Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

Central European Institute of Technology Brno University of Technology 616 00 Brno Czech Republic

Department of Biology Institute of Molecular Health Sciences ETH Zurich Zurich CH 8093 Switzerland

Department of Biotechnology and Biosciences University of Milano Bicocca 20126 Milano Italy

Department of Craniofacial Development and Stem Cell Biology King's College London Dental Institute Guy's Hospital London SE1 9RT UK

Department of Information Technology Uppsala University Uppsala SE 751 05 Sweden

Department of Molecular Neurosciences Medical University of Vienna Vienna 1190 Austria

Department of Neuroscience Karolinska Institutet Stockholm SE 171 77 Sweden

Department of Physiology and Pharmacology Karolinska Institutet Stockholm SE 171 77 Sweden

Department of Physiology and Pharmacology Karolinska Institutet Stockholm SE 171 77 Sweden ; Department of Molecular Neurosciences Medical University of Vienna Vienna 1190 Austria

Department of Physiology and Pharmacology Karolinska Institutet Stockholm SE 171 77 Sweden ; Research Center of Neurology 125367 Moscow Russia

Division of Molecular Neurobiology Medical Research Council National Institute for Medical Research London NW7 1AA UK

Hubrecht Institute of the Royal Netherlands Academy of Arts and Sciences Princess Maxima Centre and University Medical Centre Utrecht 3584 Utrecht Netherlands

Science for Life Laboratory Royal Institute of Technology Solna 17121 Sweden

Unit of Molecular Neurobiology Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm SE 171 77 Sweden

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