# Differential Expression of MITF, WNT3A, SLC7A11, and EDN3 in the Shoulder ‘Bider Marking’ of Dun Mongolian Horses

## TL;DR

This study identifies genes involved in the pigmentation pattern called 'Bider marking' in Mongolian horses, focusing on MITF and WNT3A.

## Contribution

The study reveals specific upregulation and localization of MITF and WNT3A proteins in the pigmented 'Bider marking' of Mongolian horses.

## Key findings

- MITF and WNT3A protein levels are significantly higher in the dark 'Bider marking' area compared to non-marked skin.
- SLC7A11 and EDN3 show complex and inconsistent expression patterns unrelated to pigmentation.
- MITF protein is localized to specific layers of the epidermis and hair follicles in the Bider marking region.

## Abstract

The ‘Bider marking’ on the shoulder of Dun Mongolian horses is a unique symmetrical pigmented patch with unclear formation mechanisms. This study analyzed the expression and localization of the MITF, WNT3A, SLC7A11, and EDN3 genes at both mRNA and protein levels by comparing skin tissues from the dark-colored area of the ‘Bider marking’ shoulder (BIDC), the light-colored area of the ‘Bider marking’ shoulder (BILC), and the non-Bider-marked shoulder area (NBIS). Results indicated that the protein expression of both MITF and WNT3A in the BIDC area was significantly higher than that in the NBIS area. The expression patterns of SLC7A11 and EDN3 are both constantly changing and complex; they do not demonstrate a clear association with pigmentation. Studies have shown that the upregulation of MITF and WNT3A at the protein level, along with their spatial distribution specificity, is closely related to the formation of the dark patches in ‘Bider markings.’ These findings provide important insights into the molecular mechanisms underlying this trait.

(1) Background: The “Bider marking” on the shoulder of the Dun Mongolian horse represents a unique pigmentation pattern, the molecular formation mechanism of which remains incompletely understood. This study investigates the differential expression and protein localization of pigment-related genes—specifically the core transcription factor MITF, as well as EDN3, SLC7A11, and WNT3A—in the skin. The analysis focuses on three distinct regions: the dark-colored area of the ‘Bider marking’ shoulder (BIDC), the light-colored area of the ‘Bider marking’ shoulder (BILC), and the non-Bider-marked shoulder area (NBIS). The aim is to clarify their correlation with the formation of this distinctive pigmentation pattern. (2) Methods: Skin tissue samples from both the “Bider marking” and non-Bider-marked shoulder regions were collected (n ≥ 3). The mRNA expression levels were quantified using RT-qPCR, protein levels were analyzed through Western blotting, and protein localization was assessed via immunohistochemistry and immunofluorescence. (3) Results: Compared to the NBIS group, both the BIDC and BILC groups exhibited significantly elevated protein expression of MITF and WNT3A. Further immunofluorescence showed that the distribution of MITF protein exhibits regional specificity in the epidermis and hair follicles. In the BIDC region, the protein is localized specifically to the stratum corneum of the epidermis, the dermal papilla, and the outer root sheath of hair follicles. In contrast, the mRNA and protein expression levels of SLC7A11 and EDN3 did not display consistent patterns among the three groups, and no specific differences were observed in tissue localization. (4) Conclusions: The findings show that the specific pigmentation in dark “Bider marking” regions is closely linked to the upregulated protein levels and unique spatial patterns of MITF and WNT3A; SLC7A11 and EDN3 may not be primary regulators of this trait.