. Note that injection of Stat3 MO blocks Hairy2 ability to induce Delta1 (a, 70% induced, n = 22; b, 20%, induced, n = 20) and to upregulate Sox10 (c, 55% increased, n = 30; d, 12% increased, n = 26) but not its ability to induce SoxD (e, 90% increased, n = 20; f, 82% increased, n = 22). Dorso-anterior views of neurula embryos (a,b) with the injected side on the right or lateral views of embryos (c–f) with control and injected sides are shown. LacZ mRNA was co-injected as a lineage tracer to identify the injected side. (B) Percentage of embryos injected with Hairy2-GR mRNA (500 pg), Stat3 MO7mis (15 ng) or Stat3 MO (15 ng) as indicated showing increased pH3 staining. Note that the co-injection of the Stat3 MO reduces Hairy2 ability to stimulate cell proliferation (−, Hairy2-GR mRNA injected minus DEX). Number of embryos examined is indicated. (C) Whole-mount in situ hybridization of embryos injected with Hairy2-GR (150 pg) or Stat3-GR (500 pg) mRNA as indicated. Note that Hairy2-GR or Stat3-GR mRNA injection has no effect on Delta1 expression (a, none affected n = 12; b, 0%, n = 15). When combined, Delta1 is however strongly upregulated (c, 71% induced, n = 21). Hairy2-GR (50 pg/blastomere) with Stat3-GR (250 pg/blastomere) also strongly increases Delta1 in NC induced animal caps. The amount of myc-tagged Hairy2-GR and Stat3-GR proteins detected using anti-myc and anti-Stat3 proteins produced in each condition is shown (d). Dorsal views of neurula embryos with the injected side on the right are shown. LacZ was co-injected to reveal the injected side. |
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Figure S1. Bcl2 rescues apoptosis but not the reduction of cell proliferation observed in Hairy2 depleted embryos. (A) Phosphohistone H3 immunohistochemistry of neurula embryos injected with Hairy2 MOs (10 ng) with or without Bcl2 mRNA (50 pg) as indicated. The percentage of embryos with reduced pH3 staining observed in each condition is shown in (c). Note the reduction of mitotic cells in the Hairy2 depleted embryos with or without Bcl2 mRNA (-, rhodamine dextran injected alone). Number of embryos examined is indicated. (B) TUNEL staining of neurula embryos injected with Hairy2 MOs (10 ng) with or without Bcl2 mRNA (50 pg) as indicated. The percentage of embryos with increased TUNEL staining is shown in (c). Note the increase of TUNEL staining in Hairy2 depleted embryos and the rescue of the phenotype with Bcl2 mRNA (-, rhodamine dextran injected alone). Number of embryos examined is indicated. In all panels, embryos at neurula stage are shown in dorsal view, anterior down, and the injected side revealed by rhodamine dextran coinjection (red fluorescence) is on the right. |
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Figure S2. Hairy2, Hes2 and ESR10 effects on Notch pathway genes. A) Hairy2-GR mRNA (500 pg) injection is unable to induce the expression of the Notch ligands Serrate1 (a), Delta2 (b) or the Notch receptor (c) in neurula embryos (none induced, n = 16, 18 and 15, respectively). B) Injection of the Hairy 2 MOs (10 ng) leads to decreased Delta1 expression in the migrating NC and in the eye at tailbud stages (a, 75% reduced, n = 20) while injection of a Hairy2 MO5mis has no effect (b, 75% unaffected, n = 16). Injection of either the Hairy2 MOs, like Hairy2 MO5mis, has no effect on Delta2 in tailbud embryos (c, 70% unaffected, n = 25; d, 86%, n = 14). (C) Injection of ESR10- GR or Hes2-GR mRNA (500 pg each) does not affect Delta1 expression (none induced n = 13 and 15, respectively). In all panels, embryos at neurula stage are shown in dorsal view. In the case of embryos at tailbud stage, lateral views of the injected sides are shown. LacZ mRNA was coinjected as a lineage tracer. |
