Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

Center for Brain Research Medical University Vienna Vienna Austria

Center for Regenerative Therapies Technische Universität Dresden Dresden Germany

Central European Institute of Technology Brno University of Technology Brno Czech Republic

Department of Biologic and Materials Sciences University of Michigan School of Dentistry Ann Arbor United States

Department of Genetics Cell Biology and Anatomy University of Nebraska Medical Center Omaha United States

Department of Histology and Embryology Medical Faculty Masaryk University Brno Czech Republic

Department of Information Technology Uppsala University Uppsala Sweden

Department of Medicine Karolinska Institutet Stockholm Sweden

Department of Neuroscience Karolinska Institutet Stockholm Sweden

Department of Physiology and Pharmacology Karolinska Institutet Stockholm Sweden

Institute for Regenerative Medicine Sechenov 1st Moscow State Medical University Moscow Russia

John Innes Centre Norwich United Kingdom

National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda United States

Science for Life Laboratory Royal Institute of Technology Solna Sweden

Unit of Molecular Neurobiology Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden

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Abad V, Meyers JL, Weise M, Gafni RI, Barnes KM, Nilsson O, Bacher JD, Baron J. The role of the resting zone in growth plate chondrogenesis. Endocrinology. 2002;143:1851–1857. doi: 10.1210/endo.143.5.8776. PubMed DOI

Afsharpaiman S, Saburi A, Waters KA. Respiratory difficulties and breathing disorders in achondroplasia. Paediatric Respiratory Reviews. 2013;14:250–255. doi: 10.1016/j.prrv.2013.02.009. PubMed DOI

Ben Amar M, Jia F. Anisotropic growth shapes intestinal tissues during embryogenesis. PNAS. 2013;110:10525–10530. doi: 10.1073/pnas.1217391110. PubMed DOI PMC

Campinho P, Heisenberg CP. The force and effect of cell proliferation. The EMBO Journal. 2013;32:2783–2784. doi: 10.1038/emboj.2013.225. PubMed DOI PMC

Chagin AS, Vuppalapati KK, Kobayashi T, Guo J, Hirai T, Chen M, Offermanns S, Weinstein LS, Kronenberg HM. G-protein stimulatory subunit alpha and gq/11α G-proteins are both required to maintain quiescent stem-like chondrocytes. Nature Communications. 2014;5:3673. doi: 10.1038/ncomms4673. PubMed DOI PMC

Chong HJ, Young NM, Hu D, Jeong J, McMahon AP, Hallgrimsson B, Marcucio RS. Signaling by SHH rescues facial defects following blockade in the brain. Developmental Dynamics. 2012;241:247–256. doi: 10.1002/dvdy.23726. PubMed DOI PMC

De Luca F. Impaired growth plate chondrogenesis in children with chronic illnesses. Pediatric Research. 2006;59:625–629. doi: 10.1203/01.pdr.0000214966.60416.1b. PubMed DOI

Díaz-Flores L, Madrid JF, Gutiérrez R, Varela H, Valladares F, Alvarez-Argüelles H, Díaz-Flores L. Adult stem and transit-amplifying cell location. Histology and Histopathology. 2006;21:995–1027. doi: 10.14670/HH-21.995. PubMed DOI

Eames BF, Schneider RA. The genesis of cartilage size and shape during development and evolution. Development. 2008;135:3947–3958. doi: 10.1242/dev.023309. PubMed DOI PMC

Feil R, Wagner J, Metzger D, Chambon P. Regulation of cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochemical and Biophysical Research Communications. 1997;237:752–757. doi: 10.1006/bbrc.1997.7124. PubMed DOI

Fletcher AG, Osborne JM, Maini PK, Gavaghan DJ. Implementing vertex dynamics models of cell populations in biology within a consistent computational framework. Progress in Biophysics and Molecular Biology. 2013;113:299–326. doi: 10.1016/j.pbiomolbio.2013.09.003. PubMed DOI

Foppiano S, Hu D, Marcucio RS. Signaling by bone morphogenetic proteins directs formation of an ectodermal signaling center that regulates craniofacial development. Developmental Biology. 2007;312:103–114. doi: 10.1016/j.ydbio.2007.09.016. PubMed DOI PMC

