On Tuesday this week, I was literally seeing spots, as I inspected health papers and checked Coggins (EIA) test diagrams and photos, for Paint Horses arriving in advance of their Horse Show events. The American Paint Horse is a versatile breed, used for both English and Western events, as well as in halter classes and other equine competitions. The two major spotting patterns in Paints are referred to as Overo, in which the white is scattered and splashy, and usually does not cross the back of the horse between withers and tail, and Tobiano, in which the spots are regular and distinct, and the dark color covers one or both flanks. In a 2007 paper, Brooks and colleagues provide evidence that the Tobiano color pattern is due to a large inversion on Equus caballus chromosome 3 (ECA3), which may alter the expression of the KIT receptor tyrosine kinase.
The genetics of dominant white spotting patterns, similar to Tobiano, have been studied extensively in mice, originally as part of the Victorian “mouse fancy” culture, and more recently in the context of melanocyte (pigment cell) biology. The black-eyed Dominant White Spotting class of mutants, initially characterized by Elizabeth Russell, is particularly relevant to the Tobiano pattern in horses, as these mice exhibit defects in the expression or function of the KIT protein. In mammalian embryos, KIT is expressed early by migrating neural crest cells, which are the precursors for melanocytes, and signaling through this receptor, initiated by binding of the KIT ligand (Steel factor) is essential for melanocyte precursor migration, localization, and survival. KIT signaling also has roles in germ cell migration and differentiation, and in the development of certain hematopoietic cells. Humans who are heterozygous for a mutation in the KIT gene have the pigmentation disorder piebaldism, with unpigmented forehead and belly (Wehrle-Haller, 2003).
Tobiano Paint Horse
Photo: Jean-Pol Grandmont, via Wikipedia
Brooks et al. (2007) recognized the similarities in spotting patterns between Tobiano horses and W mice, some variations of which result from inversions or deletions, within 200 bp upstream of the mouse KIT gene, and disrupt expression of the receptor during embryogenesis. Previous work from this group demonstrated linkage of Tobiano and KIT, and indicated no mutations in the KIT cDNA of Tobiano horses. Therefore, fluorescence in situ hybridization (FISH) was used to examine the positions of several genes on ECA3, in samples from Tobiano and non-Tobiano horses (I was a bit disappointed that chromosome Paint techniques weren’t appropriate for this study). This analysis revealed an inversion on ECA3 of Tobiano horses, with the distal end located between the KDR and KIT genes; the inversion is very large, but was not thought to disrupt any genes.
The authors developed a PCR test to detect the ECA3 inversion associated with the Tobiano spotting pattern, and then looked for the inversion in cells from 13 breeds, including 12 with the Tobiano spotting pattern, and one (Thoroughbred) that does not exhibit this trait. The inversion was detected in all Tobiano pattern horses, including those of Shetland Pony, Draft, Saddlebred, and Welsh Cross breeds. Since the paracentric chromosome 3 inversion does not affect the coding sequence of any known genes, the authors speculate that noncoding regulatory regions of the KIT gene may be disrupted. Moreover, the health of Tobiano horses is not affected by the inversion, though a reduction in fertility of heterozygotes, due to production of genetically unbalanced gametes, may have gone unnoticed, in the context of selecting for the spotting pattern. Brooks et al. (2007) conclude that the Tobiano inversion preceded divergence of horse and pony breeds, and may in fact have predated domestication of the horse, 6000 years ago.
Figure 4. Brooks et al., 2007
“Special Horse Show Edition” of Weekend Animal Disease Models blog posts
Wehrle-Haller, B. (2003). The role of Kit-Ligand in melanocyte development and epidermal homeostasis. Pigment Cell Res. 16, 287-296.
Brooks, S., Lear, T., Adelson, D., Bailey, E. (2007). A chromosome inversion near the KIT gene and the Tobiano spotting pattern in horses. Cytogenetic and Genome Research, 119(3-4), 225-230. DOI: 10.1159/000112065