Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Identification of Mandibular Growth Center via Clonal Cell Analysis
Irving TC Ling, MBBS, MRCSEd, Lucie Rochard, PhD, Eric C. Liao, MD, PhD.
Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

PURPOSE:
The growth centers of craniofacial bones have long been hypothesized but never directly visualized. Patterning of the mandible requires migration of cranial neural crest (CNC) cells and involves progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described the CNC migration in the morphogenesis of the mandible but the details of the chondrocytes organization in growth and extension of this facial skeleton remains unclear. Identifying the location of the growth centers of the mandible and how the cells move during maturation of the Meckel’s cartilage is critical to understanding mandibular growth and pathogenesis of congenital malformations affecting mandibular size.
METHODS:
Taking advantage of the Zebrabow transgenic line and a chemically (tamoxifen) inducible sox10:ErtCre transgenic line (Fig 1A), we are able to label developing chondrocytes during jaw morphogenesis using confocal live imaging. The Zebrabow-M transgenic line of zebrafish carries different fluorophores under the ubi promoter separated by lox sites (Fig 1A). The Zebrabow embryo expresses Red Fluorescent Protein (RFP), but after induction of Cre expression the zebrabow construction undergoes recombination and cells express colored fluorophore in a stochastic fashion, so a combination of different fluorophores (Red (RFP), Cyan (CFP) and Yellow (YFP)) but each progeny cell retains the color obtained at the time of recombination event. This technique enables clonal lineage mapping in the live embryo.
RESULTS:
Multispectral cell labeling demonstrated unidirectional extension of the Meckel’s chondrocytes in a proximal to distal direction, emanating from specified paired growth centers near the condyles that coalesce from the leading domains of the mandibular prominence, derived from the first pharyngeal arch. During extension phase of the Meckel’s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single cell layered row (Fig 1B, 1C, 1D). Failure of this organized intercalating process may have implications in disease process causing mandibular malformations.
CONCLUSION:
Using drug inducible and lineage restricted Cre recombinase in the transgenic and sequentially floxed multispectral transgenic, we are able to carry out multispectral clonal cell labeling. This advanced technology enabled us to for the first time demonstrate cellular proliferation and intercalation in the embryonic mandible precursor. This work explains the embryonic origin of the mandible and helps us consider craniofacial malformations that involve the mandible.


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