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Figure 1.
XREST/NRSF is required for morphogenesis and neuronal gene expression during early stages of neurogenesis. A, Anterodorsal views and transversal sections of embryos fixed at late neurula, injected with 500 pg of dnXREST or 5 pmol of MoXREST (indicated on top) in one cell at the 2-cell stage; the injected side in all figures is to the left. Embryos exposed to dexamethasone at late blastula [Dex(+) st. 9] and embryos injected with MoXREST exhibit an apparent loss of expression of neuronal genes in neural tube (arrows) and in the trigeminal placode (arrowheads). Late gastrula induction [Dex(+) st. 12] results in less marked effects. Transversal sections show altered morphology of neural tube and confirm the diminished expression of neuronal genes (arrows). Probes used for ISH are indicated to the left of each row. B, Lateral views of stage 37 embryo injected in one cell at the 2-cell stage with 500 pg of dnXREST and induced at late blastula stage. Note the defect of eye development in the injected side (bottom, arrow).
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Figure 2.
Loss of XREST/NRSF function disturbs neural patterning. Dorsal views of stage 15 embryos injected unilaterally with 500 pg of dnXREST. Anterior is down, and the injected side is to the left. The stage of dexamethasone addition is indicated on top, and probes used for ISH are indicated to the left. Embryos induced at stage 9 [Dex(+) st. 9] displays inhibition of N-tubulin, X-ngnr-1, and X-Delta-1 (arrowheads) and ventral displacement of neurogenic domains (brackets). Induction at stage 12 [Dex(+) st. 12] results in subtler effects. In contrast, embryos induced at both stages exhibit a precocious and ectopic expression of NaV1.2 at stage 14 (arrowheads). Induction of dnXREST at stage 9 associates the expansion of non-neurogenic domain (brackets) marked by Zic2 and surrounded by X-Delta-1 (arrowheads). Activation of dnXREST at stage 12 did not result in the expansion of Zic2.
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Figure 3.
XREST/NRSF loss of function induces expansion of neural tissue. A, Transversal sections (a–c, e–g), dorsal (d, h–j, m–t) and anterior (k, l) views of neural plate stage embryos injected with 500 pg of in vitro transcribed dnXREST mRNA or 5 pmol of MoXREST (indicated on top). Probes used for ISH are indicated to the left of each row. Embryos injected with MoXREST or with dnXREST and induced at late blastula [Dex(+) st. 9] exhibit expansion of the neural markers SoxD and Sox2 (a–h, brackets) and a diminished expression of epidermal keratin (j, l), msx-1 (n, p), and Slug (r, t) (arrows). Less severe effects are observed in embryos induced at late gastrula: SoxD (c), Sox2 (g), keratin (k), msx-1 (o) and Slug (s). Dex(+) st. 12, Embryos exposed to dexamethasone at stage 12. B, Anterior views of stage 18 embryos injected with 200 pg of dnXREST (a, b, e, f) or in combination with 1 ng of XREST/NRSF in one cell at the 2-cell stage (c, d, g, h) (indicated on top). Embryos injected with dnXREST induced at stage 9 exhibit enlargement of anterior neural plate, marked by Sox2 expression (b, arrows) and the lateral displacement and loss of keratin expression (f, arrows). Non-induced dnXREST/XREST embryos do not display noticeable defects (c, g). dnXREST/XREST embryos induced at stage 9 do not display an expanded domain of Sox2 expression (d) and decreased keratin expression (arrows).
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Figure 4.
Decreased XREST/NRSF function inhibits the acquisition of epidermal fate and does not result in ectopic neural induction. A, Lateral views of stage 16 embryos and animal ectoderm explants (animal caps) injected at the 8-cell stage in one of the most ventral animal blastomeres with 500 pg of dnXREST (a–c, g–i) or in the four animal cells with GFP and 1 ng of dnXREST (d–f, j–l). Animal caps were dissected at stage 9 and cultured until stage 16. Induction of dnXREST at stage 9 resulted in the increase of Sox2 expression in animal caps (e) but not in whole embryos (b) and in the loss of keratin expression both in animal caps (arrowheads, h) and whole embryos (arrowheads, k). The activation of dnXREST at stage 12 results in subtler effects on Sox2 (f) and keratin (i, l) expression. B, RT-PCR analysis of ectodermal explants (animal caps) from embryos injected animally with 1 ng of dnXREST and either treated (+) or untreated (−) with dexamethasone (Dex) from stage 9 onward. Animal caps were collected at stage 10.5 (indicated on top) and analyzed for expression of the indicated RNAs.
