Ciliary length regulation by intraflagellar transport in zebrafish

Reviewer #1 (Public Review):

Among the many challenges in the cilia field, is the question of how multicellular organisms assemble a variety of structurally and functionally specialized cilia, including cilia of different lengths. This study addresses the important question of how ciliary length differences are established in vertebrates. Specifically, the authors analyzed the role of intraflagellar transport (IFT) in ciliary length regulation in zebrafish, exploiting the transparency of the embryos. Zebrafish possess functionally specialized motile and non-motile cilia in a variety of tissues. Expression of GFP-tagged IFT88, a component of the IFT-B subcomplex, in a corresponding mutant, allowed the authors to image IFT in five distinct types of cilia. They note that IFT moves faster in longer cilia. Tagging and imaging of the IFT-A protein IFT43 further support this observation. IFT speed was largely unaffected in knock-out and morphants targeting the BBSome, various kinesin-2 motors, and the posttranslational modifications of tubulin polyglycylation and polyglutamylation. Using high-resolution STED imaging, the authors observe that IFT signals (likely, corresponding to IFT trains) are smaller in the shorter spinal cord cilia compared to the long cristae cilia. Based on these observations, the authors test the hypothesis that larger IFT trains recruit more motors or coordinate the motors better, resulting in faster trains, and causing cilia to be longer. This is further tested using partial knock-down of IFT88-GFP, which resulted in shorter crista cilia, reduced IFT particle number, size, and velocity. Some parts of the manuscript show "negative" data (e.g., ciliary length and IFT are not affected by the loss of BBS4) but these add beautifully to the overall story and allow for additional conclusions such as the minor role of ttll3 and ccp knockouts on ciliary length in this model. This is an excellent study, which documents IFT in a vertebrate species and explores its regulation. The data are of high quality and support most of the conclusions.

(1) The main hypothesis/conclusion is summarized in the abstract: "Our study presents an intriguing model of cilia length regulation via controlling IFT speed through the modulation of the size of the IFT complex." The data clearly document the remarkable correlation between IFT velocity and ciliary length in the different cells/tissues/organs analyzed. The experimental test of this idea, i.e., the knock-down of GFP-IFT88, further supports the conclusion but needs to be interpreted more carefully. While IFT particle size and train velocity were reduced in the IFT88 morphants, the number of IFT particles is even more decreased. Thus, the contributions of the reduction in train size and velocity to ciliary length are, in my opinion, not unambiguous. Also, the concept that larger trains move faster, likely because they dock more motors and/or better coordinating kinesin-2 and that faster IFT causes cilia to be loner, is to my knowledge, not further supported by observations in other systems (see below).

(2) I think the manuscript would be strengthened if the IFT frequency would also be analyzed in the five types of cilia. This could be done based on the existing kymographs from the spinning disk videos. As mentioned above, transport frequency in addition to train size and velocity is an important part of estimating the total number of IFT particles, which bind the actual cargoes, entering/moving in cilia.

(3) Here, the variation in IFT velocity in cilia of different lengths within one species is documented - the results document a remarkable correlation between IFT velocity and ciliary length. These data need to be compared to observations from the literature. For example, the velocity of IFT in the quite long (~ 100 um) olfactory cilia of mice is similar to that observed in the rather short cilia of fibroblasts (~0.6 um/s). In Chlamydomonas, IFT velocity is not different in long flagella mutants compared to controls. Probably data are also available for C. elegans or other systems. Discussing these data would provide a broader perspective on the applicability of the model outside of zebrafish.

Reviewer #2 (Public Review):

Summary:

In this study, the authors study intraflagellar transport (IFT) in cilia of diverse organs in zebrafish. They elucidate that IFT88-GFP (an IFT-B core complex protein) can substitute for endogenous IFT88 in promoting ciliogenesis and use it as a reporter to visualize IFT dynamics in living zebrafish embryos. They observe striking differences in cilia lengths and velocity of IFT trains in different cilia types, with smaller cilia lengths correlating with lower IFT speed. They generate several mutants and show that disrupting the function of different kinesin-2 motors and BBSome or altering post-translational modifications of tubulin does not have a significant impact on IFT velocity. They however observe that when the amount of IFT88 is reduced it impacts the cilia length, IFT velocity as well as the number and size of IFT trains. They also show that the IFT train size is slightly smaller in one of the organs with shorter cilia (spinal cord). Based on their observations they propose that IFT velocity determines cilia length and go one step further to propose that IFT velocity is regulated by the size of IFT trains.

Strengths:

The main highlight of this study is the direct visualization of IFT dynamics in multiple organs of a living complex multi-cellular organism, zebrafish. The quality of the imaging is really good. Further, the authors have developed phenomenal resources to study IFT in zebrafish which would allow us to explore several mechanisms involved in IFT regulation in future studies. They make some interesting findings in mutants with disrupted function of kinesin-2, BBSome, and tubulin modifying enzymes which are interesting to compare with cilia studies in other model organisms. Also, their observation of a possible link between cilia length and IFT speed is potentially fascinating.

Weaknesses:

The manuscript as it stands, has several issues.

(1) The study does not provide a qualitative description of cilia organization in different cell types, the cilia length variation within the same organ, and IFT dynamics. The methodology is also described minimally and must be detailed with more care such that similar studies can be done in other laboratories.

(2) They provide remarkable new observations for all the mutants. However, discussion regarding what the findings imply and how these observations align (or contradict) with what has been observed in cilia studies in other organisms is incomprehensive.

(3) The analysis of IFT velocities, the main parameter they compare between experiments, is not described at all. The IFT velocities appear variable in several kymographs (and movies) and are visually difficult to see in shorter cilia. It is unclear how they make sure that the velocity readout is robust. Perhaps, a more automated approach is necessary to obtain more precise velocity estimates.