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Figure S3. Effect of the modulation of Notch signaling on the development of NC and surrounding tissues. (A) Design of the Delta1 MO. The Delta1 translation start site is indicated in red (a). Delta1 MO inhibits the translation of both Delta1 pseudoalleles (b). In vitro transcription-translation reactions were performed by using the Sp6 Rabbit Reticulocyte Lysate System (Promega) in the presence of 35S methionine. Translation products were analysed by SDS-PAGE/autoradiography. (B) Whole-mount in situ analysis of embryos injected with Delta1 mRNA (1 ng), DeltaStu mRNA (1 ng) or Delta1 MO (7,5 ng) as indicated. The antisense probe used are indicated on the left. Note that Delta1 overexpression represses N-tubulin and increases Snail2 and Sox10. Conversely, N-tubulin is increased in Delta1Stu mRNA or Delta1 MO injected embryos (4 ng) and Snail2 and Sox10 is reduced. With the exception of Msx1 which is slightly reduced by the injection of DeltaStu mRNA or Delta1 MO, the other tested markers were unaffected by modulation of Delta1. Frequency of embryos with the indicated phenotype: a, 89% ectopic, n = 19; b, 57% reduced, n = 21; c, 60% reduced, n = 16; d, 45% increased, n = 20; e, 46% reduced n = 22; f, 67% reduced n = 18; g, 100% normal, n = 17; h, 82% normal n = 22; i, 72% normal, n = 22; j, 75% normal, n = 32; k, 77% normal, n = 22; l, 80% normal, n = 20 ; m, 100% normal, n = 26; n, 35% slightly reduced, n = 20; o, 40% slightly reduced, n = 24; p, 100% normal, n = 20; q, 100% normal, n = 18 ; r, 91% normal, n = 11; s, 100% normal, n = 10; t, 100% normal, n = 14 ; u, 100% normal, n = 16; v, 63% decreased, n = 19 ; w, 73% increased, n = 11; x, 38% increased, n = 8. Neurula stage embryos are viewed from the dorsal side, anterior down, with the injected side oriented to the right. Control and injected sides of tailbud embryos are shown. LacZ was used as a lineage tracer. |
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Figure S3. Effect of the modulation of Notch signaling on the development of NC and surrounding tissues.(C) Q-PCR analysis of the expression of Snail2, Sox10, Sox3, Six1 and N-tubulin in NC induced caps analysed at the indicated stages and derived from embryos injected with Delta1 mRNA (500 pg/blastomere), DeltaStu mRNA (1 ng/blastomere) or Delta1 MO (5 ng/blastomere). Note that Delta1 overexpression upregulates Snail2 and Sox10 and that Delta1 inhibition diminished their expression. Note also that the expression of Sox3, Ep keratin, Six1 and N-tubulin was not significantly affected. (D) Phosphohistone H3 immunohistochemistry of neurula embryos injected with Delta1 mRNA (500 pg), Delta1Stu mRNA (1 ng) or Delta1 MO (7,5 ng) (top panels). Note the increase of pH3 staining in Delta1 injected embryos and the reduction of mitotic cells in Delta1Stu mRNA or Delta1 MO injected embryos (a, 38% increased, n = 21; b, 47% decreased, n = 19; c, 43% decreased, n = 21). Embryos are shown in dorsal view, anterior down, and the injected side is oriented to the right. Rhodamine dextran (red fluorescence) was used as a lineage tracer (bottom panels). |
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Figure S7. Hairy2-GR is unable to induce Delta1 expression in Stat3 depleted NC induced explants. (A) Stat3 MO (Ohkawara et al., 2004) inhibits the translation of Stat3. In vitro transcription-translation reactions were performed by using the Sp6 Rabbit Reticulocyte Lysate System (Promega) in the presence of 35S methionine. Translation products were analysed by SDS-PAGE/autoradiography. (B) Injection of Stat3 MO (15 ng/blastomere) but not of a control Stat3 MO (Stat3 MOmis, 15ng/blastomere) blocks Hairy2 ability to induce Delta1 in NC induced explants as analysed by Q- PCR. Similar amount of the myc-tagged Hairy2-GR protein was produced in each condition as determined by western blot analysis. |