Knock-down studies in zebrafish

To study the function of CFAP43 in zebrafish, various morpholino oligonucleotides directed againstCfap43were designed. First, two morpholinos (i1 morpholino, MO i1; i4 morpholino,

2.1 Analysis of CFAP43 in Danio rerio

Figure 2.1: Expression ofCfap43inDanio rerio. ACfap43was detected in zebrafish embryos from 4 to 32 hpf.BCdh17andFoxJ1awere detected in whole embryos as well as in isolated pronephric ducts.

Cdh17expression was restricted to pronephric ducts, thus isolation of pronephric ducts was success-ful (Thisse et al., 2001). In addition Cfap43 can be detected in isolated pronephric ducts (asterisk).

CWhole mountin situhybridization with five distinct probes (a-e) resulted each in overall staining of the embryos, partly with a stronger signal in the head. A probe againstMyl7 (f, red arrowhead), which localizes to the heart, was used as positive control.

MO i4), which bind the splice acceptor sites between intron 1 and exon 2 or intron 4 and exon 5, and should prevent splicing from exons 1 to 2 or exons 4 to 5. The resulting transcripts contain premature stop-codons and should therefore result in nonsense-mediated decay of the mRNA (for review see Nicholson and Mühlemann, 2010). Effective splice inhibition as well as RNA degradation was tracked by RT-PCR. Figure 2.2 A shows the Cfap43 mRNA containing 38 exons and primer pairs, which were used to control the outcome of splice mor-pholino (MO i1 and MO i4) injection. The first two primer pairs (#256+#257 and #258+#259) were used to investigate the direct effect of the morpholino i1 on the transcript. The PCR spanning intron 1 resulted in an upward shift of the PCR product subsequent to injection of MO i1, as expected if the intron is included in the product. In contrast, the PCR spanning exons 2 to 4 showed a downward shift after injection. Cloning and sequencing of this new and unexpected product revealed a transcript, which was shortened by 90 base pairs. This deletion led to a protein, which is shortened by 30 amino acids (1612 instead of 1642 amino acids) without any premature stop codons (figure 2.2 B, asterisk). In addition quantitative RT-PCR revealed no knock-down of CFAP43 in fish injected with morpholino i1 compared to non-injected individuals, and no morphological abnormalities were observed subsequent

Figure 2.2: Knock-down and ectopic expression ofCfap43inDanio rerio. AThe effect of splice site morpholino injection was determined by RT-PCR using primer pairs #256+#257, #258+#259 (MO i1) and #276+#277 (MO i4). Primers #266+#267 were designed for quantitative RT-PCR.BRT-PCR from exon 1 to 2 resulted in a band of 247 base pairs in control embryos and an additional band of 520 base pairs in the injected individuals, demonstrating splice inhibition. RT-PCR spanning exons 2-4 resulted in a shortened product (asterisks) in the injected fish compared to the control, indicating a possible alternative splice event. CRT-PCR spanning exons 4-6 reveals bands of 293 bp (exons 4, 5 and 6) and 418 bp (exons 4, 5, 6 and intron 4), matching the expected PCR products. Thus the injected morpholino prevented splicing from exon 4 to 5. D Quantitative RT-PCRs of eight independent injections revealed knock-down of complete Cfap43 mRNA to about 60% compared to uninjected embryos.EMurineCfap43could be detected at least until 32 hpf after injection of 60 ng mRNA.

to injection with MO i1 in developing zebrafish embryos until 96 hpf. Thus injection with the i1 morpholino did not disrupt functional CFAP43. In contrast, after injection of MO i4 the PCR spanning exons 4-6 (primers #276+#277) revealed only the upward shift expected when including intron 4, but no unexpected additional bands (see figure 2.2 C). The higher band resulting from splice inhibition became more prominent with increasing amounts of injected morpholino. Since injection of high amounts of i4 morpholino led to death of the embryos by toxic side effects of the morpholino, only 5 ng of this morpholino were injected in the knock-down experiments. After each injection quantitative RT-PCR was used to mon-itor the knock-down effect. The average knock-down effect determined in nine independent

2.1 Analysis of CFAP43 in Danio rerio

experiments was approximately 60% (figure 2.2 E; table 5.1, appendix).

