Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog.

The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.

Sonic hedgehog negatively regulates pre-TCR-induced differentiation by a Gli2-dependent mechanism.

Hedgehog signaling regulates differentiation, survival, and proliferation of the earliest double-negative (DN) thymocytes, but its importance at later stages of T-cell development is controversial. Here we use loss- and gain-of-function mouse models to show that Shh, by signaling directly to the developing thymocyte, is a negative regulator of pre-TCR-induced differentiation from DN to double-positive (DP) cell. When hedgehog signaling was reduced, in the Shh(-/-) and Gli2(-/-) thymus, or by T lineage-specific transgenic expression of a transcriptional-repressor form of Gli2 (Gli2DeltaC(2)), differentiation to DP cell after pre-TCR signal transduction was increased. In contrast, when Hh signaling was constitutively activated in thymocytes, by transgenic expression of a constitutive transcriptional-activator form of Gli2 (Gli2DeltaN(2)), the production of DP cells was decreased. Gene expression profiling showed that physiologic Hh signaling in thymocytes maintains expression of the transcription factor FoxA2 on pre-TCR signal transduction.

Indian hedgehog (Ihh) both promotes and restricts thymocyte differentiation.

We show that Indian Hedgehog (Ihh) regulates T-cell development and homeostasis in both fetal and adult thymus, controlling thymocyte number. Fetal Ihh(-/-) thymi had reduced differentiation to double-positive (DP) cell and reduced cell numbers compared with wild-type littermates. Surprisingly, fetal Ihh(+/-) thymi had increased thymocyte numbers and proportion of DP cells relative to wild type, indicating that Ihh also negatively regulates thymocyte development. In vitro treatment of thymus explants with exogenous recombinant Hedgehog protein promoted thymocyte development in Ihh(-/-) thymi but inhibited thymocyte development in Ihh(+/-), confirming both positive and negative regulatory functions of Ihh. Analysis of Rag(-/-)Ihh(+/-) thymi showed that Ihh promotes T-cell development before pre-T-cell receptor (pre-TCR) signaling, but negatively regulates T-cell development only after pre-TCR signaling has taken place. We show that Ihh is most highly expressed by the DP population and that Ihh produced by DP cells feeds back to negatively regulate the differentiation and proliferation of their double-negative progenitors. Thus, differentiation from double-negative to DP cell, and hence the size of the DP population, is dependent on the concentration of Ihh in the thymus. Analysis of Ihh conditional knockout and heterozygote adult mice showed that Ihh also influences thymocyte number in the adult.

The Gli3 transcription factor expressed in the thymus stroma controls thymocyte negative selection via Hedgehog-dependent and -independent mechanisms.

The Hedgehog (Hh) responsive transcription factor Gli3 is required for efficient thymocyte development in the fetus. In this study we show that Gli3, not detected in adult thymocytes, is expressed in the murine fetal and adult thymus stroma. PCR array analysis revealed Cxcl9, Rbp1, and Nos2 as novel target genes of Gli3. We show that Gli3 positively regulates the expression of these genes, most likely by suppressing an intermediate repressor. Deletion of autoreactive thymocytes depends on their interactions with the thymus stroma. Repression of the proapoptotic gene Nos2 in Gli3 mutants coincides with reduced apoptosis of double positive thymocytes undergoing negative selection in vitro and in vivo, and the production of autoreactive thymocytes. Taken together these data indicate that Gli3 controls thymocyte apoptosis and negative selection possibly via the regulation of Nos2. Defective Gli3 expression in the thymus stroma also resulted in decreased CD5 expression on mature thymocytes and inappropriate production of MHC class I-selected CD4(+) cells, both consistent with reduced TCR signal strength. Overall our data indicate that Gli3 expressed in the thymus stroma regulates negative selection and TCR signal strength via Hh-dependent and -independent mechanisms, with implications for autoimmunity.

The T-lineage-affiliated CD2 gene lies within an open chromatin environment in acute promyelocytic leukemia cells.

The nature of hemopoietic progenitors subject to leukemic transformation in acute myeloid leukemia (AML) has not been clearly defined. To address this issue, we have used DNase I hypersensitivity assays to study the chromatin structure surrounding the T-lineage-affiliated CD2 gene in the acute promyelocytic subtype of AML (APL). Upstream and downstream flanking regions of CD2 were found to be hypersensitive to DNase I in primary APL blasts, with an identical pattern of hypersensitive sites to those detected in cells of T-lineage. All of the sites were confirmed to be inaccessible to DNase I in B-lineage leukemia cells. The demonstration of T-cell-associated chromatin features in primary APL blasts has implications for the origin of APL that may arise in more primitive progenitors than previously considered to be the case.

Non-redundant role for the transcription factor Gli1 at multiple stages of thymocyte development.

The Hedgehog (Hh) signaling pathway influences multiple stages of murine T-cell development. Hh signaling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse development and can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and nonredundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4⁻CD8⁻ double negative (DN) thymocytes before pre- TCR signal transduction, and a negative regulatory function after pre-TCR signaling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage.

KLF13 influences multiple stages of both B and T cell development.

