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Fig. 3. Alterations to the mlf following ectopic expression of Sax1. (A-I) Lateral view the ventral mesencephalon of embryos, stained for neurofilament protein (brown) and eGFP mRNA (A-F, blue), analysed 1 day (A-C, HH18) or 2 days (D-F, HH21; G-I, HH19) after electroporation. The line marks the MFB. (A,D,G) After electroporation of the GFP-expressing control construct, the axon tracts appear normal. (B,E,H) Following ectopic expression of pCAß-Sax1-IRES-GFP, the mlf is enlarged (arrows in B,E; also compare brackets in G and H), and the pc is no longer detectable (arrowhead in E). (C,F,I) Ectopic expression of the VP16Sax1 constructs results in a diminished mlf. (J,K) The nucleus of the mlf revealed by retrograde labelling with DiI from the hindbrain, demonstrating enlargement of the mlf after ectopic Sax1 expression (K) compared with an embryo expressing the control construct (J). The caudodorsal and rostroventral subnuclei of the mlf (arrowheads in J) are not distinct in Sax1-expressing embryos (K). (L) Map of the pCAß-LINK-IRESeGFPm5-ClaI expression vector, outlining the CMV enhancer/chick ß-actin promoter (CAß), the multiple cloning site (MCS) into which genes of interest can be inserted and the coding region for GFP linked by the ECMV internal ribosome entry site (IRES). (M,N) Lateral view of the ventral mesencephalon stained for phospho-Histone H3 as proliferation marker. Embryos expressing the control construct (M) or the Sax1 construct (N) show no obvious difference. (O) Schematic representation of the pCAß-Sax1-IRES-GFP and pCAß-VP16Sax1-IRES-GFP expression vectors that are based on pCAß-LINK-IRESeGFPm5-ClaI. Also depicted is the domain swap where the eh1-like transrepression domain of Sax1 (red) is replaced by the transactivation domain of Herpes simplex VP16 (green) to yield the constitutive transcriptional activator VP16Sax1. mlf, medial longitudinal fascicle; nIII, oculomotor nucleus; pc, posterior commissure.





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