Limited column formation in the embryonic growth plate implies divergent growth mechanisms during pre- and postnatal bone development
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eLife assessment
The study presents a valuable finding on quantifying the orientation and organization of chondrocyte columns in the prenatal and postnatal growth plate cartilage using advanced 3D imaging and a sophisticated image analysis pipeline. The evidence supporting the authors' conclusions regarding the lack of columns in the fetal growth plate is considered inadequate due to technical caveats, inconsistencies in the data and corresponding model, and failure to correctly put the findings in context.
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Abstract
Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. In this work, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Finally, correlation between the temporal dynamics of the ratios between clusters and columns and between bone elongation and expansion suggests that clusters may promote expansion, whereas columns support elongation. Overall, our findings support the idea that modulations of division plane rotation of proliferating chondrocytes determines the formation of either clusters or columns, a multifunctional design that regulates morphogenesis throughout pre- and postnatal bone growth. Broadly, this work provides a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.
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eLife assessment
The study presents a valuable finding on quantifying the orientation and organization of chondrocyte columns in the prenatal and postnatal growth plate cartilage using advanced 3D imaging and a sophisticated image analysis pipeline. The evidence supporting the authors' conclusions regarding the lack of columns in the fetal growth plate is considered inadequate due to technical caveats, inconsistencies in the data and corresponding model, and failure to correctly put the findings in context.
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Reviewer #1 (Public Review):
Rubin et al. study chondrocyte columns in the prenatal and postnatal growth plate in 3D for the first time, using a novel analysis pipeline in which Confetti clones in the murine growth plate are analysed morphometrically. Prenatal chondrocytes were found not to be organised in columns parallel to the main orientation of the long bone, but rather, prenatal chondrocytes were commonly organised perpendicular to the main direction of growth. In the postnatal (P40) growth plate there was a diverse arrangement of columns, but more of the columns were vertically aligned
I enjoyed reading the work and the analysis is rigorous. However, I think that it is not valid to state that columns do not form in the embryo. The data only supports the finding that strictly vertical columns do not form in the embryo, as the …
Reviewer #1 (Public Review):
Rubin et al. study chondrocyte columns in the prenatal and postnatal growth plate in 3D for the first time, using a novel analysis pipeline in which Confetti clones in the murine growth plate are analysed morphometrically. Prenatal chondrocytes were found not to be organised in columns parallel to the main orientation of the long bone, but rather, prenatal chondrocytes were commonly organised perpendicular to the main direction of growth. In the postnatal (P40) growth plate there was a diverse arrangement of columns, but more of the columns were vertically aligned
I enjoyed reading the work and the analysis is rigorous. However, I think that it is not valid to state that columns do not form in the embryo. The data only supports the finding that strictly vertical columns do not form in the embryo, as the cells are still organised into columns, albeit with a range of orientations. I do not like the term "typically" aligned, as how can we know what is "typical" when orientation has never before been assessed in 3D... And the authors' data demonstrates that it is certainly not "typical" for chondrocyte to organise into vertical columns prenatally.
It would be very interesting to delve deeper into the reason for the change in orientation of columns between pre- and post-natal. For example, does more circumferential growth happen prenatally as compared to postnatally? Is the rate of circumferential vs longitudinal growth different between prenatal and postnatal, and could the change in column orientation be responsible for a (possible) shift in the balance between longitudinal vs circumferential growth before vs after birth? The first sentence of the Discussion refers to the role of chondrocyte columns in driving bone elongation, but aren't they also involved in driving bone morphology?
I feel describing the activity of the cells as "mis-rotations" which implies the orientations are not intentional. It is likely not accidental or mistaken that the chondrocytes align in the ways they do- the diaphysis is largely for longitudinal growth while the epiphyses, and lateral expansion of the joint is also important. I find the data in Figure 4 fascinating, especially the variation in orientations between the regions of the growth plate (from proximal to distal), with the most lateral orientation at the most proximal and distal ends- it would be nice to see more discussion of these variations and what they may be contributing to.
The abstract focuses solely on the analysis of columns prenatally and would benefit from the inclusion of the data from the postnatal growth plate and from the chondrocyte rotations.
