Plastic Surgery Research Council

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ALX1 Regulates PAX3 to Enable Cranial Neural Crest Migration During Craniofacial Development
Yiyuan Hu, B.A.1, Jonathan Pini, Ph.D.1, Janina Kueper, M.D.1, Brahm Agnihotri1, Richard Maas, M.D., Ph.D.2, Eric C. Liao, M.D., Ph.D.1.
1Massachusetts General Hospital, Boston, MA, USA, 2Brigham and Women's Hospital, Boston, MA, USA.

PURPOSE: Migration and fusion of the frontonasal and paired maxillary prominences are crucial to the formation of the midface. Disruption of this developmental process leads to severe craniofacial anomalies, such as frontonasal dysplasia (FND). Using induced pluripotent stem cells (iPSCs) derived from FND subjects with a heritable ALX1 L165F gene variant, we leveraged a novel method of cranial neural crest cell (CNCC) differentiation to study ALX1 function and interaction with PAX3, delineating a transcriptional regulatory pathway crucial to CNCC differentiation.
METHODS: Wildtype and ALX1-/- iPSCs were differentiated into the CNCC lineage through the addition of epithelial growth factor into DMEM F12 medium. Subsequent cells were assayed for their CNCC properties and ability to multi-lineage differentiate into adipocytes, Schwann cells, chondrocytes, and osteoblasts. We then accessed surface marker expression, and sensitivity to apoptosis between wildtype and ALX1-/- CNCCs using FACS and migration assay. Differential gene expression in iPSC and alx1-/- zebrafish embryos was analyzed using qPCR. For in vivo analysis of CNCC migration, wild-type, alx1-/- and pax3 injected zebrafish embryos were analyzed using sox10:KAEDE transgenic line.
RESULTS: Through analysis of CNCC markers CD90, CD73, CD105, CD57, cellular morphology, gene expression data, we demonstrate that our protocol was able to differentiate iPSCs into CNCCs. Both ALX1-/- and wildtype cells were capable of differentiating into adipocytes, Schwann cells, chondrocytes, and osteoblasts. However, after CNCC differentiation, while wildtype cells were able to further engage in the CNCC lineage through CD57 down-regulation, ALX1-/- NCCs maintained a high level of CD57. ALX1-/- neural crest cells were also more sensitive to apoptotic stress and experienced migratory impairment. qPCR analysis of ALX1-/- iPSCs and alx1-/- zebrafish embryos revealed an overexpression of PAX3 during CNCC differentiation and embryonic development. Conversely, overexpression of PAX3 into control CNCCs impaired migration in iPSC model. Overexpression of pax3a/b mRNA in zebrafish embryos phenocopied the craniofacial anomalies seen in alx1 -/- mutants, and injection of a dominant-negative alx1 (alx1DN) variant impaired migration of CNCCs to the central midface.
CONCLUSION: Studies of FND using complementary iPSC and zebrafish models revealed that ALX1 down-regulates PAX3 to modulate CNCC migration and cell maturation. Disruption of ALX1 resulted in unsuppressed PAX3 expression, which caused NCCs to be unable to persist in a progenitor state, more sensitive to apoptosis, and unable to properly migrate. Discordant neural crest cell differentiation and migration resulted in a decreased contribution of NCC to the frontonasal prominences and midface deformities. These studies revealed requirement of ALX1 in transcriptional regulation of midface development. This study elucidated the molecular and cellular basis of FND pathogenesis, advancing craniofacial malformations from description of affected anatomy to fundamental understanding.


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