DEV01772 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Conservation of polaris and hippi protein sequences in different species. Sequence comparison using clustalW (Thompson et al., 1994). The Accession numbers are the following: polaris/Tg737 human (NM_006531); mouse (NM_009376); hippi human (AF245220); mouse (AF274590); C. elegans (AF520567). (Zebrafish IFT sequences identified in this work have been submitted to GenBank). The sequence comparison shows homology between zebrafish and human with 71% identity and 84% similarity for polaris and with 60% identity and 74% similarity for hippi, respectively.

• Supplemental Figure 2 -

  Fig. S2. Ubiquitous expression of polaris and hippi in the developing zebrafish embryo. In situ expression pattern of polaris at 21-somite (A) and hippi at 24 hpf. (B). Both genes are ubiquitously expressed, with polaris expression enriched along the forming pronephric duct (arrow, A). (B) hippi expression can be seen in the anterior and posterior parts of the pronephric duct (arrows).

• Supplemental Figure 3 -

  Fig. S3. Rescue of IFT morphant phenotype by hippi mRNA injection. Overexpression of zebrafish hippi by injection of capped RNA (2.3 ng) does not cause a visible phenotype, but is capable of rescuing the phenotype caused by injection of 4.6 nl of 0.1 mmol/l hippiSP morpholino. All 55 morpholino-injected embryos showed pronephric cyst formation (arrow and * in histological cross section) and some degree of hydrocephalus (arrowhead and ** in histological cross section), with 48 of 55 also having a ventrally bent body axis, whereas 6 appeared normal. Of the 44 co-injected embryos, 4 had a bent body axis, and hydrocephalus, but no renal cyst formation. Four had a straight body axis and no hydrocephalus, but kidney cysts, and 36 appeared wild type, which was confirmed by histology.

• Supplemental Figure 4 -

  Fig. S4. Topographical anatomy of supplemental quicktime movies. The numbered areas specify the anatomical region in the zebrafish embryo/larva examined by videomicroscopy in quicktime movies. (A) Wild-type embryo: (1) Movie 1; (2) Movie 2; (3) Movie 3; (4) Movie 4. (B) Morphant embryo: (5) Movie 5; (6) Movie 6; (7) Movie 7. (C) Wild-type embryo, 10-somite stage: (8) Movie 8; (9) Movie 9; (10) Movie 10. Movies 1, 2, 4, 5, 6, 7 and 8 are in slow motion (163); 9 and 10 are real-time; and 3 is in fast motion (153).

• Supplemental Figure 5 -

  Fig. S5. Splice defects caused by morpholinos. The effect of morpholinos on mRNA splicing was determined by sequencing altered RT-PCR products. The morpholino targeting the splice donor of the second coding exon of polaris causes a non-splicing of the adjacent intron with a premature stop. Injection of the hippi splice morpholino targeting the second coding exon results in an in-frame deletion of the second and third coding exon together and an out-of-frame deletion of the second coding exon only. For the dynein heavy chain 9, the morpholino causes an out-of-frame deletion of the P1-domain coding exon or a non-splicing of the adjacent intron leading to a truncation allele. Wt, wild type; mo, morpholino; SP, splice junction targeting morpholino; numbers give the size of the RT-PCR amplicon.

• Movie 1 -

  Movie 1. Wild-type pronephric tubule at 2.5 dpf. Cilia in the pronephros appear to be motile in all segments of the nephron, including the pronephric tubules.

• Movie 2 -

  Movie 2. Wild-type pronephric duct cilia in a 2.5 dpf embryo. This movie was acquired at 250 frames/second. The portion shown here is 125 frames or 500 milliseconds. The cilia can be seen to beat 9-10 times in this period, corresponding to a beat rate of about 18-20 Hz.

• Movie 3 -

  Movie 3. Functional assessment of urine output from a 3.5 dpf wild-type pronephros. Injected fluorescent dextran in the circulation is visible soon after following introduction via the cardinal vein. Time is sped up 15�. Over a period of several minutes, fluorescent urine is detectable at the cloaca.

• Movie 4 -

  Movie 4. Spinal canal cilia in a 2.5 dpf wild-type embryo. Relatively short cilia in the spinal canal are 9+0 and move in a rotary pattern.

• Movie 5 -

  Movie 5. The junction of the pronephric duct and tubule adjacent to the site of cyst formation at the anterior end of the pronephros of a 2.5 dpf embryo lacking functional hippi/IFT57. Cilia movement is stiff and uncoordinated.

• Movie 6 -

  Movie 6. The pronephric duct lumen and cilia of a 2.5 dpf embryo lacking functional hippi/IFT57. Some duct lumenal distension is observed and the cilia indicated with the arrow in the first frame has a stiff, uncoordinated movement as opposed to the rhythmic undulations observed in wild-type pronephric duct cilia.

• Movie 7 -

  Movie 7. Reduction in beat rate caused by disruption of dhc9 expression. This movie was acquired at 250 frames/second. The portion shown here is 102 frames or 408 milliseconds. The cilia can be seen to beat 4-5 times in this period, corresponding to a beat rate of about 9 to 12 Hz. Cilia length and beat waveform is not different from those of wild-type cilia.

• Movie 8 -

  Movie 8. Beating cilia in a 10-somite Kupffer�s vesicle. Cilia, outlined in the first frame, beat in a counterclockwise direction viewed from the dorsal aspect of the embryo.

• Movie 9 -

  Movie 9. A small piece of debris in a 10-somite Kupffer�s vesicle is caught moving in ciliary currents. Overall movement is counterclockwise.

• Movie 10 -

  Movie 10. Focusing through the posterior tailbud region to Kupffer�s vesicle in a 10-somite embryo. Fluorescent beads injected into Kupffer�s vesicle have aggregated and move in an overall counterclockwise direction.