DEV02806 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Splicing defects induced by jagged2 and notch3 morpholinos. (A) Injection of jagged2 exon20 splice donor morpholino results in the deletion of the highly conserved exon 20, the out of frame fusion of exons 19 and 21, and the introduction of a stop codon immediately following the exon 19 sequence. (B) Injection of notch3 exon27 splice donor morpholino results in the deletion of exon 27, the out of frame fusion of exons 26 and 28, and the introduction of a stop codon after a string of missense codons following exon 26 sequence.

• Supplemental Figure 2 -

  Fig. S2. her9 expression in the pronephros and effect of Jagged2/Notch3 knockdown. (A) Expression of her9 in wild-type embryos. her9 is expressed in the neural tube, somites and bilaterally in the pronephros (arrowheads) at 34 hpf. (B) DAPT treatment (100 μM) starting at 9.5 hpf results in a reduction in her9 expression. (C) her9 expression in mind bomb homozygotes is reduced in general and absent from the pronephros. (D) jagged2 exon 20 morpholino injection, (E) notch3 exon 27 morpholino, or (F) a combination of jagged2 exon 20 and jagged2 atg morpholino injection results in loss of her9 expression in the pronephros (arrowheads in F).

• Movie 1 -

  Movie 1. 2.5 dpf wildt-ype pronephros. Two isolated cells have cilia bundles emanating from their apical surface while most cells display a single motile cilium. To clearly demonstrate cilia beat and distribution, the pronephros of this embryo was partially obstructed at the cloaca to expand the pronephric lumen. Movie frames were acquired at 250 fps and played back at 15 fps.

• Movie 2 -

  Movie 2. 2.5 dpf jagged2 exon20 morphant pronephros. Injection of jagged2 morpholino caused a dramatic increase in the number of motile cilia in the pronephros compared to wild-type embryos (Movie 1). To clearly demonstrate cilia beat and distribution, the pronephros of this embryo was partially obstructed at the cloaca to expand the pronephric lumen. Movie frames were acquired at 250 fps and played back at 15 fps.

• Movie 3 -

  Movie 3. 2.5 dpf dbb-heterozygote pronephros. Wild-type dbb siblings show normal cilia motility in the pronephros. Compared with Movies 1 and 2, the lumen is seen here as its normal narrow diameter, because the cloaca of this embryo was not obstructed. Movie frames were acquired at 250 fps and played back at 15 fps.

• Movie 4 -

  Movie 4. 2.5 dpf dbb-homozygote pronephros. The dbb mutant pronephros is dilated as part of the mutant phenotype and lacks detectable motile cilia. Movie frames were acquired at 250 fps and played back at 15 fps.

• Movie 5 -

  Movie 5. 2.5 dpf dbb homozygote + jagged2 ATG MO pronephros. dbb-mutant embryo injected at the one-cell stage with jagged2 ATG morpholino shows recovery of motile bundles of cilia. The pronephric lumen in this mutant embryo exhibits a narrow diameter similar to wild-type embryos (Movie 3). The pronephric opening of this embryo was not obstructed and the recovery of normal lumen diameter correlates with the reappearance of motile cilia. Movie frames were acquired at 250 fps and played back at 15 fps.