Figure 2. Generation and phenotypic analysis of nobo^KO homozygous mutant embryos.

(a) Genomic and exon-intron structures of the nobo (CG4688) loci of the wild-type and nobo knock-out (nobo^KO) strains. White and black boxes indicate the coding sequence and the untranslated regions, respectively. CG4649 is a gene located next to nobo. Dashed arrows indicate orientations of the genes. In the nobo^KO allele, 1,033 bp of the nobo gene region was replaced with a gene-targeting construct including the hsp70::mini-white marker cassette with loxP sites, resulting in a 680 bp deletion in the entire (696 bp) nobo coding sequence. Arrows ‘F' and ‘R' indicate the positions of the genotyping primers used in Supplementary fig. 2 (also see Methods and Supplementary table S3). (b,c) Dark-field images of embryonic cuticles from (b) nobo^KO heterozygous (nobo^KO/+) and (c) homozygous (nobo^KO/nobo^KO) embryos. (d–g) Anti-FasIII antibody staining to visualise overall embryo morphology. (d,f) nobo^KO/+ embryos. (e, g) nobo^KO/nobo^KO embryos. (e) The bracket indicates defective head involution. (g) The arrow and the arrowhead indicate the dorsal open phenotype and abnormal gut looping, respectively. (h–k) Expression patterns of (h, i) IMP-E1 and (j, k) IMP-L1 in stage 14 embryos. (h, j) nobo^KO/+ embryos. (i, k) nobo^KO/nobo^KO embryos. These data show that the nobo mutant exhibited severe reductions of these 20E-inducible genes. Scale bars: 100 µm for all images.