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Figure 4


Fig. 4. Overexpression of Cyp1B1 alters DV patterning and motor neuron specification. (Aa) Full-length Cyp1B1 (1629 nt) with a modified Kozak sequence was subcloned into the pCAB IRES-GFP vector (Gilthorpe et al., 2002) to generate Cyp1B1-IRES-GFP. (Ab,Ac) Following electroporation of Cyp1B1-IRES-GFP into HH11 embryos, both a high level of GFP (Ab, white arrowhead) and Cyp1B1 (Ac, blue arrowhead) transcripts are detected in the left-hand side of neuroepithelium. (Ad) Endogenous expression of cCyp1B1 at HH12 in the hindbrain. (B-E) Embryos were electroporated with Cyp1B1-IRES-GFP at HH10-12, cultured for 16-20 hours and analysed by in situ hybridisation for (Ba-Bf) Shh, (Ca-Cf) Nkx6.1, (Da-Dh) Isl1 and (Ea-Ed) Gata2. Wild-type expression of each gene is shown in the `normal' column and embryos electroporated with the pCAB IRES-GFP vector alone in the `control' column. Isolated neural tubes from either the control or Cyp1B1-IRES-GFP-electroporated embryos are shown in Bc,Bd, Cc,Cd and Dc,Dd, respectively. Arrows in Bd and Cd indicate domains of loss of expression. (Be,Bf) Comparison of hindbrain flat-mounts of a normal and a Cyp1B1-IRES-GFP-electroporated embryo analysed for Shh showing that expression is reduced in the floorplate cells (Bf, green arrow). (Ce,Cf) Comparison of flat-mount hindbrain preparation of a normal and a Cyp1B1-IRES-GFP-electroporated embryo analysed for Nkx6.1 shows alteration of expression domains in the electroporated embryo (Cf, green arrow). The blue arrow in Cf indicates the region of downregulation. (De,Df) Comparison of hindbrain flat-mount preparation of a normal and a Cyp1B1-IRES-GFP-electroporated embryo analysed for Isl1 expression shows downregulation (Df, green arrow). (Dg) Lateral view of the non-electroporated control side of an embryo processed for Isl1 expression (white arrow, placodal expression). (Dh) Expression of Isl1 in the placodes is reduced on the Cyp1B1-IRES-GFP-electroporated side (white arrows). (Ec,Ed) Gata2 expression is reduced in hindbrain of Cyp1B1-IRES-GFP-electroporated embryo. The location of the facial motor nuclei is indicated by VII (green arrow).





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