Three types of hedgehog (Hh) ligands, Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh) have been found in mammals exhibiting different expression patterns and biological functions (1). Shh is extensively expressed in a number of tissues in both the embryo and adult. However, Ihh is specifically expressed in hematopoietic cells, bones, cartilage and the eyes, whereas Dhh is restricted to the gonads, external genitalia, eyes and peripheral nerves (2–5). Hh ligands that bind to a transmembrane protein, patched (Ptch), transduce an intracellular signal that is widely involved in the differentiation, proliferation and survival of numerous cells at a series of developmental stages from embryogenesis to adulthood (1). It has been reported that Hh signaling spatially and temporally paves the way for the development and differentiation of hematopoietic stem and progenitor cells (HSPCs) and blood cells in embryonic and adult hematopoietic system developing tissues. Defining the role of Hh signaling in hematopoiesis, and the potential impact in embryonic hematopoiesis and the development of multi-lineage blood cells in adults may provide a novel theoretical basis for improving regeneration following injury or hematopoietic stem cell (HSC) transplantation.

In mammals, canonical Hh signaling initiates from the binding of Hh ligands to a 12-transmembrane domain receptor protein, Ptch. This binding leads to alleviation of the Ptch-induced suppressive effects on smoothened (Smo). Smo is subsequently uninhibited, and activates zinc finger transcription factor glioma-associated oncogene (Gli) proteins, which are dissociated from a suppressive complex-containing scaffold protein, suppressor of fused (SuFu), and translocate into the nucleus to promote or depress target gene transcription (5,6), including that of Gli1, CCND, Ptch, BCL2 and Hhip. Three Gli proteins (Gli1, Gli2 and Gli3) are expressed in vertebrates with overlapping and partially redundant domains. Only Gli1 usually acts as a transcriptional activator, while Gli2 and Gli3 act as either activators or repressors, which is determined by post-transcriptional and post-translational modifications (7–9). Lack of SuFu leads to a decrease in the stability of Gli2/3 protein (10). Kinesin-like protein KIF7 (Kif7) negatively regulates Smo; however, it enhances Hh signaling in certain cases (Fig. 1) (11).

Hh signaling is predominantly involved in the later stages of embryonic hematopoiesis, which includes two spatially and temporally distinguishable hematogenic waves, namely pro-definitive and definitive hematopoiesis, to individually give rise to bipotent hepatic progenitor cells (HPCs) or HSCs (12). Mutants in the Hh pathway (the shh mutant and the slow muscle-omitted, smu, mutant) or treatment with Hh signaling inhibitor, cyclopamine, in zebrafish embryos severely reduced the number of runt-related transcription factor (runx1)⁺ definitive blood cells, while the number of βE1⁺ primitive erythrocytes were unaffected. This requirement for Hh signaling coincides with the time of three consecutive steps in dorsal aorta formation and intersomitic vessel sprouting, indicating that Hh signaling is required for HSC formation (13). Using mouse embryonic explants, Dyer et al (14) demonstrated that Ihh secreted by visceral endoderm and mature yolk sacs alone are sufficient in inducing endothelial and hematopoietic differentiation, following increased Ptch, Smo and Gli1, as well as Bmp4 within anterior epiblasts (15). HSC numbers in the aorta-gonad-mesonephros region (AGM) increased, leading to increased colony-forming units spleen day 11 (CFU-S₁₁) in the AGM with ventral tissues explant culture and in vivo transplantation compared with AGM explants, suggesting an important role of ventral tissues in the AGM HSC activity. Considering the increased level of Gli1 expression in AGM (c-Kit⁻CD34⁻) mesenchymal cells, positively regulating HSC activity by both Ihh and Shh proteins, the Hh signaling pathway is identified as a HSC inducing signal for definitive hematopoietic development in embryonic day 10 AGM (16).

Through a combined analysis of differentiating mouse embryonic stem cells (ESCs), mouse embryo cultures and zebrafish embryos, Kim et al (17) constructed a model of later embryonic hematopoietic development from mesoderm stage to hematopoietic cell differentiation. This hierarchy of differentiation consists of Flk1⁺ mesoderm patterning to the endothelium with arterial identity (VE-cadherin⁺CD41⁻CD45⁻) through the activation of Notch signaling, and promoting blood formation (CD41/45⁺) from hemogenic endothelial cells upon upregulation of the protein, stem cell leukemia.

Expression of transcriptional factors that promote hemogenic endothelium differentiation, such as BRACHYURY, GATA2 and RUNX1, was increased in human ESCs following treatment with a Smo agonist, purmorphamine, for 6 days (18,19). However, treatment with a Smo antagonist, SANT-1, reduced the expression of RUNX1 and BRACHURY in hESCs, and increased the expression of markers of endocardiogenic endothelium differentiation, suggesting a crucial role of Shh for differentiation of hESCs toward a hemogenic lineage (19,20).

