Expression and Analysis of TBX3 Gene in the Skin from Three Locations on Dun Mongolian Bider Horse

The Mongolian horse, one of the oldest and most genetically diverse breeds, exhibits a wide variety of coat colors and patterns, including both wild-type and unique features. A notable characteristic of dun Mongolian horses is the presence of Bider markings-symmetrical, black-mottled patterns observed on the shoulder blades. These markings are also seen in Przewalski's horses. The dun coat color, a common wild-type phenotype in domestic horses, is characterized by pigment dilution with distinct dark areas and is regulated by mutations in the TBX3 gene. This study aimed to investigate the role of TBX3 in the development of Bider markings in dun Mongolian horses.

Skin tissue samples were collected from three key anatomical regions of dun Mongolian horses with Bider markings: the croup, dorsal midline, and shoulder. Histological staining was conducted to examine the skin and hair follicle structure and pigment distribution. RT-qPCR was used to measure TBX3 mRNA expression, while immunoblotting and immunohistochemistry were employed to analyze TBX3 protein levels and localization.

Hematoxylin and eosin staining revealed the skin and hair follicle structures, including the epidermis, hair shaft, and hair bulb across different stages of the hair growth cycle. Differences in pigmentation were observed across the sampling sites. The croup and the light-colored area of the shoulder showed asymmetrical pigmentation, while the dorsal midline and dark-colored area of the shoulder displayed symmetrical pigmentation. TBX3 mRNA expression levels were significantly higher in the croup compared to the shoulder and dorsal midline; however, corresponding TBX3 protein expression did not show significant differences. Immunohistochemical analysis localized TBX3 protein predominantly in the hair bulb and epidermis.

This study demonstrates region-specific differences in TBX3 expression that correlate with pigmentation patterns in dun Mongolian Bider horses. These findings provide valuable insights into the molecular mechanisms underlying Bider markings, offering a deeper understanding of the genetic regulation of coat color and primitive markings in equines.