Figure 2

Enhancer screening in Zic1 and Zic3 CNEs. (A) Structure of human Zic4/Zic1 and Zic3. Top indicates the scales in kilobase, centered at the transcription initiation of Zic1 or Zic3 the + (plus) direction is that of transcription. Blue thin vertical lines below gene names indicate the protein coding exons. Clustered green vertical lines indicate the presence of sequence similarity with the human sequence at each position of the sequences of animals indicated at the left side (mouse, chicken, Xenopus, and zebrafish). Extent of conservation is indicated as the height of bars. The extent of conservation among the 100 vertebrate species selected at UCSC genome browser is indicated by 100 vert. Black vertical lines in the CNE line indicate the position of tested CNEs in this study. Ol-a-d and Dr-e1, known regulatory elements identified in teleost fishes^30,31. (B) Structure of the reporter vector. TK, herpes simplex virus thymidine kinase promoter (minimal activity by itself); CMV, human cytomegalovirus immediate early enhancer and promoter (strong ubiquitous activity). Pictures, representative results of the reporter expression in chicken embryos at HH11 for CMV, TK, Zic1-NE, Zic1-ME, Zic3-NE1, Zic3-NE2, and Zic3-ME. (Top) Bright field views. (Middle) Reporter GFP signal at HH11 (24 h after electroporation). (Bottom) β-galactosidase activity staining for co-electroporated EF-LacZ to indicate the transfected cells. Scale bar, 1 mm. Frequencies of the reporter GFP expression/Zic1 or Zic3 expressing region are indicated above the pictures. Numbers indicate those of tested embryos. (C) Reporter GFP signals at HH9 (12 h after electroporation). Expression patterns at additional time points are shown in Supplementary Fig. S2. Scale bar, 1 mm. (D) Spatial distribution of enhancer activity in a summary.