Patterns of proliferation and apoptosis during murine hair follicle morphogenesis

In this study, we have correlated cutaneous apoptosis and proliferation in neonatal mice during hair follicle morphogenesis, We have applied a novel t riple-staining technique that uses Ki67 immunoreactivity as a marker of pro liferation as well as TUNEL and Hoechst 33342 staining as apoptosis markers . We have also assessed the immunoreactivity of interleukin-1 beta -convert ing enzyme, caspase 1, a key enzyme in the execution of apoptosis, and of P -cadherin, which has been suggested as a key adhesion receptor in segregati ng proliferating keratinocytes. The TUNEL data were systematically compared with high resolution light microscopy and transmission electron microscopy data. Virtually all keratinocytes of the developing hair bud were strongly Ki67(+), suggesting that the hair bud is not an epidermal invagination but primarily the product of localized keratinocyte proliferation. As hair fol licle development advanced, three distinct foci of proliferation became app arent: the distal outer root sheath around the hair canal, the mid outer ro ot sheath, and the proximal hair matrix. Of these proliferating hair follic le keratinocytes only defined subsets expressed P-cadherin. TUNEL+ cells in the hair follicle were not found before stage 5 of murine hair follicle mo rphogenesis. During the early stages of hair follicle development, interleu kin-1 beta -converting enzyme immunoreactivity was present on all keratinoc ytes, but virtually disappeared from the proximal hair follicle epithelium later on. High resolution light microscopy/transmission electron microscopy revealed scattered and clustered apoptotic keratinocytes in all epithelial hair follicle compartments throughout hair follicle development, including its earliest stages. This highlights striking differences in the demarcati on of apoptotic hair follicle keratinocytes between the TUNEL technique and high resolution light microscopy/transmission electron microscopy and sugg ests a role for apoptosis in sculpting the hair follicle even during early hair follicle development.