Primary cilia in the growing limb are preferentially orientated, uncoupled from centriolar position

Reviewer #1 (Public review):

Summary:

This manuscript provides fundamental insight into ciliary biology, specifically, how ciliary axoneme orientation is governed by microenvironmental bending or intrinsic cytoskeletal steering rather than strictly basal body docking coordinates.

Strengths:

There are three major strengths in this manuscript. First, combining high-resolution imaging with deep-tissue sectioning yields impressive lateral resolution, enabling robust separation of dual centrioles within the crowded chondrocyte extracellular matrix. Second, the authors established an automated pipeline that evaluates thousands of individual cells across multiple anatomical regions and differentiation zones, lending strong statistical weight to positional and volumetric measurements. Lastly, they demonstrate that ciliation peaks in the peripheral resting zone and ciliary length peaks in hypertrophic cells, providing a compelling cellular explanation for why Ift88 deletion impacts peripheral growth plate geometry and hypertrophic expansion.

Weaknesses:

There are three major weaknesses in this manuscript. First, the paper lacks explicit descriptions of data mentioned in the Abstract and Methods, including the RNA-seq differential expression, WGCNA modules, and immobilization/ciliary alignment data. Second, while the Methods section mentions correcting for "Z-blur" / point-spread function distortion in 3D spherical coordinate calculations, further detail is required on how orientations are disambiguated from optical sectioning depth artifacts. Lastly, the RNA-seq analysis demonstrates that ambulatory unloading alters hedgehog and primary cilia gene signatures, yet axoneme orientation itself remains static. The narrative requires a clearer mechanistic synthesis regarding how mechanical loading modulates ciliary signaling if physical alignment of the cilia is refractory to mechanical force in chondrocytes.

Reviewer #2 (Public review):

Summary:

The aim of this work was to characterise in detail the cellular organisation of the growth plate in the growing mouse limb, the effect of mechanical loading due to physical activity, and the role of primary cilia in mediating this effect as putative mechanosensors. Primary cilia are generally thought to function as mechanosensors in a range of different organs and tissues, e.g. in the kidney. Exposure to mechanical loading is a normal part of post-natal limb growth, and the central hypothesis underlying this work was that primary cilia will play an important role in transducing the effects of mechanical loading into cellular responses during this process. To investigate this, the authors used a mouse model encoding fluorescent markers for primary cilia, and also allowing conditional knockout of IFT88, a protein that plays a key role in primary cilium biogenesis. The authors compared the effects of mechanical loading by comparing tissue from mice with normal or surgically immobilised limbs. To investigate the role of primary cilia, the authors performed the same experiments with mice treated with tamoxifen to induce IFT88 knockout.

Strengths:

A major strength of this work is that it studied primary cilia in a fully in vivo system. The authors employed cutting-edge imaging approaches and image analysis pipelines to rigorously study cellular organisation, centriole positioning, cilium length, and orientation across thousands of cells in limbs from 18 animals. The imaging results presented are of an exceptionally high-quality. The analysis of imaging data is extremely quantitative and robust and utilised appropriate statistical analyses, which were clearly stated throughout. The imaging data were complemented with transcriptomic data and analyses, which provided an orthogonal dimension for understanding cellular responses. An interesting and unexpected outcome from this is that the primary cilia are oriented in the same direction, and inclined at a roughly 45{degree sign} angle to the mediolateral and proximal-distal axes of the limb. The authors speculate as to how this might arise and the role it may play in sensing.

Weaknesses:

Overall, the work reported in this study was of a very high quality, and I could not find any significant shortcomings. However, I did feel that the paper was not very well written in many places, which made it difficult to read.

Overall, I think that the authors did achieve their aims in this study. It will be interesting to unpick the cellular mechanisms that lead to alignment of the cilia, the role of this alignment in mechanosensing, and the molecular mechanisms by which the cilia sense mechanical strain. Thus, this work provides a fertile ground for future studies, which will have important consequences for the study of primary cilia in vivo.

Reviewer #3 (Public review):

Summary:

This study aims to characterize the three-dimensional organization of primary cilia in the postnatal mouse growth plate and to determine how ciliary prevalence, length, position, and orientation vary across anatomical regions and stages of chondrocyte differentiation. The authors combine volumetric imaging and automated image analysis with conditional disruption of IFT88, limb immobilization, and bulk transcriptomics. The work provides a valuable anatomical dataset and identifies several interesting spatial patterns, including increased ciliation in the lateral resting zone, longer cilia in hypertrophic chondrocytes, and, most notably, a non-random orientation of ciliary projections despite a much broader distribution of centriole positions.

