Fig. 3. Site-directed mutagenesis of E-boxes of the ACEL-like element and LAG-1 binding sites in egl-43. (A) egl-43 gene structure. The ACEL-like element that contains two E-boxes is predicted in the first intron, and eight LAG-1 binding sites are predicted in the 5' regulatory and the first intronic regions. (B,C) egl-43 (wild type)::YFP is expressed (green) at early L2 in the pre-AC/pre-VU cells located in two different focal planes. (D,E) Either the Z1.ppp or Z4.aaa cell moves to the central position on the ventral surface of the gonad to become the AC (green, arrow), which is located (D) in a focal plane different from the two focal planes containing the three VU cells (green). Only one VU focal plane is shown in E. (F,G) The AC begins to express cdh-3::CFP at late L2. The AC (arrow) is appears yellow in F because of the co-localization of cdh-3::CFP (red) and egl-43::YFP (green). (H,I) The egl-43 (mutated E-boxes)::YFP is not expressed in the pre-AC/pre-VU cells, AC and VU cells until late L2. (J,K) egl-43 (mutated E-boxes)::YFP is expressed in the three VU cells (green), and not in the AC (arrow), when the cdh-3::CFP begins to be expressed in the AC (J, red) at late L2. (L-Q) The egl-43 (mutated LAG-1 sites)::YFP is expressed in the pre-AC/pre-VU cells at early L2 (L,M, green). One cell (Z4.aap) expresses YFP very weakly in this animal (L). At mid-L2, YFP expression is retained in the AC (N, in green, arrow), but is lost or decreased in the VU cells (O). At late L2 when the cdh-3::CFP (red) begins to be expressed in the AC (P, arrow), three VU cells regain the YFP signal (Q). The AC expresses both cdh-3::CFP (red) and egl-43 (mutated LAG-1 sites)::YFP (green) at this stage, resulting in the merged yellow colour (P). The egl-43 (mutated LAG-1 sites)::YFP (green) begins to be expressed in two DU cells (yellow arrow) right after the three VU cells regain YFP expression (Q). Only one VU focal plane is shown. For each construct, about 100 animals were examined from six to ten transgenic lines. The pictures represent typical expression patterns at specific stages. The lines include syEx895 and syEx896 for the wild type; syEx891 and syEx892 for the mutated E-boxes; syEx897, syEx898, syEx899 and syEx900 for the mutated LAG-1 binding sites.