Figure 3.

Histological and gene expression analysis of skin biopsies from GEA and control animals. (a–c) Hematoxylin-eosin-stained sections of ear skin biopsies. (a) Black skin from a control black and white Holstein animal. Melanocytes are located on the basal layer of epidermis (indicated by arrowheads) and brown melanin granules are disseminated within keratinocytes. (b) Melanocytes are visible, but not melanin in white skin from a GEA animal. (c) Black skin from a GEA animal with a revertant phenotype. Melanin is present in the epidermis. Note the presence of both black and white hair. (d and e) Hematoxylin-eosin-safran stained sections of eyes from GEA and control animals. Note the complete lack of pigmentation of the choroid from the GEA animal (d) as compared with control (e). (f–h) Transmission Electronic Microscopy pictures of skin from wild-type (f) and from a GEA animal (g). Melanocytes (arrowheads) are easily distinguished from the surrounding keratinocytes. Numerous melanosomes lie in the wild-type keratinocytes, while only a few abnormal ones are observed in the mutant skin. (h) Detail of a GEA keratinocyte cytoplasm containing abnormal melanosomes (arrowhead). (i) Relative expression quantification of melanogenic genes in the skin of wild-type and GEA Holstein animals. MITF-M expression is similar in black skin from control animals (n = 3) and in unpigmented (white) and revertant skin (black) from GEA animals (n = 3), showing that the regulation of expression of the melanogenic genes is not a consequence of a downregulation of MITF-M in mutant animals. Melanogenic genes (TYR, TYRP1, and PMEL) are not expressed in GEA white skin, with the exception of DCT which has already been shown to be regulated by MITF-independent mechanisms. In all black skins sampled from GEA revertant animals, the expression of melanogenic genes is partially restored. Scale bars represent 50 µM in (a) and (b); 100 µM in (c–e); 5 µM in (f,g); and 1 µM in (h).