Aberrant cell segregation in craniofacial primordia and the emergence of facial dysmorphology in craniofrontonasal syndrome

Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder characterized by craniofacial, skeletal, and neurological anomalies and caused by mutations in EFNB1. Heterozygous females are more severely affected by CFNS than hemizygous male patients, a phenomenon called cellular interference that is correlated with cell segregation resulting from EPHRIN-B1 mosaicism. Efnb1 heterozygous mutant mice also exhibit more severe phenotypes than Efnb1 hemizygous males as well as cell segregation, but how craniofacial dysmorphology arises from cell segregation is unknown and CFNS etiology therefore remains poorly understood. Here, we couple geometric morphometric techniques with temporal and spatial interrogation of embryonic cell segregation in mouse models to elucidate mechanisms underlying CFNS pathogenesis. By generating ephrin-B1 mosaicism at different developmental timepoints and in specific cell populations, we find that ephrin-B1 regulates cell segregation independently in early neural development and later in craniofacial development, correlating with the emergence of quantitative differences in face shape. Whereas specific craniofacial shape changes are qualitatively similar in Efnb1 heterozygous and hemizygous mutant embryos, heterozygous embryos are quantitatively more severely affected, indicating that Efnb1 mosaicism exacerbates loss of function phenotypes rather than having a neomorphic effect. Notably, tissue-specific disruption of Efnb1 throughout neural development does not appear to contribute to CFNS dysmorphology, but its disruption within neural crest cell-derived mesenchyme results in phenotypes very similar to widespread loss. Ephrin-B1 can bind and signal with EphB1, EphB2, and EphB3 receptor tyrosine kinases, but the signaling partner(s) relevant to CFNS are unknown. Geometric morphometric analysis of an allelic series of Ephb1; Ephb2; Ephb3 mutant embryos indicates that EphB2 and EphB3 are key receptors mediating Efnb1 hemizygous-like phenotypes, but the complete loss of EphB1-3 does not recapitulate CFNS-like Efnb1 heterozygous severity. Finally, by generating Efnb1+/-; Ephb1; Ephb2; Ephb3 quadruple knockout mice, we determine how modulating cumulative receptor activity influences cell segregation in craniofacial development and find that while EphB2 and EphB3 play an important role in craniofacial cell segregation, EphB1 is more important for cell segregation in the brain; surprisingly, complete loss of EphB1-EphB3 does not completely abrogate cell segregation. Together, these data advance our understanding of the morphogenetic etiology and signaling interactions underlying CFNS dysmorphology.Author SummaryCraniofacial anomalies are extremely common, accounting for one third of all birth defects, but even when the responsible genes are known, it often remains to be determined exactly how development has gone wrong. Craniofrontonasal syndrome (CFNS), which affects multiple aspects of craniofacial development, is a particularly mysterious disorder because it is X-linked, but affects females more severely than males, the opposite situation of most X-linked diseases. The responsible gene has been identified as EFNB1, which encodes the EPHRIN-B1 signaling molecule that regulates cellular position. Why EFNB1+/- heterozygous females exhibit severe stereotypical CFNS phenotypes is not well understood, but it is related to the fact that X chromosome inactivation generates mosaicism for EPHRIN-B1. Using mice harboring mutations in the Efnb1 gene in different embryonic tissues, and in receptor genes Ephb1-3, together with quantitative methods to measure craniofacial structures in developing embryos, we establish the tissue-specific contributions of ephrin-B1 mosaicism to craniofacial dysmorphology. We also examine when ephrin-B1 regulates cellular position during different stages of craniofacial development and which EphB receptors are involved. Our results reveal the specific cellular context and signaling interactions that are likely to underlie CFNS, and provide new understanding of how EPHRIN-B1 may regulate normal craniofacial development.