Craniofacial anomalies are disfiguring, debilitating birth defects that afflict a large portion of the world's population. These malformations can be caused by genetic and/or environmental factors, and one such factor, prenatal hypoxia, is of particular interest in our research on craniofacial dysmorphology. There is experimental and clinical evidence for a correlation between hypoxia and craniofacial malformations, however, the mechanisms underlying such defects are not yet understood. The goal of our research has been to understand the mechanisms whereby hypoxia causes abnormal craniofacial morphology in early embryonic development.

Chick embryos were incubated in either normoxic (21% O2) or hypoxic (7%-19% O2) conditions and collected on days 2-6 for morphological and cellular analyses. Embryos were photographed for morphological analyses, and facial shape variation was quantified via two-dimensional geometric morphometrics. 13-day embryos were cleared and stained for analysis of skeletal and cartilaginous development. Cell proliferation was assessed via BrdU staining, and apoptosis was assessed by whole-mount and section TUNEL and caspase 3 immunoassay. Embryos were examined for oxidative stress using a phospho-AMPK immunoassay.

In morphometric analyses of normal growth, older embryos (HH22-28) showed greater shape variation among embryos than younger embryos (HH16-21). All the embryos fell along a well-defined nonlinear curve of normal facial growth in relation to size, and displayed variation in relation to chronological age and discrete morphological stage. Within stages, older embryos displayed greater variation within stages than younger embryos. In studies of hypoxic embryos, their survival was reduced in a step-wise manner in comparison to normoxic control embryos. Hypoxic embryos showed a wide range of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. They also displayed delayed skull bone development, with some skeletal defects. Abnormal facial shape variation occurred in relation to centroid size and age among individuals in hypoxic groups versus the normoxic population. Hypoxia disrupted cell proliferation and caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed increased metabolic stress response.