Transcriptome-based comparison reveals key genes regulating allometry growth of forelimb and hindlimb bone in duck embryos

Abstract

Allometric growth of the forelimb and hindlimb is a widespread phenomenon observed in vertebrates. As a typical precocial bird, ducks exhibit more advanced development of their hindlimbs compared to their forelimbs, enabling them to walk shortly after hatching. This phenomenon is closely associated with the development of long bones in the embryonic stage. However, the molecular mechanism governing the allometric growth of duck forelimb and hindlimb bones is remains elusive. In this study, we employed phenotypic, histological, and gene expression analyses to investigate developmental differences between the humerus (forelimb bone) and tibia/femur (hindlimb bones) in duck embryos. Our results revealed a gradual increase in weight and length disparity between the tibia and humerus from E12 to E28 (embryo age). At E12, endochondral ossification was observed solely in the tibia but not in the humerus. The number of differentially expressed genes (DEGs) gradually increased at H12 vs. T12, H20 vs. T20, and H28 vs. T28 stages consistent with phenotypic variations. A total of 38 DEGs were found across all 3 stages. Protein-protein interaction network analysis demonstrated strong interactions among members of HOXD gene family (HOXD3/8/9/10/11/12), HOXB gene family (HOXB8/9), TBX gene family (TBX4/5/20), HOXA11, SHOX2, and MEIS2. Gene expression profiling indicated higher expression levels for all HOXD genes in the humerus compared to tibia while opposite trends were observed for HOXA/HOXB genes with low or no expression detected in the humerus. These findings suggest distinct roles played by different clusters within HOX gene family during skeletal development regulation of duck embryo's forelimbs versus hind limbs. Notably, TBX4 exhibited high expression levels specifically in tibia whereas TBX5 showed similar patterns exclusively within humerus as seen previously across other species' studies. In summary, this study identified key regulatory genes involved in allometric growth of duck forelimb and hindlimb bones during embryonic development. Skeletal development is a complex physiological process, and further research is needed to elucidate the regulatory role of candidate genes in endochondral ossification.