Abstract
The wide diversity of skeletal proportions in mammals is evident upon a survey of any natural history museum's collections and allows us to distinguish between species even when reduced to their calcified components. Similarly, each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation1,2,3,4,5,6,7. However, the mechanisms of chondrocyte volume enlargement have remained a mystery8,9,10,11. Here we use quantitative phase microscopy12 to show that mammalian chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types13, this is a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through a mechanism dependent on insulin-like growth factor. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify and establish a volume set point.
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Acknowledgements
We would like to thank T. J. Mitchison, C. E. Farnum and members of the Developmental Bone Morphogenesis program project grant (National Institutes of Health (NIH)) for helpful discussions. We also thank the Nikon Imaging Center at Harvard Medical School for technical support, A. Luyten and R. Shivdasani for providing mouse megakaryocytes and P. Ramirez for jerboa care. This work was supported by NIH grants P01DK056246 to C.J.T.; R01GM026875 to M.W.K.; and by NIH grant P41RR02594, National Science Foundation (NSF) grant DBI0754339 and support from the Hamamatsu Corporation to R.R.D.
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K.L.C. and S.O. conceived the project and carried out most of the experiments. Y.S. and R.R.D. carried out critical tomographic experiments validating the primary approaches taken. C.J.T. and M.W.K. supervised the project. K.L.C., S.O., M.W.K. and C.J.T. wrote the manuscript.
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This file contains Supplementary Figures 1-14, Supplementary Methods, and Data , Supplementary Table 1 and Supplementary References. (PDF 3883 kb)
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Cooper, K., Oh, S., Sung, Y. et al. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions. Nature 495, 375–378 (2013). https://doi.org/10.1038/nature11940
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DOI: https://doi.org/10.1038/nature11940
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