a, K15-CrePR;Ldhafl/fl animals treated with Mifepristone during telogen (day 50) were allowed to develop for 6 months. None of the K15-CrePR;Ldhafl/fl mice showed complete hair regrowth, compared to control animals that all grew their hair coats back completely. Images are representative of at least 12 animals per genotype. b, Histological examination of the long term K15-CrePR;Ldhafl/fl mice showed that Ldha-null HFSCs remained in telogen while WT HFSCs went through anagen and then returned to telogen. This is apparent from thick sections (50 micron, right) that show an increased number of club hairs in the WT relative to Ldha-null follicles. Scale bars indicate 100 micrometers (left), and 20 micrometers (middle and right). c, IHC for HFSC marker Sox9 showed that deletion of Ldha from HFSCs does not affect their presence in the bulge even after 6 months. In addition, IHC and Ldh activity assay demonstrate that the deletion of Ldha was sustained. Because of the mosaicism of the deletion, in some portions of K15-CrePR;Ldhafl/fl skin Ldha was not deleted. Shown on the bottom row is tissue from hair bearing skin in the K15-CrePR;Ldhafl/fl mice where Ldha was still expressed, showing that new hair growth in K15-CrePR;Ldhafl/fl mice was due to lack of deletion of Ldha caused by the mosaic approach used to mediate Cre recombination. Scale bars indicate 20 micrometers. d, To determine how various signaling pathways previously linked to the hair cycle are affected by loss of Ldha in HFSCs, we performed IHC for markers that indicate activity of these pathways in telogen and telogen-anagen transition. Note that pStat5 appears to be suppressed in normal telogen-anagen transition, and this does not seem to occur in Ldha-null HFSCs. pStat1 and pStat3 did not seem to be affected by loss of Ldha. Expression of Gli3, a target of Shh signaling, is typically induced in an activated hair germ derived from HFSCs, but Ldha-null HFSCs do not make an active hair germ. Activation of the Wnt pathway is indicated by nuclear localization of β-catenin, and very little nuclear β-catenin was detected in Ldha-null HFSCs. Scale bars indicate 6 micrometers.