A Ciliary Protein EVC2/LIMBIN Plays a Critical Role in the Skull Base for Mid-Facial Development

Abstract

Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplastic disorder. Affected patients present a wide spectrum of symptoms including short stature, postaxial polydactyly, and dental abnormalities. We previously disrupted Evc2, one of the causative genes for EvC syndrome, in mice using a neural crest-specific, Cre-mediated approach (i.e., P0-Cre, referred to as Evc2 P0 mutants). Despite the fact that P0-Cre predominantly targets the mid-facial region, we reported that many mid-facial defects identified in Evc2 global mutants are not present in Evc2 P0 mutants at postnatal day 8 (P8). In the current study, we used multiple Cre lines (P0-Cre and Wnt1-Cre, respectively), to specifically delete Evc2 in neural crest-derived tissues and compared the resulting mid-facial defects at multiple time points (P8 and P28, respectively). While both Cre lines indistinguishably targeted the mid-facial region, they differentially targeted the anterior portion of the skull base. By comprehensively analyzing the shapes of conditional mutant skulls, we detected differentially affected mid-facial defects in Evc2 P0 mutants and Evc2 Wnt1 mutants. Micro-CT analysis of the skull base further revealed that the Evc2 mutation leads to a differentially affected skull base, caused by premature closure of the intersphenoid synchondrosis (presphenoidal synchondrosis), which limited the elongation of the anterior skull base during the postnatal development of the skull. Given the importance of the skull base in mid-facial bone development, our results suggest that loss of function of Evc2 within the skull base secondarily leads to many aspects of the mid-facial defects developed by the EvC syndrome.

Human–chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution

“…6 , Supplementary Tables 21 – 23 ). Importantly, the key phenotypes that are often used to describe pronounced differences in facial shape between humans and chimpanzees (specifically, midfacial retrusion with a more downward facial trajectory 1 ) are observed in both the current and previous Evc2 KO studies 64 , 65 , 68 , as well as in Ellis-van Creveld patients 47 , 65 ( Fig. 6 , Extended Data Fig.…”

“…Studies in cattle and mice have shown that the role of EVC2 is conserved in these mammals 55 , 63 – 68 . In humans, homozygous loss-of-function mutations in either EVC2 or EVC cause the Ellis-van Creveld syndrome.…”

“…This leads to a shortened cranial base, which in turn drives midfacial retraction 72 . Interestingly, EVC2 plays a key role in the development of these cartilaginous joints 61 , 68 . Indeed, Evc2 loss in mouse CNCCs causes early cessation of growth in the cranial base joints, leading to a shortened cranial base and a retracted midface.…”

Molecular and Cellular Pathogenesis of Ellis-van Creveld Syndrome: Lessons from Targeted and Natural Mutations in Animal Models

“…Many elements within the craniofacial region, specifically the neurocranium and viscerocranium, arise from neural crest cells (NCCs), a special population of cells that maintain their identity despite divergence from the ectoderm in early gastrulation [52]. Abnormal craniofacial morphology (i.e., midfacial depression) in both global and NCC-specific Evc2/Limbin deficient mice (referred to as Evc2/Limbin-KO and Evc2/Limbin-cKO, respectively) is a consequence of abnormalities within NCC-derived elements of the neurocranium and viscerocranium [50,51,53,54]. As with patients with EVC, midfacial depression or retrusion in mice becomes more pronounced with age and can be attributed to positional differences between the nasal bone, jaws, and cranial base [51,53].…”

“…Abnormal craniofacial morphology (i.e., midfacial depression) in both global and NCC-specific Evc2/Limbin deficient mice (referred to as Evc2/Limbin-KO and Evc2/Limbin-cKO, respectively) is a consequence of abnormalities within NCC-derived elements of the neurocranium and viscerocranium [50,51,53,54]. As with patients with EVC, midfacial depression or retrusion in mice becomes more pronounced with age and can be attributed to positional differences between the nasal bone, jaws, and cranial base [51,53]. Though this suggests a critical, NCC-specific role for Evc2/Limbin in determining postnatal craniofacial morphology, it does not indicate which structure (i.e., maxilla vs. skull base) is responsible for the resultant phenotype.…”