Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2% to 7.4% in at least 12 chicken strains worldwide. To reveal the intrinsic factors causing crossed beaks, genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present studies. Crossed beak was induced by short length of unilateral mandibular ramus, and a total of 110 differentially expressed genes were up- or down-regulated in the affected (short) mandibular condyle side as compared to the normal side. Carbonic anhydrase 2 (CA2) and Carbonic anhydrase 13 (CA13) were enriched in the carbonate dehydratase activity, and high-expressed in mandibular condyle and osteoblasts (P < 0.05). However, both were low-expressed in short mandibular condyle side of affected birds (P < 0.05). The carbonate dehydratase inhibitor experiments confirmed that there is positive association between the calcification and carbonic anhydrase isoenzymes. Quantitative analysis with cetylpyridinium chloride showed a decrease in calcification when the cells were transfected with an anti-CA13 shRNA. Our research suggested that CA2 and CA13 are down-calcified in short-side mandibular condyle, and caused mandibular ramus to grow slowly. CA2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.