(4) They claim that IFT speeds are determined by the size of IFT trains, based on their observations in samples with a reduced amount of IFT88. If this was indeed the case, the velocity of a brighter IFT train (larger train) would be higher than the velocity of a dimmer IFT train (smaller train) within the same cilia. This is not apparent from the movies and such a correlation should be verified to make their claim stronger.

(5) They make an even larger claim that the cilia length (and IFT velocity) in different organs is different due to differences in the sizes of IFT trains. This is based on a marginal difference they observe between the cilia of crista and the spinal cord in immunofluorescence experiments (Figure 5C). Inferring that this minor difference is key to the striking difference in cilia length and IFT velocity is incorrect in my opinion.

Impact:

Overall, I think this work develops an exciting new multicellular model organism to study IFT mechanisms. Zebrafish is a vertebrate where we can perform genetic modifications with relative ease. This could be an ideal model to study not just the role of IFT in connection with ciliary function but also ciliopathies. Further, from an evolutionary perspective, it is fascinating to compare IFT mechanisms in zebrafish with unicellular protists like Chlamydomonas, simple multicellular organisms like C elegans, and primary mammalian cell cultures. Having said that, the underlying storyline of this study is flawed in my opinion and I would recommend the authors to report the striking findings and methodology in more detail while significantly toning down their proposed hypothesis on ciliary length regulation. Given the technological advancements made in this study, I think it is fine if it is a descriptive manuscript and doesn't necessarily need a breakthrough hypothesis based on preliminary evidence.

Reviewer #3 (Public Review):

Summary:

A known feature of cilia in vertebrates and many, if not all, invertebrates is the striking heterogeneity of their lengths among different cell types. The underlying mechanisms, however, remain largely elusive. In the manuscript, the authors addressed this question from the angle of intraflagellar transport (IFT), a cilia-specific bidirectional transportation machinery essential to biogenesis, homeostasis, and functions of cilia, by using zebrafish as a model organism. They conducted a series of experiments and proposed an interesting mechanism. Furthermore, they achieved in situ live imaging of IFT in zebrafish larvae, which is a technical advance in the field.

Strengths:

The authors initially demonstrated that ectopically expressed Ift88-GFP through a certain heat-shock induction protocol fully sustained the normal development of mutant zebrafish that would otherwise be dead by 7 dpf due to the lack of this critical component of IFT-B complex. Accordingly, cilia formations were also fully restored in the tissues examined. By imaging the IFT using Ift88-GFP in the mutant fish as a marker, they unexpectedly found that both anterograde and retrograde velocities of IFT trains varied among cilia of different cell types and appeared to be positively correlated with the length of the cilia.

For insights into the possible cause(s) of the heterogeneity in IFT velocities, the authors assessed the effects of IFT kinesin Kif3b and Kif17, BBSome, and glycylation or glutamylation of axonemal tubulin on IFT and excluded their contributions. They also used a cilia-localized ATP reporter to exclude the possibility of different ciliary ATP concentrations. When they compared the size of Ift88-GFP puncta in crista cilia, which are long, and spinal cord cilia, which are relatively short, by imaging with a cutting-edge super-resolution microscope, they noticed a positive correlation between the puncta size, which presumably reflected the size of IFT trains, and the length of the cilia.

Finally, they investigated whether it is the size of IFT trains that dictates the ciliary length. They injected a low dose (0.5 ng/embryo) of ift88 MO and showed that, although such a dosage did not induce the body curvature of the zebrafish larvae, crista cilia were shorter and contained less Ift88-GFP puncta. The particle size was also reduced. These data collectively suggested mildly downregulated expression levels of Ift88-GFP. Surprisingly, they observed significant reductions in both retrograde and anterograde IFT velocities. Therefore, they proposed that longer IFT trains would facilitate faster IFT and result in longer cilia.

Weaknesses:

The current manuscript, however, contains serious flaws that markedly limit the credibility of major results and findings. Firstly, important experimental information is frequently missing, including (but not limited to) developmental stages of zebrafish larvae assayed (Figures 1, 3, and 5), how the embryos or larvae were treated to express Ift88-GFP (Figures 3-5), and descriptions on sample sizes and the number of independent experiments or larvae examined in statistical results (Figures 3-5, S3, S6). For instance, although Figure 1B appears to be the standard experimental scheme, the authors provided results from 30-hpf larvae (Figure 3) that, according to Figure 1B, are supposed to neither express Ift88-GFP nor be genotyped because both the first round of heat shock treatment and the genotyping were arranged at 48 hpf. Similarly, the results that ovl larvae containing Tg(hsp70l:ift88 GFP) (again, because the genotype is not disclosed in the manuscript, one can only deduce) display normal body curvature at 2 dpf after the injection of 0.5 ng of ift88 MO (Fig 5D) is quite confusing because the larvae should also have been negative for Ift88-GFP and thus displayed body curvature. Secondly, some inferences are more or less logically flawed. The authors tend to use negative results on specific assays to exclude all possibilities. For instance, the negative results in Figures 4A-B are not sufficient to "suggest that the variability in IFT speeds among different cilia cannot be attributed to the use of different motor proteins" because the authors have not checked dynein-2 and other IFT kinesins. In fact, in their previous publication (Zhao et al., 2012), the authors actually demonstrated that different IFT kinesins have different effects on ciliogenesis and ciliary length in different tissues. Furthermore, instead of also examining cilia affected by Kif3b or Kif17 mutation, they only examined crista cilia, which are not sensitive to the mutations. Similarly, their results in Figures 4C-G only excluded the importance of tubulin glycylation or glutamylation in IFT. Thirdly, the conclusive model is based on certain assumptions, e.g., constant IFT velocities in a given cell type. The authors, however, do not discuss other possibilities.