Kishi M et al. (2000), Requirement of Sox2-mediated signaling for diff...

XB-ART-11592

Development 2000 Feb 01;1274:791-800. doi: 10.1242/dev.127.4.791.

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Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.

Kishi M , Mizuseki K , Sasai N , Yamazaki H , Shiota K , Nakanishi S , Sasai Y .

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From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

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Species referenced: Xenopus
Genes referenced: actl6a ag1 chrd egr2 en2 fgf4 foxd3 foxl3 hesx1 hoxb9 krt12.4 myod1 ncam1 neurog2 otx2 snai2 sox15 sox2 sox9 tubb2b zic2

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	Fig. 1. Dominant-negative effects of Sox2BD(-) and Sox2-EnR on wild-type Sox2. (A) Structures of two dnSox2 constructs. (Top) Wild type; (middle) Sox2BD(-), lacking most of the DNA-binding HMG domain (black box). (Bottom) Sox2-EnR, the Sox2 HMG domain was fused to the Drosophila engrailed repressor region (grey box) at the carboxyl terminus. (B-L) Neural differentiation of the animal caps was analyzed with the pan-neural N-CAM marker at stage 19. Injection of the FGF4 expression plasmid (B) or control plasmid (inset) did not induce N-CAM expression in the explants. Coinjection of wild-type Sox2 mRNA with the FGF4 plasmid caused neural differentiation (C). This neural differentiation was suppressed by coinjecting Sox2BD(-) mRNA (D) or Sox2-EnR mRNA (G) in the caps injected with Sox2 mRNA and the FGF4 plasmid. This suppression was reversed by expressing Sox2-GR in the presence of 10 mM Dex (E,H) but not in its absence (insets). Sox9-GR did not rescue N-CAM expression even with Dex (F,I). SoxD mRNA injection caused neural differentiation of animal caps (J). This neuralization was not inhibited by coinjection of Sox2BD(-) or Sox2-EnR mRNA (K,L). In each injection, the total amounts of RNA and DNA were made constant by adding an adequate amount of control lacZ mRNA and plasmid DNA. The injected amounts were 10 pg for FGF4 plasmid, 100 pg for Sox2 and SoxD, 400 pg for Sox2BD(-), 50 pg for Sox2-EnR, 200 pg for Sox2-GR and 200 pg for Sox9-GR mRNA.
	Fig. 2. Injection of dnSox2 mRNAs suppresses neural differentiation of animal cap explants caused by dnBMPR. (A) Expression of N-CAM in normal embryos at stage 19, when the animal caps below were harvested. (B-I) Effects of dnSox2 were examined with the N-CAM marker in the animal cap assay. Injection of dnBMPR (C) but not of control lacZ (B) mRNA induced N-CAM expression. The neural differentiation caused by dnBMPR was inhibited by coinjecting Sox2BD(-) mRNA (D) or Sox2-EnR mRNA (G). This inhibition was reversed by Sox2-GR coinjection in the presence of Dex (E,H) but not in its absence (not shown). The inhibitory effect of Sox2BD(-) or Sox2-EnR was not counteracted by Sox9-GR even with Dex treatment (F,I). In each injection, the total amounts of RNA were made constant by adding an adequate amount of control mRNA. The injected amounts of mRNA were 50 pg for dnBMPR, 400 pg for Sox2BD(-), 50 pg for Sox2-EnR, 200 pg for Sox2-GR, 200 pg for Sox9-GR.
	Fig. 3. Injection of Sox2BD(-) inhibits neural differentiation in vivo. Whole-mount in situ hybridization analyses at the open neural plate stage (dorsal view; upside, anterior). (A-C) Both embryos were injected with synthetic mRNAs into each animal blastomere at the 8-cell stage: control lacZ mRNA (600 pg; A), Sox2BD(-) mRNA (400 pg + 200 pg lacZ; B) or Sox2BD(-) + Sox2-GR (400 pg + 200 pg; C). N-CAM expression in the neural plate was severely suppressed in Sox2BD(-)-injected embryos while Sox2-GR coinjection rescued N-CAM expression in the presence of Dex. (D-I) 400 pg of Sox2BD(-) mRNA (right) or control mRNA (left) were injected into each animal blastomere of the 8-cell embryo. Injection of Sox2BD(-) had little effect on the expression of the dorsal mesodermal markers Chd or MyoD (D,E) while the proneural gene Xngnr1 (F), the neuronal marker Ntubulin (G), the neural crest markers Slug (H) and fkh6 (I) were significantly suppressed. Note that the fkh6 expression was inhibited in presumptive head crest regions (arrow) but not in posterior mesoderm (arrowhead).