Figure 2.3 (column "MO i4") shows the result of Cfap43 knock-down by the splice site morpholino. An overview over a batch of fish (A) shows the usual straight body axis of non-injected individuals (a, f), which is indistinguishable from the appearance of i4 mor-pholino injected fish (b, g). Strong body axis curvature as observed after knock-down of other cilia-related genes (e.g. Kramer-Zucker et al., 2005; Ryan et al., 2013) was not achieved in this situation. Pericardial edemas were found in 14% of the individuals (figure 2.3 B (g), representative picture of one individual, arrowhead). Pericardial edemas were described to develop due to defective water homeostasis in cases of disturbed pronephros or pronephric duct function in the absence of cilia motility (reviewed in Swanhart et al., 2013). In addition to pericardial edema few individuals from knock-down experiments displayed hydrocephali (4%), which are visible in magnifications of the heads (figure 2.3 C (l)). Miss-arranged otoliths were observed after injection of the i4 morpholino in 11% of the individuals (figure 2.3 D (u)), which can be explained by the failure of proper otolith positioning by motile cilia. For anal-ysis of the pronephric ducts zebrafish expressing murine ARL13B-GFP were used, which allowed imaging of the cilia without the need of additional staining procedures. For better visualization of the pronephric ducts fish were stained with fluorescently labeled phalloidin, which binds to F-actin and thus marks among others the apical surface of polarized epithel cells. Confocal Z-stacks were taken for evaluation of the pronephric ducts and their cilia and overlays generated using ImageJ. Ducts of the knock-down fish appeared slightly dilated and cilia orientation, which was very uniform in the non-injected controls, seemed to be randomized (figure 2.3 E). To quantify the effect of knock-down, the pronephric duct width was measured at several points along the duct and averaged for each individual. The results are shown as a dot for each fish in a scatter plot (figure 2.3 G; for raw data see table 5.3, appendix).

To validate the knock-down results described above, another morpholino directed against the start-codon was used to block translation (ATG morpholino, MO ATG), without affecting transcript structure. Antibodies detecting CFAP43 in zebrafish were not available, therefore this knock-down could not be proven. However, ATG morphants recapitulated the pheno-types observed in i4 morphants, suggesting that MO ATG effectively blocked translation.

In addition to the phenotypes observed in i4 morpholino injected animals, many ATG mor-pholino injected fish (84%) displayed a curved body axis (figure 2.3 A (d), B (i) and F (dark blue column)). As for i4 morpholino injected individuals, pericardial edema (78%) and hy-drocephali (25%) were observed after injection of the ATG morpholino (B (i) and C (n)), but occurred with a higher frequency. Magnifications of the otic vesicles reveal malformations as fused or surplus otoliths (66%, figure 2.3 D (y-a’)). As already observed for body axis

curva-ture, otoliths defects appeared to be more severe and occurred more often in animals, which were injected with the ATG morpholino, as compared to the i4 morpholino. Also dilation of the pronephric ducts and miss-orientation of its cilia was more prominent in comparison to the splice morpholino injected animals (figure 2.3 E). In addition to the phenotypes depicted in figure 2.3, laterality was investigated in 48 hpf embryos by evaluation of the direction of heart looping in clutches of three independent injections. No randomization of heart loop-ing could be monitored, neither in fish injected by i4 morpholino nor by ATG morpholino.

Thus, in zebrafish CFAP43 seems not to be necessary for determination of the left-right axis.

Although cilia of the Kupffer’s vesicle seem to perform their function normally, all the other phenotypes observed after injection of the morpholinos have been linked to defects of cili-ogenesis or cilia motility before (Grimes et al., 2016; Kramer-Zucker et al., 2005; Swanhart et al., 2013; Wu et al., 2011).

To control the specificity of the knock-down effects, rescue experiments were performed by co-injection of murine Cfap43 mRNA with each of the morpholinos. The murine tran-script was chosen, since the morpholinos were specific for the sequence of zebrafishCfap43.

Therefore the morpholinos could neither promote a knock-down of the murine mRNA nor could the RNA promote a rescue effect by binding and titration of the morpholinos. Figure 2.2 D shows the presence of the murine mRNA until 32 hpf, thus a potential rescue effect should be visible at least until this stage of development. Body axis curvature of fish, which were injected with the i4 morpholino and murine mRNA, was comparable to the results seen after injection of the i4 morpholino alone (figure 2.2 A (b, c) and F). In contrast, the curved body axis seen in ATG morpholino injected embryos appeared to be milder in the rescue situation (ATG morpholino +Cfap43 mRNA) and fewer individuals were affected (84% vs.

