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Dev Dyn
2007 Jun 01;2366:1475-83. doi: 10.1002/dvdy.21152.
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Xenopus hairy2 functions in neural crest formation by maintaining cells in a mitotic and undifferentiated state.
Nagatomo K
,
Hashimoto C
.
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The neural crest is a population of mitotically active, multipotent progenitor cells that arise at the neural plate border. Neural crest progenitors must be maintained in a multipotent state until after neural tube closure. However, the molecular underpinnings of this process have yet to be fully elucidated. Here we show that the basic helix-loop-helix (bHLH) transcriptional repressor gene, Xenopus hairy2 (Xhairy2), is an essential early regulator of neural crest formation in Xenopus. During gastrulation, Xhairy2 is localized at the presumptive neural crest prior to the expression of such neural crest markers as Slug and FoxD3. Morpholino-mediated knockdown of Xhairy2 results in the repression of neural crest marker gene expression while inducing the ectopic expression of the cell cycle inhibitor p27(xic1) in the presumptive neural crest. We also found that ectopic p27(xic1) disturbs neural crest formation. Furthermore, the depletion of Xhairy2 leads to the apoptosis of mitotic cells. Our results suggest that Xhairy2 functions in neural crest specification by maintaining cells in the mitotic and undifferentiated state.
Figure 1. Xhairy2 is required for neural crest marker gene expression. A: Double in situ hybridization with Xhairy2 (magenta) and Slug (blue) at stage 13. B,E: Control-MO injection has no effect on any of the neural crest markers examined at stage 13: Slug (B) and FoxD3 (E). C,F: In embryos injected with Xhairy2-MO, the expression of Slug (C) and FoxD3 (F) is inhibited on the injected side (red arrowhead) at stage 13. D,G: Loss of neural crest marker expression in embryos injected wit Xhairy2-MO is rescued by injecting 20 pg of Xhairy2 mRNA (yellow arrowhead): Slug (D) and FoxD3 (G). H,I: In embryos injected with Xhairy2-MO, the expression of Xsox2 (H) and Keratin (I) is not apparently affected at stage 13. White brackets indicate the width of the neural plate. J,K: In embryos injected with Xhairy2-MO, Xbmp4 expression is not affected at stages 11.5 (J) and 12.5 (K). A, lateral views with anterior to the left. B, dorsal views with anterior to the bottom. Injected area is the right side. Turquoise staining is lineage tracer β-gal.
Figure 2. Depletion of Xhairy2 leads to loss of neural crest derivatives. A,B: Control-MO injection has no effect on any of the neural crest markers examined at stage 17: Slug (A) and FoxD3 (B). C,D: In embryos injected with Xhairy2-MO, the expression of Slug (C) and FoxD3 (D) in inhibited on the injected side (red arrowhead) at stage 17. E,F: Compared with the control side (E), the Xhairy2-MO injected side shows reduced migratory neural crest cells (F) as depicted by Xtwist expression (red arrowhead) at stage 24. G,H: Compared with the control side (G), the Xhairy2-depleted embryo shows no cranial sensory ganglia on the injected side (H) as depicted by n-tubulin expression (red arrowhead) at stage 25. I: Schematic diagram depicting ventral cranial cartilages of a stage 45 embryo. J: Cartilage staining of Control-MO injected embryo shows normal structures at stage 45. K: Cartilage staining of Xhairy2-MO injected embryo shows partial loss of cartilage on the injected side (red arrowhead) at stage 45. L,M: Phenotypes of stage 35 embryos whose bilateral trunk neural crest was injected with Control-MO or Xhairy2-MO. Melanocytes of Xhairy2-MO injected embryo are reduced (M; yellow arrowhead) compared with Control-MO injected embryo (L). L: The dorsal fin of Control-MO injected embryo shows normal structures M: The dorsal fin of Xhairy2-MO injected embryo shows partial loss (red arrowhead). A, dorsal views with anterior to the bottom. E,G, lateral views with anterior to the right. F, H, L, M, lateral views with anterior to the left. J, K, ventral views with anterior to the top. A,J,K, injected area is the right side. Turquoise staining is lineage tracer β-gal.
Figure 3. Depletion of Xhairy2 induces ectopic p27xic1 and X-Delta-1 expression in presumptive neural crest. A,B: Xhairy2-MO induces slight increases of n-tubulin expression at stage 15 (A) and X-ngnr-1 expression at stage 13 (B) in trigeminal ganglia. C: Control-MO injection has no effect on p27xic1 expression at stage 13. D: In embryos injected with Xhairy2-MO, ectopic expression of p27xic1 is induced on the injected side (red arrowhead) at stage 13. E: The ectopic expression of p27xic1 is repressed by injecting 20 pg of Xhairy2 mRNA (yellow arrowhead). F: Control-MO injection has no effect on X-Delta-1 expression at stage 13. G: Xhairy2-MO induces a modest increase of X-Delta-1 expression in the cranial neural crest region (red arrowhead) at stage 13. H: The modest increase of X-Delta-1 expression is repressed by injecting 20 pg of Xhairy2 mRNA (yellow arrowhead). I: Double in situ hybridization with Xhairy2 (blue) and p27xic1 (magenta) at stage 12. J: Double in situ hybridization with Xhairy2 (blue) and p27xic1 (magenta) at stage 13. Dorsal views with anterior to the bottom. Injected area is the right side. Turquoise staining is lineage tracer β-gal.
Figure 4. Repression of p27xic1 expression by Xhairy2 is involved in early neural crest specification. A,B: Embryo injected with 10 pg of p27xic1 mRNA shows repression of Slug (A) and Foxd3 (B) expression (red arrowhead). C: The injection of 10 pg of p27xic1 mRNA produces a slightly increase of X-Delta-1 expression on the lateral side of the neural plate (red arrowhead). D: p27xic1-MO injection has no effect on X-Delta-1 expression (F) or any of the neural crest markers examined: Slug (D) and Foxd3 (E). G,H,J,K: The repression of early neural crest marker expression by Xhairy2-MO seems to be rescued by p27xic1-MO injection (yellow arrowhead): Slug (J) and Foxd3 (K), but not by Control-MO injection (red arrowhead): Slug (G) and Foxd3 (H). I,L: The mild increase of X-Delta-1 expression by Xhairy2-MO seems to be rescued by p27xic1-MO injection (L) but not by Control-MO injection (I). Dorsal views with anterior to the bottom at stage 13. Injected area is the right side. Turquoise staining is lineage tracer β-gal.
Figure 5. Xhairy2 is required for cell proliferation and survival in neural crest. A: Phosphohistone H3 immunohistochemistry of Xhairy2-MO or Control-MO injected embryos. A,C: There is no detectable difference in the number of actively cycling cells between the side injected with Control-MO and the uninjected side at stages 13 (A) and 18 (C). B: There is no detectable difference in the number of actively cycling cells between the side injected with Xhairy2-MO and the uninjected side at stage 13. D: The number of actively cycling cells is decreased in the neural crest region injected with Xhairy2-MO (red arrowhead), compared with the uninjected side at stage 18. E: TUNEL staining of Xhairy2-MO or Control-MO injected embryos. E,G: There is no detectable difference in the number of apoptotic cells between the side injected with Control-MO and the uninjected side at stages 13 (E) and 18 (G). F: There is no detectable difference in the number of apoptotic cells between the side injected with Xhairy2-MO and the uninjected side at stage 13. H: A significant increase in the number of apoptotic cells is observed on the side injected with Xhairy2-MO (red arrowhead), compared with the uninjected side at stage 18. Dorsal views with anterior to the bottom. Injected area is the right side.
