Activation of the Hedgehog (Hh) signalling pathway by loss of function mutations of the Ptch1 receptor promotes stem or progenitor cell proliferation in several cell types, most notably the basal cells of the skin and granule cells of the cerebellum. We have intercrossed MxCre transgenic mice with conditional Ptch1 knockout mice to study the effects of deleting Ptch1 on adult hematopoiesis, with the hypothesis that loss of Ptch1 would activate the Hh pathway leading to increased hematopoietic stem cells (HSC). Within 4 weeks after deletion of Ptch1 with administration of poly(I:C), MxCrePtch1-null mice developed apoptosis of bone marrow pre-B cells and double positive thymocytes. Overall, MxCrePtch1-null mice have 10-fold less pre-B cells and thymocytes. MxCrePtch1-null mice also develop a 3-fold increase in lineage negative c-kit+ Sca-1+ (LKS) bone marrow cells, a cell fraction enriched for HSCs. Despite increased numbers of LKS, loss of Ptch1 did not increase the numbers of HSCs as measured by competitive repopulation assays. MxCrePtch1-null mice also developed typical Ptch1-related tumours including basal cell carcinomas and cerebellar tumours, which was consistent with the ability of the MxCre transgene to delete loxP-flanked genes in cell types other than hematopoietic cells. To determine if the hematopoietic changes observed in the MxCrePtch1-null mice were cell intrinsic or due to loss of Ptch1 on cells of the microenvironment, we intercrossed conditional Ptch1 mice with hematopoietic specific Cre transgenic mice. Surprisingly, HSC-specific deletion of Ptch1 using tamoxifen-inducible SCLert(2)Cre mice did not lead to any increase in LKS numbers. Similarly, lymphoid specific deletion of Ptch1 with the B-cell specific CD19Cretransgene or the T-cell specific LckCre transgene did not lead to any lymphoid defects. The lack phenotype in hematopoietic-specific Ptch1-null mice indicates that Ptch1 is redundant on hematopoietic cells including HSCs. Furthermore, the lack of phenotype also suggests that the defects observed in the MxCrePtch1-null mice were due to loss of Ptch1 in the microenvironment. To prove that Ptch1 regulates the hematopoietic microenvironment, we performed reciprocal transplant experiments whereby lethally irradiated MxCrePtch1- null mice were reconstituted with wild-type bone marrow cells. Remarkably, wild-type hematopoiesis grown within the MxCrePtch1-null microenvironment developed the identical hematopoietic defects with increased LKS and apoptosis of pre-B cells and thymocytes. Conversely, the MxCrePtch1-null hematopoietic defects could be completely rescued by transplant into lethally irradiated wild-type mice. Histological examination of bones from MxCrePtch1-null mice showed marked alterations in trabecular and cortical bone. Given the recent demonstration that the Hh pathway regulates adult bone homeostasis, we hypothesize that the increased LKS and loss of pre-B cells observed in Ptch1-null mice are secondary to changes within the bone marrow cell niche.
Disclosures: No relevant conflicts of interest to declare.