Y. Hu1, J. Pini1, E. Liao1 1Massachusetts General Hospital,Center For Regenerative Medicine,Boston, MA, USA
Introduction:
Frontonasal dysplasia (FND) is a class of congenital disorders caused by incomplete growth and fusion of the central frontonasal protrusion with the paired maxillary facial prominences. Genetic etiologies of FND are unclear, but mutations in ALX1 are associated with FND. ALX1 is expressed in the facial mesenchyme of vertebrate embryos, and in cranial neural crest cells (NCCs) and its cartilage derivatives. However, little is known about this transcription factor’s role during craniofacial development.
Methods:
Using a human pedigree of 4 subjects in a family with heritable FND, we defined an ALX1 gene variant (A, B). Control and patient blood samples were used to generate induced Pluripotent Stem Cells (iPSCs) using Sendai virus transfection of Yamanaka factors. iPSCs were characterized based on their embryonic stem cells (ESCs) properties. Moreover, karyotype, and ALX1 gene sequencing were performed to finalize the iPSCs characterization. Further, iPSCs were differentiated to neural crest cells to study the role of ALX1 in craniofacial formation. To study the requirement of ALX1 in an in-vivo context, CRISPR Cas9 mutagensis and dominant-negative alx1 mRNA overexpression were employed to create alx1 mutant zebrafish. Cell migration, differentiation and gene expression were examined in the mutant alx1 embryos.
Results:
Control and ALX1-/- iPSCs displayed embryonic stem cells properties, such as self-renewal and ESC markers expression. iPSCs from both genotypes displayed normal karyotype. Whole exome sequencing revealed a pathogenic L165F variant. The iPSC were differentiated into neural crest cells (C), and demonstrated phenotypes that reveal their mechanism of developmental anomaly. Notably, we found abnormal expression of neural crest markers and an increase in the sensitivity of these differentiating cells to apoptotic stress (D).
In studying our zebrafish models, we were able to recapitulate our findings in-vitro and observed phenotypes of palate and eye malformation (E).
Conclusion:
Studies of FND using complementary iPSC and zebrafish models revealed that ALX1 is dispensable for iPSC differentiation into neural crest, but exhibit increased apoptosis during the differentiation process, leading to a fewer number of NCC progenitor cells that contribute to craniofacial morphogenesis. These studies revealed the requirement of ALX1 in craniofacial regulatory networks and provide a framework for how patient derived iPSC can be used to model craniofacial anomalies. Further study of ALX1 function and identification of its target genes will elucidate frontonasal development and innovate future treatments.