The descriptive evidence is largely solid, and the imaging dataset should be useful to researchers studying primary cilia, skeletal development, and tissue mechanobiology. However, the evidence is incomplete for several of the central mechanistic conclusions. In particular, the current analyses do not establish that ciliary orientation is uncoupled from basal-body position, and they leave unresolved how ciliary orientation relates to cell orientation. The immobilization experiment also supports a narrower conclusion than the proposed cell-intrinsic orientation program, while the final model linking ciliary angle to multidirectional signal integration remains speculative. Overall, the authors succeed in identifying an interesting and reproducible anatomical pattern, but the mechanism underlying that pattern remains largely open.

Strengths:

A major strength is the scale and anatomical context of the imaging. Quantifying thousands of cilia and tens of thousands of centrioles in three dimensions while preserving information about growth-plate zone and position across the limb is technically demanding. This allows the authors to identify regional differences that would be lost in dissociated cells or bulk tissue measurements.

The study also provides several potentially useful observations. Ciliation is higher in the lateral periphery, particularly in the resting zone, cilia are longer in hypertrophic chondrocytes, and ciliary projections show a reproducible non-random orientation. The latter is the most interesting result of the study and provides a useful foundation for asking how organelle orientation is established within a developing tissue.

I also appreciated that the authors place these observations in several biological contexts rather than stopping at a descriptive atlas. IFT88 disruption alters ciliation and growth-plate cell organization, while immobilization changes growth-plate dimensions, cell morphology and orientation, and the transcriptome even though the measured ciliary properties remain comparatively stable. These perturbations give the anatomical observations useful biological context.

Weaknesses

The main concern is that the claim that ciliary orientation is uncoupled from basal-body position is not directly demonstrated. The manuscript shows that centriole positions are broadly distributed and, separately, that ciliary projections have a preferred tissue-level orientation. Different population-level distributions, however, do not establish independence within individual cells. For example, basal bodies could be broadly distributed while their position determines which of two opposite directions along a common tissue axis the cilium adopts. This possibility is particularly relevant because Centrin-2 labels both centrioles, whereas only one serves as the basal body. The current data therefore support a difference between the population distributions of position and orientation, but not yet the stronger claim that the two are uncoupled.

A related gap is the relationship between cell orientation and ciliary orientation. The manuscript measures both, and cell orientation changes with growth-plate region, IFT88 deletion, and immobilization, while ciliary orientation appears relatively stable. Yet the two measurements are never directly related within the same cells. It is therefore unclear whether cilia adopt a reproducible angle relative to the major axis of their own cell, whether this relationship changes between the center and periphery, or whether changes in cell organization can occur independently of local ciliary alignment. This seems important for interpreting the tissue-level orientation pattern.

The statistical treatment of orientation also deserves caution. These are circular or spherical measurements, yet much of the analysis relies on linear distributions and Kolmogorov-Smirnov tests. This is particularly problematic around the 0{degree sign}/360{degree sign} boundary, where values near 350{degree sign} and 30{degree sign} are geometrically close but appear separated in a linear representation. The manuscript also does not clearly distinguish between a preferred axis, where opposite directions are equivalent, and a preferred polarity, where one direction is favored. In addition, thousands of cilia are nested within a much smaller number of mice, so the apparent statistical power should not be driven primarily by pooled object counts. Given that regional differences are a central theme of the paper, it would also be useful to know more clearly whether the preferred axis or the strength of the orientation bias differs between the middle and lateral growth plate at the animal level. I also could not identify a formal comparison of the centriole or ciliary distributions with an appropriate uniform circular or spherical null. The reported tests mainly compare zones and regions, so the claims that centriole position is non-preferential and ciliary orientation is non-random are not yet statistically established in the form presented.

The IFT88 conditional knockout is not carried through to the principal orientation question. The authors examine cell size, cell-axis organization, ciliation, and cilium length, but do not report whether the remaining cilia retain the preferred orientation or whether centriole positioning changes. Given the central role of this genetic perturbation in the manuscript, this leaves the genetic and orientation arms of the study somewhat disconnected.

