The demonstration of tissue clonality by X-linked enzyme histochemistry

We report here the first demonstration in normal, as opposed to chimaeric mice, of the clonal architecture of the small intestine, oesophagus, breast, and thyroid, using polymorphism of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) as a clonal marker. Reproducible results using X-linked histochemistry were found to depend upon the simultaneous study of tissue from normal mice, mice homozygous for abnormal enzyme levels, and heterozygous mice. In tissues from homozygous animals where levels of enzyme activity normally vary, the use of a physiological stimulus to reduce this metabolic heterogeneity allowed interpretation of clonality in the heterozygotes. The details of the histochemical technique used require modification for each of the different tissues studied. A dual population of cells was seen in the heterozygous animals in all tissues studied. Oesophageal epithelium showed sharply separated alternating patches of positive and negative cells. Small intestinal crypts were always monophenotypic, while villi were polyphenotypic, indicating that crypts are monoclonal and villi derive from more than one crypt, confirming work in allophenic animals. In contrast, thyroid follicles and breast acini showed a mixture of mono- and polyphenotypia, consistent with a polyclonal origin. These results, which have important implications for the study of the clonal origins of normal and neoplastic growth, require the direct in situ demonstration of the cellular phenotype.