Abstract

The Hedgehog (Hh) signaling pathway plays a critical role in embryonic development and adult tissue homeostasis and has emerged as an important therapeutic target in many cancers, including leukemia and myeloproliferative diseases. Our mechanistic understanding of Hh pathway signaling and regulation comes primarily from developmental studies in neural and limb development. Studies of Hedgehog signaling in the hematopoietic system have produced contradictory results, and no clear consensus regarding Hh signaling in normal hematopoiesis is available to inform the role of Hedgehog signaling in hematologic malignancies. In our work we have focused on understanding the downstream effectors of Hedgehog signaling, the Gli transcription factors. The three Gli proteins, Gli1, Gli2 and Gli3 have both transcriptional activator and repressor functions, which allow for regulation and fine-tuning of Hedgehog pathway output. Previous studies from our group have revealed that Gli1null HSCs had no defects in self-renewal, however myeloid differentiation and stress hematopoiesis were severely impaired (Merchant, et al., Blood 2010). In normal tissues, Hh pathway activation via Ptch/Smo causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor Gli3R. Our recent studies demonstrated that GLI3R has a tumor suppressor role in human acute myeloid leukemia by directly repressing AKT expression (Chaudhry et al., AACR Annual Meeting 2015). To date nothing is known about the role of Gli3 in normal hematopoiesis. In the present study, we crossed Vav-Cre transgenic mice to Gli3fl/fl mice to generate mice with a conditional loss of Gli3 (Gli3null) in the hematopoietic system. HSC self-renewal was analyzed by serial transplant. In comparison to HSCs from Gli3 wild type (Gli3WT) mice bone marrow (BM), HSCs from Gli3null BM showed decreased long-term engraftment and self-renewal. In addition, quantification of long-term HSC (LT-HSC, CD34neg Flt3neg KSL), short-term HSC (ST-HSC, CD34+ Flt3neg KSL), and multi-potent progenitor (MPP, CD34+ Flt3+ KSL) revealed that the frequency of LT-HSCs in Gli3null BM (0.004-0.007%) was lower compared to Gli3 WT BM (0.008-0.02%). In mice transplanted with Gli3null BM, myeloid expansion was observed with a block in T and B cell lineage differentiation. Analysis of the c-Kit+ Sca1neg Linneg (KL) myeloid progenitor compartment revealed a two-fold increase in the FcRγhigh CD34+ KL granulocyte-monocyte progenitors (GMPs) in Gli3null BM, suggesting an expansion of granulocytic compartment. Since Gli3R is a key negative regulator of Gli1, these are consistent with decrease in GMP and myeloid differentiation previously seen in Gli1null mice. In summary, our studies reveal a previously unknown function for Gli3 in regulating HSCs and myeloid differentiation, and help to elucidate the complex regulation of Hh signaling in the hematopoietic system.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.