Ford-Hutchinson AF, Ali Z, Lines SE, Hallgrímsson B, Boyd SK, Jirik FR. Inactivation of Pten in osteo-chondroprogenitor cells leads to epiphyseal growth plate abnormalities and skeletal overgrowth. Journal of Bone and Mineral Research. 2007;22:1245–1259. doi: 10.1359/jbmr.070420. PubMed DOI

Fritz JA, Brancale J, Tokita M, Burns KJ, Hawkins MB, Abzhanov A, Brenner MP. Shared developmental programme strongly constrains beak shape diversity in songbirds. Nature Communications. 2014;5:3700. doi: 10.1038/ncomms4700. PubMed DOI

Fukuda T, Scott G, Komatsu Y, Araya R, Kawano M, Ray MK, Yamada M, Mishina Y. Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2. Genesis. 2006;44:159–167. doi: 10.1002/dvg.20201. PubMed DOI

Gao B, Song H, Bishop K, Elliot G, Garrett L, English MA, Andre P, Robinson J, Sood R, Minami Y, Economides AN, Yang Y. Wnt signaling gradients establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2. Developmental Cell. 2011;20:163–176. doi: 10.1016/j.devcel.2011.01.001. PubMed DOI PMC

Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. Journal of Cellular Biochemistry. 2006;97:33–44. doi: 10.1002/jcb.20652. PubMed DOI

Graner F, Glazier JA. Simulation of biological cell sorting using a two-dimensional extended Potts model. Physical Review Letters. 1992;69:2013–2016. doi: 10.1103/PhysRevLett.69.2013. PubMed DOI

Green AA, Kennaway JR, Hanna AI, Bangham JA, Coen E. Genetic control of organ shape and tissue polarity. PLoS Biology. 2010;8:e1000537. doi: 10.1371/journal.pbio.1000537. PubMed DOI PMC

Guenther C, Pantalena-Filho L, Kingsley DM. Shaping skeletal growth by modular regulatory elements in the Bmp5 gene. PLoS Genetics. 2008;4:e1000308. doi: 10.1371/journal.pgen.1000308. PubMed DOI PMC

Hari L, Miescher I, Shakhova O, Suter U, Chin L, Taketo M, Richardson WD, Kessaris N, Sommer L. Temporal control of neural crest lineage generation by wnt/β-catenin signaling. Development. 2012;139:2107–2117. doi: 10.1242/dev.073064. PubMed DOI

Hayes AJ, MacPherson S, Morrison H, Dowthwaite G, Archer CW. The development of articular cartilage: evidence for an appositional growth mechanism. Anatomy and Embryology. 2001;203:469–479. doi: 10.1007/s004290100178. PubMed DOI

Hellander A. multicell. e7edeefhttps://github.com/ahellander/multicell Github. 2015

Hu D, Young NM, Xu Q, Jamniczky H, Green RM, Mio W, Marcucio RS, Hallgrimsson B. Signals from the brain induce variation in avian facial shape. Developmental Dynamics : An Official Publication of the American Association of Anatomists. 2015 doi: 10.1002/dvdy.24284. PubMed DOI PMC

Jarjour M, Jorquera A, Mondor I, Wienert S, Narang P, Coles MC, Klauschen F, Bajénoff M. Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells. The Journal of Experimental Medicine. 2014;211:1109–1122. doi: 10.1084/jem.20132409. PubMed DOI PMC

Kaucka M, Ivashkin E, Gyllborg D, Zikmund T, Tesarova M, Kaiser J, Xie M, Petersen J, Pachnis V, Nicolis SK, Yu T, Sharpe P, Arenas E, Brismar H, Blom H, Clevers H, Suter U, Chagin AS, Fried K, Hellander A, Adameyko I. Analysis of neural crest-derived clones reveals novel aspects of facial development. Science Advances. 2016;2:e1600060. doi: 10.1126/sciadv.1600060. PubMed DOI PMC

Kennaway R, Coen E, Green A, Bangham A. Generation of diverse biological forms through combinatorial interactions between tissue polarity and growth. PLoS Computational Biology. 2011;7:e1002071. doi: 10.1371/journal.pcbi.1002071. PubMed DOI PMC

Kerney RR, Brittain AL, Hall BK, Buchholz DR. Cartilage on the move: cartilage lineage tracing during tadpole metamorphosis. Development, Growth & Differentiation. 2012;54:739–752. doi: 10.1111/dgd.12002. PubMed DOI PMC