51%, figure 2.3 A (d, e), B (i, j) and F). Pericardial edema (MO i4: 14% vs. 6%, MO ATG: 78%

vs. 51%), hydrocephali (MO i4: 4% vs. 1%, MO ATG: 25% vs. 8%) and otolith defects (MO i4:

11% vs. 3%, MO ATG: 66% vs. 27%) were observed with lower frequencies after co-injection of the Cfap43 RNA compared to the knock-down situations described above. Furthermore dilation of the pronephric ducts could be partially rescued by co-injection of the mRNA with the morpholinos. After rescue of the i4 morpholino the pronephric ducts had an appearance similar to wild type ducts, and also treatment with the ATG morpholino together with the RNA led to slimmer ducts than injection of the ATG morpholino alone. The frequencies of the observed phenotypes after morpholino-mediated knock-down and the corresponding rescue experiments are summarized in the graph in figure 2.3 F and the averages of the mea-sured pronephric duct widths are shown in 2.3 G. The corresponding numbers are shown in table 5.2 in the appendix. Body axis curvature, pericardial edema and hydrocephali were evaluated in five independent experiments using between 20 and 100 individuals for each

2.1 Analysis of CFAP43 in Danio rerio

Figure 2.3: Effects ofCfap43knock-down in Danio rerio. AOverview images of 100 hpf zebrafish embryos show straight body axes in non-injected as well as most of the splice-morpholino (+ murine Cfap43mRNA) injected embryos. In contrast, many of ATG-morpholino injected embryos displayed a curved body axis, which appeared to be milder after injection of Cfap43 mRNA. B Pictures of representative embryos allow show the body axis and reveal pericardial edema (arrowhead).C Mag-nifications of the heads show hydrocephali seen in some i4- and ATG-morpholino injected animals. D Examples of otoliths are shown for differently treated zebrafish embryos. Untreated embryos formed two otoliths in each otic vesicle, in the knock-down situation fused, split or miss-arranged otoliths could be observed (arrowheads). EConfocal images of the pronephric duct of 32 hpf embryos show a slim tube with uniformly oriented cilia in non-injected fish. Pronephric ducts of splice- or ATG-morpholino injected animals were dilated and cilia seemed to be more randomly oriented. These phenotypes appeared milder after co-injection ofCfap43mRNA.F Quantitative analysis of observed phenotypes is depicted in a bar diagram. GWidth of pronephric duct was determined by measuring the distance between phalloidin-marked cell surfaces at several points in each confocal image. The resulting plot indicates that knock-down ofCfap43led to dilated pronephric ducts, which could partly be rescued by co-injection with murineCfap43mRNA.

condition. Otolith formation was investigated in three experiments and pronephric ducts of 16 to 23 individuals were imaged in three (controls, morpholino knock-down) to four (rescue) independent experiments.

In addition to the above mentioned tests, the length of cilia in the spinal central canal and pronephric duct were measured using confocal images of both organs. In this experiment non-injected fish were compared to animals treated with i1 morpholino or ATG morpholino.

Both types of injected fish displayed cilia shortened about 10%. During analysis of those ex-periments it became apparent, that i1 morpholino did not lead to knock-down ofCfap43(see figure 2.2 B), thus shortened cilia were an artifact produced by injection of the morpholinos, rather than a true phenotype ofCfap43knock-down.

Figure 2.4: Effect of CFAP43 on cilia length. A Average length of ponephric duct cilia of 24 hpf zebrafish embryos was reduced after morpholino injection. BAverage length of spinal central canal cilia of 72 hpf zebrafish embryos was reduced after morpholino injection.

In summary, knock-down of CFAP43 could be achieved using two different morpholi-nos, whereas the third morpholino led to alternative splicing products that can generate functional protein. CFAP43 knock-down resulted in developmental defects that have been associated with defects in cilia motility before and could be partially rescued by expression of murine CFAP43. Thus, using the zebrafish as a model system not only evidence for CFAP43 function in motile cilia could be shown, but also the functional conservation between CFAP43 proteins from fish and mouse.