	Fig. 4. Injection of Sox2BD(-) inhibits expression of both anterior and posterior neural markers. Whole-mount in situ hybridization analyses were performed with regional neural markers in neurula embryos. (A) SoxDBD(-) mRNA (right) or control lacZ mRNA (left) was injected into each animal blastomere of the 8-cell embryo (400 pg of mRNA/cell). DIG-labeled Otx2 and HoxB9 probes were used simultaneously for double-label in situ hybridization. In the embryo injected with SoxDBD(-), the expression of the forebrain marker Otx2 (arrow) was suppressed whereas that of the spinocaudal marker HoxB9 (arrowhead) was not reduced. (B-F) Embryos were injected with Sox2BD(-) mRNA (right) or control mRNA (left) in a manner comparable to that for SoxDBD(-) mRNA injections. Injection of Sox2BD(-) inhibited expression of Otx2 (B), Xanf1 (C), En2 (D), Krox20 (E), and HoxB9 (F). Note that inhibition of HoxB9 expression was detected only in the posterior neural tube (arrowhead) but not in the posterior mesoderm (arrow).
	Fig. 5. Effects of Sox2BD(-) on early neural marker genes. (A-F) Embryos were injected with Sox2BD(-) mRNA (right) or control mRNA (left) as described for Fig. 4. Whole-mount in situ hybridization was performed with Zic2 (A,C,E) and Sox2-5¢UTR (B,D,F) probes. No significant inhibition of Zic2 or Sox2 expression was detected at stage 11 (A,B). In embryos at stage 12 and 14, injection of Sox2BD(-) reduced expression of both early neural markers (C-F). (G) Accumulation of Sox2BD(-) protein (FLAGtagged) shown by western blot analysis. Embryos injected with FLAG-tagged Sox2BD(-) mRNA were harvested at stages indicated above each lane. Western blotting with anti-FLAG antibody (upper panel) gave a single band of the expected size (25 kDa) in each lane. This 25 kDa band was not detected when control lacZ-injected embryos were used (not shown). CBB staining of SDS-PAGE (lower panel) is presented as loading controls.
	Fig. 6. Temporal requirement of Sox2-mediated signaling during neural differentiation. Sox2BD(-)-GR was generated by adding the GR sequence to the carboxyl terminus of Sox2BD(-) (A, bottom). (B) N-CAM expression in animal caps neuralized with dnBMPR. Application of Dex did not affect N-CAM mRNA expression either in the animal caps injected with dnBMPR (C) or in the embryo injected with control LacZ mRNA (D). In animal cap assays (E-H), neural differentiation triggered by dnBMPR was not inhibited by coinjecting Sox2BD(-)-GR mRNA in the absence of Dex (E) while the induction of N-CAM in the injected caps was significantly suppressed when Dex was added to the culture medium from stage 9 onwards (F), stage 12 (G) or stage 14 onwards (H). (I-L) 200 pg of Sox2BD(-)-GR mRNA was injected into each animal blastomere of the 8-cell embryo. Without Dex treatment, injection of Sox2BD(-)-GR had little effect on N-CAM expression (I). Significant suppression of N-CAM was observed when the injected embryos were treated with Dex from stage 9 onwards (J), stage 12 (K) or stage 14 (L) onwards. The extent of suppression of (H) and (L) tended to be less complete than that found in Dex treatment from earlier stages (F,G,J,K).
	Fig. 7. Sox2BD(-) does not promote epidermogenesis at the cost of neural tissues. (A) RT-PCR analysis of stage 19 animal caps injected with control (lane 2), dnBMPR (lane 3), dnBMPR + Sox2BD(-) (lane 4) or Sox2BD(-) (lane 5) mRNA. Primers specific for N-CAM, epidermal Keratin, XAG-1, muscle-actin (M-actin) and the control EF-1a were used. (B,C) Double-labeled in situ hybridization with N-CAM (purple) and Keratin (brown) probes. The embryos were injected with control mRNA (B) or Sox2BD(-) mRNA (C) at one animal blastomere of the 8-cell embryo. The neural plate region that failed to express N-CAM (arrow) did not give Keratin signals, either. (D,E) Confocal microscopy images of the neural plate region of neurula embryos. Green fluorescence shows the progeny of the blastomere that was injected with control lacZ and EGFP mRNA (D) or Sox2BD(-) and EGFP mRNA (E). The insets are low magnification images showing the nuclear DAPI staining. The brackets indicate the areas shown in high magnification GFP images. n, notochord; arrows, cuboidal cells in the superficial layer; arrowheads, cells in the sensorial layer.