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Fig. 6. xSyndecan1 regulates dorso-ventral patterning of the ectoderm. (A–F) xSyn1 knockdown increased neural plate wide and reduced non-neural ectoderm. Embryos at the two-cell stage were microinjected in both blastomeres with (A, D) control and (B, E) xSyn1 morpholinos (34 ng), raised until neurula stage and in situ hybridization against (A, B) sox2 and (D, E) cytokeratin performed. Neural plate width was measured at the half of the antero-posterior axes with ImageJ. Statistical analysis with Kruskal–Wallis test and Dunn's post test showing that means vary significantly with (C) p < 0.01 and (F) p < 0.05, respectively. The number of embryos used for each experimental point is indicated above each bar. (G, H) xSyn1 is necessary for epidermal integrity. Embryos at the two-cell stage were microinjected in both blastomeres with control and xSyn1 morpholinos (34 ng) and raised until tailbud stage. Arrows point where epidermis is affected in xSyn1 morphant embryos. |
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Fig. 2. xSyndecan-1 rescue of dorso-ventral patterning in a cell non-autonomous manner. (A–L) Embryos at two-cell stage were microinjected with MO-Beta-cat in both blastomeres, followed by one (1x, 40 pg of total mRNA) or four (4x, 80 pg of total mRNA) microinjections of xSyn1 mRNA at the four cell stage. Embryonic development was stopped at (A–G) stage 18, (H–K) 10 or (L) 20 and molecular markers analyzed by in situ hybridization using probes against (A–D) sox2, (E–G) sizzled and (H–K) chordin or by (L) RT-PCR. The rescue of molecular markers observed by in situ hybridization analysis was very penetrant. All βcat morphant embryos are completely devoid of sox2 and chordin expression (panels B and I, N = 12–15) and showed an expansion of sizzled (panel F, N = 11). One injection of xSyn1 mRNA is 100% efficient in restoring dorso-ventral patterning (panels C, G and J, N = 5–17). 4× microinjections induce radial expression of sox2 and chordin in 18% (N = 11) and 71% (N = 7) of the embryos, respectively. (M, N) xSyn1 acts in a cell non-autonomous manner. Embryos at two-cell stage were microinjected with MO-βcat in both blastomeres, followed by one microinjection of xSyn1 (40 pg) and lacZ (50 pg) at the four-cell stage, allowed to develop, fixed at stage 27 and stained for β-galactosidase. (N) Section of one embryo, showing that of all dorsal tissues only notochord was stained with β-galactosidase (for details see the insert). (O) xSyn1 rescue of Spemann Organizer specific markers. Embryos at the two-cell were microinjected with MO-βcat in both blastomeres, followed by microinjection of xSyn1 mRNA in all blastomeres at the four-cell stage. RNA was isolated at stages 9, 10.5 and 11.5 and expression of organizer genes evaluated by RT-PCR. |