The Kruppel-like factor, KLF13, is a member of a family of transcription factors shown to be involved in haematopoietic development. Here we show that KLF13 is involved in the development of B and T cells at multiple stages. Expression of KLF13 in the thymus was maximal in the DP population and in KLF13(-/-) deficient mice there was an accumulation of DP thymocytes and reduction of CD4(+)SP cells. Cell-surface expression of CD3(high), CD8, CD5 and HSA were altered on KLF13(-/-) DP cells, consistent with a defect in TCR signalling and at the DP to SP transition in KLF13(-/-) mice. KLF13 is also expressed in peripheral T-cells and peripheral T cell activation was impaired in KLF13(-/-) mice. Analysis of early B cell development in the bone marrow (BM) revealed a partial arrest of B cells at the transition from CD43(+) to CD43(-) pre-B cell, a transition that requires signalling through the pre-BCR. The proportion of IgM(+)/IgD(+) mature B cells was also increased in the BM of the KLF13(-/-) mice. This finding is consistent with a reduction in the strength of BCR signal or an accumulation of recirculating B cells from the periphery. Analysis of splenocytes isolated from KLF13(-/-) mice revealed an increase in the expression of CD21 and CD23 on B220(+) B cells, demonstrating a negative regulatory role for KLF13 in co-regulation of expression of CD21 and CD23. Thus KLF13 is involved at multiple different checkpoints in development that require signalling through the TCR, pre-BCR or mature BCR.

Repression of hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation.

Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2DeltaC2), increased T-cell differentiation and activation in response to TCR signalling. Expression of the Gli2DeltaC2 transgene increased differentiation from CD4(+)CD8(+) to single positive thymocyte, and increased peripheral T cell populations. Gli2DeltaC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.

Splenomegaly and modified erythropoiesis in KLF13-/- mice.

To study the function of the Krüppel-like transcription factor KLF13 in vivo, we generated mice with a disrupted Klf13 allele. Although Klf13(-/-) mice are viable, fewer mice were present at 3 weeks than predicted by Mendelian inheritance. Viable Klf13(-/-) mice had reduced numbers of circulating erythrocytes and a larger spleen. The spleen contained an increased number of Ter119(med)CD71(hi), Ter119(hi)CD71(hi), and Ter119(hi)CD71(med) cells but not Ter119(hi)CD71(-) cells, indicating an increase in less mature erythroblasts. A higher proportion of the Ter119(med)CD71(hi) cells were proliferating, indicating that the mice were under a degree of erythropoietic stress. These data indicate that KLF13 is involved in the normal control of erythropoiesis.

Sonic hedgehog signalling in T-cell development and activation.

The production of mature functional T cells in the thymus requires signals from the thymic epithelium. Here, we review recent experiments showing that one way in which the epithelium controls the production of mature T cells is by the secretion of sonic hedgehog (SHH). We consider the increasing evidence that SHH-induced signalling is not only important for the differentiation and proliferation of early thymocyte progenitors, but also for modulating T-cell receptor signalling during repertoire selection, with implications for positive selection, CD4 versus CD8 lineage commitment, and clonal deletion of autoreactive cells. We also review the influence of hedgehog signalling in peripheral T-cell activation.

Beta-selection: abundance of TCRbeta-/gammadelta- CD44- CD25- (DN4) cells in the foetal thymus.

Expression of TCRbeta and pre-TCR signalling are essential for differentiation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double-positive (DP) stage. Thymocyte development in adult Rag1, Rag2 or TCRbetadelta-deficient mice is arrested at the DN3 stage leading to the assumption that pre-TCR signalling and beta-selection occur at, and are obligatory for, the transition from DN3 to DN4. We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta. These foetal icTCRbeta-/gammadelta- DN4 cells were T lineage as determined by expression of Thy1 and icCD3 and TCRbeta DJ rearrangement. In addition, in the foetal Rag1-/- thymus, a normal percentage of DN4 cells were present. In wild-type mice after hydrocortisone-induced synchronisation of differentiation, the majority of DN4 cells that first emerged did not express icTCRbeta/gammadelta, showing that adult thymocytes can also differentiate to the DN4 stage independently of pre-TCR signalling. Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis. Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.

Activation of the Hedgehog signaling pathway in T-lineage cells inhibits TCR repertoire selection in the thymus and peripheral T-cell activation.

TCR signal strength is involved in many cell fate decisions in the T-cell lineage. Here, we show that transcriptional events induced by Hedgehog (Hh) signaling reduced TCR signal strength in mice. Activation of Hh signaling in thymocytes in vivo by expression of a transgenic transcriptional-activator form of Gli2 (Gli2DeltaN(2)) changed the outcome of TCR ligation at many stages of thymocyte development, allowing self-reactive cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection; reducing the ratio of CD4/CD8 single-positive (SP) cells; and reducing cell surface CD5 expression. In contrast, in the Shh(-/-) thymus the ratio of CD4/CD8 cells and both positive and negative selection of a transgenic TCR were increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from double-positive (DP) to SP cell in a physiological situation. In peripheral T cells, Gli2DeltaN(2) expression attenuated T-cell activation and proliferation, by a mechanism upstream of ERK phosphorylation.

