Figure 6. Recent origin of the N2 motif in dipterans.

(A) Schematic representation of proposed steps giving rise to the Cic-S isoform and the N2 motif. The diagrams show the regions spanning the alternatively spliced exons of cic-S and cic-L transcripts in selected species, which are represented graphically and are not drawn to scale. The size of introns splitting the N2-L coding sequences in non-dipteran species are as follows: Bombyx mori (758 bp), Tribolium castaneum (50 bp), Apis mellifera (86 bp) and Acyrthosiphon pisum (129 bp) (see also main text). The conserved protein motifs encoded at relevant exon junctions are shaded in red, blue or black as in Fig. 3. (B) Insect phylogeny illustrating the presence of the Cic-S and N2 motifs in dipterans (red). (C) Diagram of the Cic-S, Cic-L and Cic-L^N2 proteins; Cic-L^N2 carries the N2 motif inserted within a poorly conserved sequence of Cic. Cic-L and Cic-L^N2 were expressed with an HA tag at the C-terminus. (D and E) Expression of HA-tagged Cic-L (D) and Cic-L^N2 (E) proteins in embryos stained with anti-HA antibody. (F and G) mRNA expression patterns of kni in cic¹/cic^Q474X mutant embryos expressing Cic-L (F) and Cic-L^N2 (G); only Cic-L^N2 leads to significant rescue of the central kni stripe. (H and I) Cuticle phenotypes of the same genetic backgrounds shown in F and G, respectively.