Kettunen P, Nie X, Kvinnsland IH, Luukko K. Histological development and dynamic expression of Bmp2-6 mRNAs in the embryonic and postnatal mouse cranial base. The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology. 2006;288:1250–1258. doi: 10.1002/ar.a.20402. PubMed DOI

Kobayashi S, Takebe T, Zheng YW, Mizuno M, Yabuki Y, Maegawa J, Taniguchi H. Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium. PLoS One. 2011;6:e26393. doi: 10.1371/journal.pone.0026393. PubMed DOI PMC

Kronenberg HM. Developmental regulation of the growth plate. Nature. 2003;423:332–336. doi: 10.1038/nature01657. PubMed DOI

Kuchen EE, Fox S, de Reuille PB, Kennaway R, Bensmihen S, Avondo J, Calder GM, Southam P, Robinson S, Bangham A, Coen E. Generation of leaf shape through early patterns of growth and tissue polarity. Science. 2012;335:1092–1096. doi: 10.1126/science.1214678. PubMed DOI

Laranjeira C, Sandgren K, Kessaris N, Richardson W, Potocnik A, Vanden Berghe P, Pachnis V. Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury. Journal of Clinical Investigation. 2011;121:3412–3424. doi: 10.1172/JCI58200. PubMed DOI PMC

Laurita J, Koyama E, Chin B, Taylor JA, Lakin GE, Hankenson KD, Bartlett SP, Nah HD. The Muenke syndrome mutation (FgfR3P244R) causes cranial base shortening associated with growth plate dysfunction and premature perichondrial ossification in murine basicranial synchondroses. Developmental Dynamics. 2011;240:2584–2596. doi: 10.1002/dvdy.22752. PubMed DOI

Leone DP, Genoud S, Atanasoski S, Grausenburger R, Berger P, Metzger D, Macklin WB, Chambon P, Suter U. Tamoxifen-inducible glia-specific cre mice for somatic mutagenesis in oligodendrocytes and schwann cells. Molecular and Cellular Neuroscience. 2003;22:430–440. doi: 10.1016/S1044-7431(03)00029-0. PubMed DOI

Li L, Newton PT, Bouderlique T, Sejnohova M, Zikmund T, Kozhemyakina E, Xie M, Krivanek J, Kaiser J, Qian H, Dyachuk V, Lassar AB, Warman ML, Barenius B, Adameyko I, Chagin AS. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice. The FASEB Journal. 2017;31:1067–1084. doi: 10.1096/fj.201600918R. PubMed DOI PMC

Ma W, Lozanoff S. Spatial and temporal distribution of cellular proliferation in the cranial base of normal and midfacially retrusive mice. Clinical Anatomy. 1999;12:315–325. doi: 10.1002/(SICI)1098-2353(1999)12:5<315::AID-CA2>3.0.CO;2-2. PubMed DOI

Mackie EJ, Ahmed YA, Tatarczuch L, Chen KS, Mirams M. Endochondral ossification: how cartilage is converted into bone in the developing skeleton. The International Journal of Biochemistry & Cell Biology. 2008;40:46–62. doi: 10.1016/j.biocel.2007.06.009. PubMed DOI

Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, Mackem S, Carmeliet G, Kronenberg HM. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Developmental Cell. 2010;19:329–344. doi: 10.1016/j.devcel.2010.07.010. PubMed DOI PMC

Mallarino R, Campàs O, Fritz JA, Burns KJ, Weeks OG, Brenner MP, Abzhanov A. Closely related bird species demonstrate flexibility between beak morphology and underlying developmental programs. PNAS. 2012;109:16222–16227. doi: 10.1073/pnas.1206205109. PubMed DOI PMC

McBratney-Owen B, Iseki S, Bamforth SD, Olsen BR, Morriss-Kay GM. Development and tissue origins of the mammalian cranial base. Developmental Biology. 2008;322:121–132. doi: 10.1016/j.ydbio.2008.07.016. PubMed DOI PMC

Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent cre reporter mouse. Genesis. 2007;45:593–605. doi: 10.1002/dvg.20335. PubMed DOI

Nagayama M, Iwamoto M, Hargett A, Kamiya N, Tamamura Y, Young B, Morrison T, Takeuchi H, Pacifici M, Enomoto-Iwamoto M, Koyama E. Wnt/beta-catenin signaling regulates cranial base development and growth. Journal of Dental Research. 2008;87:244–249. doi: 10.1177/154405910808700309. PubMed DOI

