Morphometric Analysis of Efnb1 Dependent Palate Growth

Abstract

Congenital craniofacial anomalies represent one of the most prevalent types of structural birth defects and include common dysmorphology such as cleft palate. While morphometric analysis of surfaces generated from micro computed tomography (micro‐CT) and magnetic resonance imaging (MRI) is a well‐established method, it has not yet been applied to understanding the three‐dimensional changes in palatal shelf morphology that result in a cleft palate phenotype. EFNB1 mutations result in craniofrontonasal syndrome, an X linked condition, consisting of a variety of craniofacial anomalies, including cleft palate. Mouse models for loss of Efnb1, have demonstrated its importance during normal development of the anterior secondary palate, with loss of Efnb1 resulting in changes in secondary palatal shelf shape including apparently reduced anterior palatal shelf extension, leading to a cleft secondary palate. Palatal shelf shape appears more disrupted in Efnb1+/− embryos, which are mosaic for EPHRIN‐B1 expression, but how this mosaicism contributes to palate dysmorphology has been difficult to understand in two dimensions. This study aims to establish a method of quantifying secondary palatal shelf outgrowth and shape that is coupled with an ability to quantify gene expression changes through the use of morphometrics. We performed whole‐mount immunofluorescent staining of E‐CADHERIN, EPHRIN‐B1, and DAPI followed by confocal microscopy to generate 3D renderings that we quantify using landmark based morphometrics. This method allows a deeper understanding of changes in tissue shape as they relate to gene expression.Support or Funding InformationThis study was supported by NIH grant R01DE023337 from NIDCR