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Reviewer #2 (Public Review):
The origin and function of proliferative chondrocyte columns in the growth plate that are generally aligned with predicted longitudinal growth vectors have been robustly debated since the implementation of clonal analysis and live cell imaging techniques more than a decade ago. In particular, live cell imaging demonstrated that in the proliferative zone, most daughter pairs rotate fully or partially after division to form columns of stacked cells and a minority of pairs fail to rotate. These observations and others led to a mechanistic model of column formation, but limitations in the live cell imaging methods that only visualize a single round of division and rotation left open an important question - what is the effect of different rotation profiles on column formation, bone growth, and morphology?
This …
Reviewer #2 (Public Review):
The origin and function of proliferative chondrocyte columns in the growth plate that are generally aligned with predicted longitudinal growth vectors have been robustly debated since the implementation of clonal analysis and live cell imaging techniques more than a decade ago. In particular, live cell imaging demonstrated that in the proliferative zone, most daughter pairs rotate fully or partially after division to form columns of stacked cells and a minority of pairs fail to rotate. These observations and others led to a mechanistic model of column formation, but limitations in the live cell imaging methods that only visualize a single round of division and rotation left open an important question - what is the effect of different rotation profiles on column formation, bone growth, and morphology?
This manuscript describes the use of an inducible lineage tracing system in the mouse combined with a novel image analysis pipeline to analyze column formation over multiple cell divisions. The main conclusion is that many clones generate single columns in postnatal mice (as expected), but clones in embryonic growth plate cartilage form clusters distributed laterally, not aligned with longitudinal growth. These findings are interpreted to suggest that column formation is not required for long bone growth in the embryo and that lateral expansion of proliferative chondrocyte clusters may drive an increase in bone width.
Although these findings are intriguing and potentially impactful, there are important caveats to the approach that generate significant uncertainty in both the measurements and the conclusions. (1) The claim that embryonic growth plate chondrocytes do not form columns conflicts with the observation of columnar stacks in the clusters. (2) Interpretation of nuclear elevation data is based on the unproven assumption that nuclei should be stacked in cell columns. (3) Clonal analysis of proliferative chondrocyte cell division and stacking behaviors is only valid if clone labeling is initiated in a proliferative chondrocyte, not when the founder cell is a resting chondrocyte. The data are insufficient to validate this absolute requirement.
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Reviewer #3 (Public Review):
The manuscript by Rubin and Agrawal et al presents a very nice imaging analysis of clonal cell organization in the fetal and late juvenile mouse growth cartilages. The authors have performed a thorough quantification of the orientations of clusters and of clones of cells with respect to the growth axis. They conclude that growth cartilage is not as strictly 'columnar' as has been commonly described, especially at the fetal stage. There is value to having such quantifications in the literature as a reminder that interpretations of phenotypes need to be rooted in the cell biology of the stage at hand, as emphasized by the authors. However, although the approach is comprehensive, aspects of the quantification methods are not described adequately to determine if they are correct for the questions. There are also …
Reviewer #3 (Public Review):
The manuscript by Rubin and Agrawal et al presents a very nice imaging analysis of clonal cell organization in the fetal and late juvenile mouse growth cartilages. The authors have performed a thorough quantification of the orientations of clusters and of clones of cells with respect to the growth axis. They conclude that growth cartilage is not as strictly 'columnar' as has been commonly described, especially at the fetal stage. There is value to having such quantifications in the literature as a reminder that interpretations of phenotypes need to be rooted in the cell biology of the stage at hand, as emphasized by the authors. However, although the approach is comprehensive, aspects of the quantification methods are not described adequately to determine if they are correct for the questions. There are also some inequivalent comparisons to prior literature and an oversight of important published observations showing that some of these conclusions have been known for decades, though not as thoroughly quantitative. There have long been observations that some growth cartilages do not have proliferative columns oriented in the axis of growth and that not all columns of a growth cartilage are perfectly organized; these facts do not negate the observations that columnar organization does exist, as re-confirmed here, and that it correlates with and contributes to rapid growth rates. Each of these points is further elaborated below.
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