Kiyota T et al. (2007), Ets-1 regulates radial glia formation during ve...

XB-ART-39388

Organogenesis 2007 Oct 01;32:93-101. doi: 10.4161/org.3.2.5171.

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Ets-1 regulates radial glia formation during vertebrate embryogenesis.

Kiyota T , Kato A , Kato Y .

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Radial glia cells are the first distinguishable glial population derived from neural epithelial cells and serve as guides for migrating neurons and as neural progenitor cells in the developing brain. Despite their functional importance during neural development, the determination and differentiation of these cells remains poorly understood at the molecular level. Ets-1 and Ets-2, Ets (E26 transformation-specific) transcription factors, are vertebrate homologues of Drosophila pointed, which is expressed in a subset of glia cells and promotes different aspects of Drosophila glia cell differentiation. However, it remains unsolved that the function of Ets genes is conserved in vertebrate glia development. Here we report that Ets-1 but not Ets-2 is necessary for Xenopus radial glia formation and the activity of Ets-1 is sufficient for radial glia formation prior to neural tube closure. Furthermore, we show that Ras-MAPK (mitogen activated protein kinase) signaling, which acts as an upstream activator of Ets-1 in other biological processes, also regulates radial glia formation. A mutant form of Ets-1, which is not responsive to Ras-MAPK signaling, inhibits radial glia formation promoted by Ras-MAPK signaling. Together, our results show that Ets-1 activated by Ras-MAPK signaling promotes radial glia formation during Xenopus embryogenesis.

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Species referenced: Xenopus
Genes referenced: ets1 ets2 gal.2 mapk1 nr3c1 nrp1 prph vim