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Figure 5.
XREST loss of function reverts the effect of Bmp4 misexpression on ectodermal patterning. Anterior views of stage 18 embryos injected with 500 pg of dnXREST, alone or in combination with 500 pg of Bmp4 mRNA (indicated on top). Probes used for ISH are indicated to the left of each row, and the stage of dexamethasone addition is indicated on top. The injected side of not induced dnXREST/BMP4 embryos exhibits the loss or decrease of neural ectoderm revealed by the domain of Sox2 expression (c, arrows, compare with a), the ectopic expression of keratin (g, arrow, compare with e), and the dorsal displacement (brackets) and contraction (arrowhead) of Slug expression domain (k, compare with i) (brackets). The activation of dnXREST at late blastula stage results in the restoration of anterior neural plate size of BMP4 misexpressing embryos. Note the recovered expression of Sox2 (d), keratin (h), and Slug (l) in relation to the neural plate size (brackets). Nevertheless, loss of keratin (f) and Slug (j) expression observed in dnXREST-injected embryos induced at stage 9 was not efficiently rescued by the BMP4 coexpression (h, l, arrows).
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Figure S2. XREST/NRSF mRNA expression pattern. XREST/NRSF is expressed in whole ectoderm at early gastrula stage, lateral view (a) and transverse section (b, arrowhead). During the early neurula stage, XREST/NRSF is restricted to the anterior and lateral neural ridge areas, anterior view (c, arrowhead ). At the neural tube stage, XREST/NRSF is expressed in the presomitic mesoderm, dorsal view (d, arrow) and transverse section (e, arrowhead). Later, it is detected surrounding the anterior nervous system and the developing eye (f, arrowhead) and slightly in the migratory neural crests (f, arrow).
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Figure S3. β-galactosidase, control morpholine, and XREST1 injected embryos do not display altered expression of N-tubulin, Sox2 and Slug. Anterior views of stage 18 embryos injected with β-galactosidase mRNA, control morpholine and XREST1 mRNA (indicated on top). Probes used for ISH are indicated to the left of each row.
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Figure S4. Interference of XREST/NRSF function does not increases Notch signaling. Anterior-dorsal views of stage 16 embryos injected with 500 pg of dnXREST in one cell at the 2-cell stage. The activation of dnXREST at stage 9 results in the decrease of XHes-1 expression (arrowhead) (b), and apparently does not disturb Notch-1 expression pattern (d).
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Figure S5. Loss of XREST/NRSF function results in a persistent alteration of the patterning of the central nervous system. Anterior views of Xenopus larvae, showing the expression of Sox2 RNA. A control embryo is shown on the left; three embryos which were injected with 500 pg of dnXREST DNA in one cell at the 2-cell stage, and exposed to dexamethasone at stage 9 are shown to the right. Note the expansion of the Sox2-expressing domain in the side derived from the injected cell (shown to the right) and the loss of the normal contour of the eye field.
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Figure S6. The expansion of neural plate associated to the interference of XREST/NRSF function is rescued by the expression of zREST/NRSF. Anterior-dorsal views of stage 18 embryos analyzed for Sox2 expression. Embryos were injected with MoXREST (a); MoXREST and 1ng of ZREST/NRSF (b); 200 pg of dnXREST/NRSF (c); 200 pg of dnXREST and 1ng of ZREST/NRSF (d). The size of neural plate is indicated with brackets and the anterior-lateral expansion is indicated with arrowheads (a, c). Coinjection of MoXREST and ZREST/NRSF RNA rescued the neural plate expansion of MoXREST injected embryos (a, b). Coinjection dnXREST RNA and the RNA encoding the full length ZREST/NRSF protein reversed the expansion of neural ectoderm caused by dnXREST alone (c, d).
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