(A and D) The 4x intron kuk-LacZ construct was a variant of the kuk-LacZ that contains four copies of kuk small transcript intron (dark gray). Each intron is separated by 201 nucleotides of an in frame Lac-Z sequence (light gray). The no intron kuk-LacZ construct has all splice sites present in the 4x intron kuk-LacZ construct mutated to thymidines. The constructs were fused to a nullo minimal promoter (blue) (A), or fused to an inducible UAS promoter (green) (D). Orientation and position of primers used for splicing analysis is indicated (arrows). (B) RT-PCR analysis showed significant splicing defects (intron retention) of the 4x intron kuk-LacZ construct when expressed under the control of an endogenous early zygotic promoter (nullo promoter). The first intron was correctly spliced, being detected mainly the PCR product corresponding to the spliced form (green dot). The remaining introns (second, third, and fourth) were completely unspliced (red dots, intron retention). In the intronless (no intron kuk-LacZ) construct, under the control of the same nullo promoter were only observed PCR bands whose sizes correspond to unspliced forms (red dots, intron retention). (C) Real-time qPCR analysis showed that the 4x intron kuk-LacZ and no intron kuk-LacZ constructs were expressed to the same extent when under the control of the nullo minimal promoter (t test p>0.05 ns.). (E) RT-PCR analysis of the 4x intron kuk-LacZ construct showed significant splicing defects (intron retention) when zygotically expressed in wild-type embryos under the control of an inducible UAS promoter. Although the most 5′-localized introns (first and second) were still partially spliced, being observed two PCR bands corresponding to the spliced (green dots, int1: 191 and int2: 385 bp), and unspliced forms (red dots, int1: 347 and int2: 541 bp). The furthest 3′-localized introns (third and fourth) were completely unspliced, being only observed one PCR band with the size corresponding to intron retention (red dots, int3: 463 and int4: 385 bp). Maternal expression of the 4x intron kuk-LacZ construct was sufficient to significantly suppress splicing defects in the four introns analyzed (green dots, spliced forms: int1: 191, int2: 385, int3: 307, int4: 229 bp; red dots, unspliced forms: int1: 347, int2: 541, int3: 463, int4: 385 bp). Zygotic and maternal expression of the no intron kuk-LacZ construct only showed PCR bands with sizes corresponding to unspliced forms (red dots, intron retention). (F) Real-time qPCR analysis showed that the 4x intron kuk-LacZ and no intron kuk-LacZ constructs were expressed to the same extent both maternally (Two-way ANOVA p>0.05 ns.) and zygotically (p>0.05 ns.) in wild-type embryos. All PCR bands shown in these panels were cloned and sequenced to confirm their identity. Green dots indicate correctly spliced transcripts, red dots indicate unspliced transcripts (intron retention).