The transcription factor Gli3 regulates differentiation of fetal CD4- CD8- double-negative thymocytes.

Glioblastoma 3 (Gli3) is a transcription factor involved in patterning and oncogenesis. Here, we demonstrate a role for Gli3 in thymocyte development. Gli3 is differentially expressed in fetal CD4- CD8- double-negative (DN) thymocytes and is most highly expressed at the CD44+ CD25- DN (DN1) and CD44- CD25- (DN4) stages of development but was not detected in adult thymocytes. Analysis of null mutants showed that Gli3 is involved at the transitions from DN1 to CD44+ CD25+ DN (DN2) cell and from DN to CD4+ CD8+ double-positive (DP) cell. Gli3 is required for differentiation from DN to DP thymocyte, after pre-T-cell receptor (TCR) signaling but is not necessary for pre-TCR-induced proliferation or survival. The effect of Gli3 was dose dependent, suggesting its direct involvement in the transcriptional regulation of genes controlling T-cell differentiation during fetal development.

Sonic hedgehog is produced by follicular dendritic cells and protects germinal center B cells from apoptosis.

The Hedgehog (Hh) signaling pathway is involved in the development of many tissues during embryogenesis, but has also been described to function in adult self-renewing tissues. In the immune system, Sonic Hedgehog (Shh) regulates intrathymic T cell development and modulates the effector functions of peripheral CD4(+) T cells. In this study we investigate whether Shh signaling is involved in peripheral B cell differentiation in mice. Shh is produced by follicular dendritic cells, mainly in germinal centers (GCs), and GC B cells express both components of the Hh receptor, Patched and Smoothened. Blockade of the Hh signaling pathway reduces the survival, and consequently the proliferation and Ab secretion, of GC B cells. Furthermore, Shh rescues GC B cells from apoptosis induced by Fas ligation. Taken together, our data suggest that Shh is one of the survival signals provided by follicular dendritic cells to prevent apoptosis in GC B cells.

Diacylglycerol kinase alpha activity promotes survival of CD4+ 8+ double positive cells during thymocyte development.

The diacylglycerol kinases (DGK) form a family of isoenzymes that catalyse the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), both powerful second messengers in the cell. DGKalpha is expressed in brain, peripheral T cells and thymocytes and has been shown to translocate to the nuclear matrix upon T-cell receptor (TCR) engagement. Here, we show that high level expression of DGKalpha is induced following a signal transmitted through the pre-TCR and the protein tyrosine kinase, lck. Activity of DGKalpha contributes to survival in CD4+ 8+ (DP) thymocytes as pharmacological inhibition of DGK activity results in death of this cell population both in cell suspension and thymic explants. DGKalpha promotes survival in these thymocytes through a Bcl-regulated pathway. A consequence of inhibition of DGKalpha is the specific down-regulation of Bcl-xl, whereas in transgenic mice that over-express Bcl-2, death induced by the inhibitor is partially blocked. Thus we report a novel activity of DGKalpha in survival of thymocytes immediately after entry into the DP stage in development.

Reduced thymocyte development in sonic hedgehog knockout embryos.

The Hedgehog family of secreted intercellular signaling molecules are regulators of patterning and organogenesis during animal development. In this study we provide genetic evidence that Sonic Hedgehog (Shh) has a role in the control of murine T cell development. Analysis of Shh(-/-) mouse embryos revealed that Shh regulates fetal thymus cellularity and thymocyte differentiation. Shh is necessary for expansion of CD4(-)CD8(-) double-negative (DN) thymocytes and for efficient transition from the earliest CD44(+)CD25(-) DN population to the subsequent CD44(+)CD25(+) DN population and from DN to CD4(+)CD8(+) double-positive cells.

Bone morphogenetic protein 2/4 signaling regulates early thymocyte differentiation.

Bone morphogenetic protein (BMP)2 and BMP4 are involved in the development of many tissues. In this study, we show that BMP2/4 signaling is involved in thymocyte development. Our data suggest that termination of BMP2/4 signaling is necessary for differentiation of CD44(+)CD25(-)CD4(-)CD8(-) double negative (DN) cells along the T cell lineage. BMP2 and BMP4 are produced by the thymic stroma and the requisite BMP receptor molecules (BMPR-1A, BMPR-1B, BMPR-II), and signal transduction molecules (Smad-1, -5, -8, and -4) are expressed by DN thymocytes. BMP4 inhibits thymocyte proliferation, enhances thymocyte survival, and arrests thymocyte differentiation at the CD44(+)CD25(-) DN stage, before T cell lineage commitment. Neutralization of endogenous BMP2 and BMP4 by treatment with the antagonist Noggin promotes and accelerates thymocyte differentiation, increasing the expression of CD2 and the proportion of CD44(-)CD25(-) DN cells and CD4(+)CD8(+) double-positive cells. Our study suggests that the BMP2/4 pathway may function in thymic homeostasis by regulating T cell lineage commitment and differentiation.