The role of Hh signaling in HSPCs remains controversial. Bhardwaj et al (21) showed that Hh signaling components, including Shh, Ptch and Smo, were expressed in primitive and mature CD19⁺, CD33⁺ and CD3⁺ cells, as well as stromal cells isolated from adult bone marrow (BM) and endothelial cells from human umbilical veins, while Gli1, Gli2 and Gli3 were absent in both myeloid and lymphoid lineages. In NOD-SCID mice transplanted with Shh-treated human CD34⁺CD38⁻Lin⁻ cells, the proliferation of CD34⁺CD38⁻Lin⁻ cells was enhanced and multi-lineage blood cells, including myeloid CD15⁺CD33⁺, lymphoid CD19⁺CD20⁺, CD34⁺ and rare CD34⁺CD38⁻ cells increased. In addition, the total number of HPCs increased when Hh signaling was blocked by an anti-Hh antibody, suggesting a positive regulatory role in HSPCs. This effect was also observed in vitro. The promoter action of Shh on HSPCs and hematopoietic reconstitution is dependent on downstream Bmp4 signaling, as noggin, a specific inhibitor of Bmp4, is capable of inhibiting Shh-induced proliferation in a similar manner to anti-Hh antibody. Together with mesenchymal stem cells, Shh protein promotes the proliferation of HSCs, which is associated with increased expression of angiogenic factor receptor Tie-2 in HSCs cells, and angiogenic factors VEGF and Ang1 in MSCs (22). Expansion of human HSCs in vitro and in vivo stimulated by Ihh in the stromal supportive culture system was observed in an independent study (23).

Using a Ptch-1^+/− mouse model, which exhibited increased Hh signaling activity, Trowbridge et al (24) found that Hh signaling induced cycling and expansion of primitive BM HPCs during homeostasis and stress at the expense of HSC exhaustion. Compared with wild-type (WT) mice, Ptch-1^+/− mice had significantly higher numbers of Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells and CFUs, and exhibited accelerated recovery of peripheral blood leukocytes after 5-fluorouracil (5-FU) treatment. Ptch-1^+/−Lin⁻ BM cells had a greater short-term regeneration capability but reduced long-term reconstitution efficiency. Downregulation of cell cycle-related genes (Tfdp2, Skp1a, CyclinA2, Rad51, CyclinH, CDC16, CDC2a, Mre11a and RPA3) in the Ptch-1^+/− group was associated with the absence of long-term hematopoiesis. The regenerative capacity of Ptch-1^+/− HSCs could be restored by cyclopamine through the upregulation of cycling Lin⁻Sca-1⁺ cells. Homozygous deletion of Gli1 (Gli1^null) increased the number of long-term HSCs, which survived for a prolonged period following engraftment. However, in vitro colony-forming assays showed that the number of granulocyte CFUs derived from Gli1^null BM was almost 2-fold lower compared with those derived from Gli1^WT mice. Furthermore, the number of neutrophils and platelets in Gli1^null mice recovered at a reduced rate following 5-FU treatment, indicating that loss of Gli1 affected myeloid progenitor function and impaired their subsequent ability to recover (25).

An independent study demonstrated that Smo deletion or overexpression exerted no significant effect on HSC self-renewal, differentiation and reconstitution ability. In Smo-deficient LSKs, <10% (70/739) of HSC gene signatures changed (upregulated or downregulated) and the expression of genes closely associated with long-term HSC activity was not altered (26). The number of T and B cells, erythroid and myeloid cells, and megakaryocytes and LSK cells in Smo^Null mice were not changed, similar to the results obtained using Smo^WT mice. Furthermore, deletion of Smo did not affect BM reconstitution after transplantation, and no significant difference was observed in hematopoietic colony formation potential, long-term survival ability of competitive transplantation and recovery ability of BM under 5-FU-induced stress (27).

Expression of Shh, Ptch-1, Smo and Gli1 was significantly reduced in mice with pesticide-induced aplasia (51). Expression of Shh, Ihh and Dhh of Hh ligands was markly reduced in BM stromal cells. Supplementation of the recombinant mouse Shh (rmShh) in vitro promoted CFUs of granulocytes, erythrocytes, monocytes and megakaryocytes, and CFUs of granulocyte-macrophage progenitors, and augmented fibroblastic colony formation, suggesting that rmShh can minimize the suppression of different pesticide mixtures on hematopoiesis and rescue stromal and hematopoietic precursors from pesticide-induced cytotoxicity in vitro (52). Downregulation of Hh signaling may be involved in hematopoietic injury and failure. Results of our previous study demonstrated that expression of Shh in the BM of patients with aplastic anemia was significantly lower than that in patients with iron deficiency anemia. The expression level of Shh was positively associated with white blood cell count, lymphocyte count and CD4⁺/CD8⁺ ratio (unpublished data).

While progress has been made in elucidating the role of Hh signaling on hematopoiesis (Fig. 2), there are still a number of questions that need to be addressed. The disagreement between results may be attributed to a set of methodological differences, such as different hematopoietic microenvironments (AGM, fetal liver or BM), developmental stages (primitive, early or late definitive hematopoiesis), species (zebrafish or mouse) and physiological state (stress damage or normal hematopoiesis). Whether the Hh signaling pathway exhibits potential as a target for the diagnosis and treatment of blood disorders requires further investigation.