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Experiment details for otx2

Zhang S et al. (2014) Assay

Fezf2 promotes neuronal differentiation through localised activation of Wnt/β-catenin signalling during forebrain development.

Gene Clone Species Stages Anatomy
otx2.S laevis NF stage 15 anterior
otx2.S laevis NF stage 20 optic vesicle , anterior
otx2.S laevis NF stage 28 to NF stage 35 and 36 retina , forebrain , eye

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  Figure S2. Comparison of expression pattern of fezf2 expression relative to various nerual markers at different stages, related to Figure 1. Anterior is to the bottom in stage 15 and 20 embryos (frontal view), and to the left in stage 28 and 35 embryos (lateral view).

Gene Clone Species Stages Anatomy
otx2 tropicalis NF stage 17 anterior neural tube
otx2 tropicalis NF stage 28 forebrain , eye

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  Figure S4 (related to Figure 1). fezf2 knockdown, but not control MO knockdown, leads to defects in forebrain neuronal differentiation. (A) fezf2 knockdown led to defects in forebrain development after initial patterning stage. Whole-mount in situ hybridisation analyses on control versus fezf2 MO injected stage 28 embryos, stained using the forebrain-specific markers, otx2 and pax6. (B) fezf2 knockdown did not cause defects in early forebrain patterning, as revealed by whole-mount in situ hybridisation on control versus fezf2 MO injected stage 17 embryos, stained using the forebrain-specific markers arx, otx2, and pax6. Anterior is to the bottom. (C-H) 1 of 2 blastomeres at 2-cell stage was injected with control MO, cultured to stage 30, fixed sectioned transversely across the forebrain and stained via immunofluorescence for the transcription factor Sox3 (marker for neural progenitors) (C-D) or MyT1 (marker for differentiated primary neurons) (F-G). (C) Merged image. (D) Sox3 staining alone. (E). Statistical analysis of Sox3+ cells in control MO injected side relative to the uninjected side of the forebrain (n=3 embryos, no significant difference). The uninjected side has been normalised to 100%. (F) Merged image. (G) MyT1 staining alone. (H) Statistical analysis of MyT1+ cells in control MO injected side relative to the uninjected side of the forebrain (n=3 embryos, no significant difference). The uninjected side has been normalised to 100%. (I-K) Transverse sections were prepared as for panels C-D and then processed for TUNEL staining. (I) Merged image. (J) TUNEL-positive cells alone. (K) Percentage of the TUNEL-positive cells in the control MO injected side compared to the uninjected side (n=3 embryos). In all cases, the FITC tag on the control MO was used to identify the injected side; DAPI was used to stain nuclei. Error bars represent ±s. e. m. Scale bar: 25 μM. ns: not significant. (L) (a-b) Immunofluorescence staining using the neuronal differentiation marker, Ntubulin, on stage 32 embryos injected with 10 ng of control or fezf2 MO. (a) control MO; (b) fezf2 MO, showing a strong reduction of N-tubulin staining in the rostral area. (c) Sections of stage 32 embryos at the prethalamus level and stained with N-tubulin antibody. Embryos were injected with 5 ng of fezf2 MO into one of the two blastomeres at the 2-cell stage. A strong reduction of N-tubulin staining was observed in the injected side. Arrowhead: injected side. DAPI was used to provide global visualisation on the head structure. In all cases, error bars represent ±s. e. m.