Tulp3 knockdown analyses of cilia-related phenotypes during zebrafish embryogenesis. (A) Exon-intron structure (drawn to scale) of tulp3 in zebrafish (Ensembl Transcript ID: ENSDART00000093236.6). Translation start codon (ATG), termination codon (TGA), TB-MO tulp3, SB1-MO tulp3 and SB2-MO tulp3 are indicated. Black and blue half arrows indicate primer pairs used for analysis of SB1-MO tulp3 and SB2-MO tulp3 efficiency, respectively. (B, C) Expression analysis of tulp3 using semi-quantitative RT-PCR on cDNA of Co-MO (1ng) or SB1-MO tulp3 (0,5 or 1ng) injected embryos (B) and Co-MO (2ng) or SB2-MO tulp3 (2ng) injected embryos, respectively (C); while an exon exclusion is not detectable upon injection of SB1-MO tulp3 or SB2-MO tulp3, respectively, the highly reduced wildtype PCR products compared to the control indicate an (partial) intron insertion through cryptic splice site activation that might not be detectable by utilized PCR conditions. Black arrows point to tulp3 and ef1α PCR products, respectively; Primer dimer (PD). H2O served as negative control and ef1α as loading control; dividing lines in (B) indicate different contrast from different parts of the same gel image. (D-G) Bright-field images of zebrafish embryos at 2dpf injected with Co-MO (2ng) (D) or TB-MO tulp3 (2ng) (E-G). In comparison to Co-MO injected embryos, injection of TB-MO tulp3 leads to different degrees of ventral body curvature. (H–K) Knockdown of Tulp3 leads to pronephric cyst formation (white stars in (J)) and otolith deposition defects (white arrows in (K)) at 2dpf as shown in a dorsal view with anterior to the left of a TB-MO tulp3 (2ng) injected li1Tg embryo (J), and an embryo shown in a bright-field image (K), respectively, in comparison to Co-MO (2ng) injected embryos (H, I); expression of EGFP fluorescence labels glomerulus (G), neck (N) and proximal convoluted tubule (PCT). (L) Quantification of pronephric cyst formation in 2dpf zebrafish embryos injected with Co-MO (2ng) (4 independent experiments; n = 31, n = 63, n = 52 and n = 82 analysed embryos, respectively), TB-MO tulp3 (2ng) (n = 35, n = 79, n = 57 and n = 81), TB-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 54, n = 71, n = 57 and n = 73) or Co-MO (0,5 ng) (3 independent experiments; n = 46, n = 46 and n = 47), SB1-MO tulp3 (0,5 ng) (n = 41, n = 44 and n = 46), SB1-MO tulp3 (0,5 ng) + HTULP3 mRNA (5pg) (n = 46, n = 46 and n = 46) or Co-MO (2ng) (3 independent experiments; n = 42, n = 39 and n = 37), SB2-MO tulp3 (2ng) (n = 31, n = 37 and n = 38), SB2-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 41, n = 41 and n = 42). (M) Quantification of otolith deposition defects in 2dpf zebrafish embryos injected with Co-MO (2ng) (4 independent experiments; n = 42, n = 70, n = 50 and n = 82 analysed embryos, respectively), TB-MO tulp3 (2ng) (n = 44, n = 79, n = 71 and n = 81), TB-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 56, n = 71, n = 56 and n = 78). (N) Quantification of ventral body curvature in 2dpf zebrafish embryos injected with Co-MO (2ng) (4 independent experiments; n = 41, n = 70, n = 50 and n = 82 analysed embryos, respectively), TB-MO tulp3 (2ng) (n = 40, n = 80, n = 72 and n = 84), TB-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 57, n = 71, n = 56 and n = 79) or Co-MO (0,5ng) (3 independent experiments; n = 46, n = 46 and n = 46), SB1-MO tulp3 (0,5ng) (n = 43, n = 46 and n = 46), SB1-MO tulp3 (0,5ng) + HTULP3 mRNA (5pg) (n = 46, n = 46 and n = 46) or Co-MO (2ng) (3 independent experiments; n = 42, n = 39 and n = 34), SB2-MO tulp3 (2ng) (n = 42, n = 40 and n = 36), SB2-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 38, n = 40 and n = 36). (O) Quantification of altered heart looping in 2dpf zebrafish embryos injected with Co-MO (2ng) (3 independent experiments; n = 41, n = 70 and n = 50 analysed embryos, respectively), TB-MO tulp3 (2ng) (n = 43, n = 79 and n = 71), TB-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 56, n = 70 and n = 55) or Co-MO (0,5ng) (3 independent experiments; n = 46, n = 46 and n = 46), SB1-MO tulp3 (0,5ng) (n = 43, n = 35 and n = 46), SB1-MO tulp3 (0,5ng) + HTULP3 mRNA (5pg) (n = 46, n = 46 and n = 46) or Co-MO (2ng) (3 independent experiments; n = 42, n = 39 and n = 34), SB2-MO tulp3 (2ng) (n = 41, n = 39 and n = 40), SB2-MO tulp3 (2ng) + HTULP3 mRNA (5pg) (n = 39, n = 40 and n = 36); total number of embryos used for analyses are shown above respective bar. Unprocessed gel images (B and C, respectively) are presented in Suppl. Figure 7.
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