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	Figure 1. The expression profiles of Ets‐1, Ets‐2 and radial glia marker, vimentin in Xenopus hindbrain. (A�I) The expressions of Ets‐1 (A�C), Ets‐2 (D�F), and vimentin (G�I) in Xenopus embryos were shown. All pictures of whole embryos are dorsal views, anterior toward left. (A, D, G) stage 17, late neurula. (B, E and H) stage 25, early tailbud. (C, F and I) stage30, tailbud. (a�f) transversal section cut at the level of prospective hindbrain or hindbrain in (A�F), respectively. Sections were prepared from double whole mount in situ hybridization with Ets‐1 or Ets‐2 (purple) and vimentin (turquoise blue). Arrow heads in (a�f) indicated the overlapping expression of Ets‐1 or Ets‐2 and vimentin. a, anterior; p, posterior; e, eye; hb, hindbrain. The original figure contains color pictures
	Figure 2. The activities of Ets‐1 and Ets‐2 are sufficient for promoting radial glia formation prior to neural tube closure. (A) DNA constructs used in this paper. PNT, pointed domain; ETS, Ets‐DNA‐binding domain; hGR, human glucocorticoid receptor ligand binding domain; MT, myc‐tag. (B) nucb‐Gal (nuclear b‐Galactosidase)‐injected stage 42 Xenopus hindbrain stained by anti‐vimentin antibody. All of the high magnification images presented in this paper were taken from a similar part surrounded by a white square in this panel. Scale bar: 100 mm. (C) High magnification view of the hindbrain indicated in (B). Arrow heads indicates a radial process, which can be traced from the ventricular zone to the brain surface. (D and E) High magnification images of the injected and uninjected side of the hindbrain stained by anti‐vimentin antibody in 100 pg Ets1‐GR‐injected (D) and 100 pg Ets2‐GR‐injected (E) embryos with DEX treatment at stage 18. Scale bar: 25 mm. (F and H) The ratio of radial processes (RPratio) between the injected and uninjected side of the same hind‐ brain. Gray bars or white bars indicate with DEX treatment or without DEX treatment, respectively. The numbers of X‐axis indicate the developmental stages when DEX was added. *p < 0.001 compared with the ration of Ets1‐GR‐injected (F) or Ets2‐GR‐injected (H) embryos without DEX treatment, respectively. Each value represents the average of 12 independent measurements. (G and I) The expression of Ets1‐GR and Ets2‐GR proteins without DEX at 18, 20 and 22. The level of tubulin protein was used for the normalization of samples. The original figure contains color pictures.
	Figure 3. Ets‐1 but not Ets‐2 is required for radial glia formation in Xenopus hindbrain. (A) Reduction of endogenous Ets proteins by Morpholino Oligo‐ nucleotides (MO) at stage 18. The level of tubulin protein was used for the normalization of samples. (B�F) High magnification images of the injected and uninjected side of the hindbrain in 40 ng ConMO‐injected (B) vimentin, 40 ng Ets1MO‐injected (C) vimentin and XIF3, (C�) vimentin and GFAP, 40 ng Ets2MO‐injected (D) vimentin and XIF3, (D�) vimentin and GFAP, Ets1MO/100 pg Ets1‐GRDATG‐injected (E) vimentin,and Ets2MO/20 pg Ets2‐GRDATG‐injected (F) vimentin embryos. DEX was added at stage 18. Scale bar: 25 mm. (G) The ratio of radial processes (RPratio) between the injected and uninjected side of the hindbrain for each injection experiment. Gray bar or white bars indicate with DEX treatment at stage 18 or without DEX treatment, respectively. The numbers of X‐axis indicate the developmental stages when DEX was added. p < 0.001 compared with the ratio of ConMO‐injected embryos. p < 0.001 compared with the ratio of Ets1MO/ Ets1‐GRDATG‐injected embryos without DEX treatment. *p < 0.001 compared with the ratio of Ets2MO/Ets2‐GRDATG‐injected embryos without DEX treatment. Each value represents the average of 12 independent measurements. (H�J) High magnification images of the injected and uninjected side of the hindbrain in 50 pg dn‐Ets1‐GR‐injected (H) vimentin and XIF3, dn‐Ets1‐GR/100 pg Ets1‐GR‐injected (I) vimentin and dn‐Ets1‐GR/100 pg Ets2‐GR‐injected (J) vimentin embryos. DEX was added at stage 18. Scale bar: 25 mm. (K) The ratio of radial processes (RPratio) between the injected and the uninjected side of the hindbrain for each injection experiment. Gray bars or white bars indicate with DEX treatment at stage 18 or without DEX treatment, respectively. p < 0.001compared with the ratio of dn‐Ets1‐GR‐injected embryos without DEX treatment. **p < 0.001 compared with the ratio of dn‐Ets1‐GR‐injected embryos with DEX treatment. Each value represents the average of 12 independent measurements. The original figure contains color pictures.
	Figure 4. The depletion of Ets‐2 but not Ets‐1 affected early neural develop‐ ment. (A) The ratio of radial processes (RPratio) between the injected and uninjected side of the hindbrain of Ets2MO/Ets2‐GRDATG‐injected embryos. Gray bars or a white bar indicate with DEX treatment or without DEX treat‐ ment, respectively. The numbers of X‐axis indicate the developmental stages when DEX was added. *p < 0.001 compared with the ratio of Ets2MO/ Ets2‐GRDATG‐injected embryos without DEX treatment. Each value represents the average of 12 independent measurements. (B and C) The expression of Et1 (B) and Ets2 (C) genes in stage 12 embryos. All panels are dorsal views, anterior toward left. (D�F) Whole‐mount in situ hybridization analysis of nrp1 expression at stage 14. (D) n = 29 (no change)/29, (E) n = 20 (reduced)/20, (F) n = 21(no change)/21. The injected side in each embryo is indicated by b‐Gal staining (red color). (G) Reduction of endogenous Ets proteins by Morpholino Oligonucleotides (MO) at stage 14. The level of tubulin protein was used for the normalization of samples. The original figure contains color picture
	Figure 5. Ras‐MAPK signaling is upstream of Ets‐1 and promotes radial glia formation. (A�E) High magnification images of the injected and uninjected side of the hindbrain in 250 pg dn‐Ras‐injected (A) vimentin and XIF3, (A�) vimentin and GFAP, 100 pg at‐MAPKK‐injected, (B) vimentin, dn‐Ras at‐MAPKK‐in‐ jected (C) vimentin, 100 pg Ets1‐GRT36A‐injected, (D) vimentin and XIF3, and at‐MAPKK/Ets1‐GRT36A‐injected, (E) vimentin embryos. DEX was added at stage 18. Scale bar: 25 mm. (F) The ratio of radial processes (RPratio) between the injected and uninjected side of the hindbrain for each injection experi‐ ment. Gray bars or white bars indicate with DEX treatment at stage 18 or without DEX treatment, respectively. p < 0.001 compared with the ratio of nucb‐Gal‐injected embryos. p < 0.001 compared with the ratio of at‐MAPKK/Ets1‐GRT36A‐injected embryos without DEX treatment. **p < 0.001 compared with the ratio of Ets1‐GRT36A‐injected embryos without DEX treatment. Each value represents the average of 12 independent measurements. The original figure contains color pictures

References [+] :

Albagli, A model for gene evolution of the ets-1/ets-2 transcription factors based on structural and functional homologies. 1994, Pubmed