Hypoxia Affects the Ethmoid Plate and Causes Clefts in Zebrafish

Method: Groups of zebrafish embryos were subjected to 30% (n=16), 50% (n=14), and 80% (n=17) hypoxia at 8 hours post fertilization for 24 hours. Control (embryos under normoxia), 30%, 50%, and 80% groups were fixed 7 days post fertilization using 4% paraformaldehyde. Zebrafish craniofacial cartilage was stained by Alcian blue to reveal the effects of low oxygen. Individual fish were visualized under a microscope following the subsequent removal of the mandible. Measurements of the length of the cleft in the anterior portion of the ethmoid plate were performed using ImageJ software and analyzed in the GraphPad Prism 5. Group data were compared using one-way ANOVA and post-hoc Tukey tests. 

Result: A significant reduction in the length of the anterior portion of the ethmoid plate, producing a cleft phenotype, were observed in the 30% (0.147 mm ± 0.011; p<0.0001), 50% (0.116 mm ± 0.024; p<0.0001), and 80% (0.140 mm ± 0.036; p<0.0001) hypoxia groups compared to the control (0.208 mm ± 0.010; p<0.0001). The size of the cleft in the ethmoid plate increased as the % hypoxia increased, except in the 80% hypoxia group when compared to the 50% hypoxia group.

Conclusion: Cleft in the medial portion of the ethmoid plate is associated with a hypoxic environment during zebrafish development. This mechanism may be similar to cleft palate formation in humans due to maternal cigarette smoking and other sources of hypoxia. This project was supported by the University of Pittsburgh Central Research Development Fund (CRDF).