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Fig. 4. Deletion analysis of AN enhancer. (A) Fine mapping of the AN enhancer. (11-7)x2 is a duplicate of the #11-#7 sequences (see B), whereas (11-10)x5 is a quintuplet of the #11-#10 sequences. Percentages indicate the sequence identity of the fragment with the counterpart region of human or Xenopus. (B) Nucleotide sequences of the mouse, human and Xenopus EB 165 bp region. Linker-scanner mutations were introduced such that each 15 bp block was replaced with a transcriptionally inert 15 bp linker. The number of ß-gal-positive embryos among transient transgenic embryos in each block is provided in parenthesis; mutations affecting enhancer activity are indicated in red. Asterisks in #8, #9 and #11 represent residual expression as shown in C, part c. (C) ß-gal expression driven by the EcoT22I/BglII(EB)165 bp subfragment (a), by the EB165 bp with the mutation at #10 (b), by the EB165 bp with the mutation at #11 (c), by the (11-7)x2 fragment (d) and by a 1.6 kb Xotx2 region (e) at E7.75. (a-d) Lateral views, anterior is leftwards; (e) a frontal view. Note the loss of expression by the #10 mutation (b), residual expression by the #11 mutation (c) and significant expression with the (11-7)x2 fragment (d) in the anterior neuroectoderm (arrowheads). Arrows indicate the expression in anterior mesoderm and endoderm by the 1.8 kb promoter. Scale bars: 100 µm.

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