Finally, the immobilization experiment and the mechanistic interpretation should be separated more carefully. Only four animals were analyzed in the offloaded and contralateral conditions, and medial and lateral regions were averaged because of the small sample size. The experiment shows that the established ciliary orientation remains relatively stable over a two-week postnatal interval despite clear changes elsewhere in the tissue. It does not exclude a role for mechanical forces earlier in establishing the axis, nor does a nonsignificant difference with four animals demonstrate equivalence. Similarly, the proposed cell-intrinsic orientation program and the final "single-axis blindness" model are interesting hypotheses, but the study does not yet identify what establishes the axis or how the observed ciliary angle would alter sensitivity to a force or biochemical gradient. Those ideas are worth discussing, but they should remain clearly separated from the observations directly supported by the data.

Author response:

In response to the valuable reviewers’ comments and suggested changes, we are finalising changes to the manuscript, in order to resubmit a revised version, and a document with full author responses, that reflects all the review comments.

These changes include adding points of clarity, improving accuracy on wording of key messages, adding additional interpretation of the data, including additional data and analyses that reflect open questions raised, and more discussion concerning these unanswered questions, which are subjects of future work.

In response to specific points we were asked to provisionally address (actions in italics):

Reviewer #1. We are pleased the reviewer sees the insight these data bring. We indeed think it likely that cilia axoneme orientation is governed by the immediate microenvironment and/or changes to the cytoskeleton and are actively looking to explore this.

To address the 3 areas of concern:

(1) Our re-writing of the abstract and the results concerning transcriptomic data seeks to overcome weaknesses in descriptions of these data.

(2) Further detail is being added on how z-distortion is corrected for, so accuracy is the same in all axis and orientation measurements are robust.

(3) We will add to the discussion to add our thoughts as to why ciliary and cilia signalling genes are regulated by immobilisation, but that immobilisation does not apparently affect cilia structure.

Reviewer #2. Thank you for such broadly positive comments, we are pleased the scale and depth of the quantitative analyses comes across, but will make sure that revisions throughout improve the quality of the writing describing these. We are actively exploring means to test ideas for how cilia axoneme become orientated in this way and what the function is. These preliminary ideas will be reflected in the discussion.

Reviewer #3. Thank you for such a detailed and thoughtful review. They will ensure the data are presented to their very best and we will address the concerns raised. This descriptive study had a hypothesis, and made discoveries which surprised us, we have tried, as you say, to put this in some context of the role of cilia and the role of mechanical forces in GP biology. Most notably we are considering that uncoupled is not the correct term here. To address areas of concern;

(1) We agree ‘uncoupled’ is not the correct word here. We cannot find a pattern of correlation between centriole position and orientation. However, the two can’t be ‘uncoupled’ and there is no proven independence on a single-cell level (that cilia position and cilia orientation are not in any way mechanistically linked). We will make changes and add more details on what we have considered in this area.

(2) Similarly, we have not correlated in each cell, cellular orientation and ciliary orientation, though we have made attempts and not yet found a relationship. However, again, this is not the same as one being absent. We might have expected ciliary orientation to change as cell orientation does (through zones or with pertubations) as we have seen in vitro but this remains to be fully explored and is one subject of follow-up work. We are considering column populations and per animal considerations of the data.

(3) We did take a cautious approach to statistics and specialist advice, but advice was not to overcomplicate things when there are 2 main messages related to centriole position and cilia orientation. Firstly, centriolar position appears random or without preference thus distribution of position on cell, is homogenous. Second, ciliary orientation angle is not a homogenous distribution, as would be expected if random with this number of measurements. We do, and will add comments to this effect, have to mindful of large dataset, but do not think this means we are looking at false discoveries due to number of comparisons. We are considering how better to reflect this. To address these important points we will add a section to the methods and discussion and will endeavour to change results to this end as it is a central point of the manuscript.

(4) We will add new data concerning IFT88cKO and centriole position and orientation.

(5) We will add a critique of the immobilisation experiments to ensure the relatively diminished power is clear. We agree force may have set things up initially, we will ensure this is discussed and we will ensure our proposal for why cilia orientation is this way is framed as a hypothesis. We have preliminary data, but this is the subject of an entire new project thus not yet supported by robust experimental evidence so is speculative at this stage and we will ensure this is clear.