Nakamura E, Nguyen MT, Mackem S. Kinetics of tamoxifen-regulated cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton. Developmental Dynamics. 2006;235:2603–2612. doi: 10.1002/dvdy.20892. PubMed DOI

Nilsson O, Marino R, De Luca F, Phillip M, Baron J. Endocrine regulation of the growth plate. Hormone Research in Paediatrics. 2005;64:157–165. doi: 10.1159/000088791. PubMed DOI

Radszuweit M, Block M, Hengstler JG, Schöll E, Drasdo D. Comparing the growth kinetics of cell populations in two and three dimensions. Physical Review E. 2009;79:051907. doi: 10.1103/PhysRevE.79.051907. PubMed DOI

Repiso A, Bergantiños C, Serras F. Cell fate respecification and cell division orientation drive intercalary regeneration in Drosophila wing discs. Development. 2013;140:3541–3551. doi: 10.1242/dev.095760. PubMed DOI

Sakamoto A, Chen M, Kobayashi T, Kronenberg HM, Weinstein LS. Chondrocyte-specific knockout of the G protein G(s)alpha leads to epiphyseal and growth plate abnormalities and ectopic chondrocyte formation. Journal of Bone and Mineral Research. 2005;20:663–671. doi: 10.1359/JBMR.041210. PubMed DOI

Schötz EM, Lanio M, Talbot JA, Manning ML. Glassy dynamics in three-dimensional embryonic tissues. Journal of the Royal Society Interface. 2013;10:20130726. doi: 10.1098/rsif.2013.0726. PubMed DOI PMC

Sheehan MJ, Nachman MW. Morphological and population genomic evidence that human faces have evolved to signal individual identity. Nature Communications. 2014;5:4800. doi: 10.1038/ncomms5800. PubMed DOI PMC

Snippert HJ, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Simons BD, Clevers H. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell. 2010;143:134–144. doi: 10.1016/j.cell.2010.09.016. PubMed DOI

Strutt D. Organ shape: controlling oriented cell division. Current Biology. 2005;15:R758–R759. doi: 10.1016/j.cub.2005.08.053. PubMed DOI

Tesařová M, Zikmund T, Kaucká M, Adameyko I, Jaroš J, Paloušek D, Škaroupka D, Kaiser J. Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule. Journal of Instrumentation. 2016;11:C03006. doi: 10.1088/1748-0221/11/03/C03006. DOI

Thompson H, Ohazama A, Sharpe PT, Tucker AS. The origin of the stapes and relationship to the otic capsule and oval window. Developmental Dynamics. 2012;241:1396–1404. doi: 10.1002/dvdy.23831. PubMed DOI

Van Liedekerke P, Palm MM, Jagiella N, Drasdo D. Simulating tissue mechanics with agent-based models: concepts, perspectives and some novel results. Computational Particle Mechanics. 2015;2:401–444. doi: 10.1007/s40571-015-0082-3. DOI

Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by indian hedgehog and PTH-related protein. Science. 1996;273:613–622. doi: 10.1126/science.273.5275.613. PubMed DOI

Wang Y, Spatz MK, Kannan K, Hayk H, Avivi A, Gorivodsky M, Pines M, Yayon A, Lonai P, Givol D. A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3. PNAS. 1999;96:4455–4460. doi: 10.1073/pnas.96.8.4455. PubMed DOI PMC

Wealthall RJ, Herring SW. Endochondral ossification of the mouse nasal septum. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology. 2006;288:1163–1172. doi: 10.1002/ar.a.20385. PubMed DOI

Yamaguchi TP, Bradley A, McMahon AP, Jones S. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development. 1999;126:1211–1223. PubMed

Young B, Minugh-Purvis N, Shimo T, St-Jacques B, Iwamoto M, Enomoto-Iwamoto M, Koyama E, Pacifici M. Indian and sonic hedgehogs regulate synchondrosis growth plate and cranial base development and function. Developmental Biology. 2006;299:272–282. doi: 10.1016/j.ydbio.2006.07.028. PubMed DOI

Yu K, McGlynn S, Matise MP. Floor plate-derived sonic hedgehog regulates glial and ependymal cell fates in the developing spinal cord. Development. 2013;140:1594–1604. doi: 10.1242/dev.090845. PubMed DOI PMC

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