| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Immunology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and a functionally rearranged TCR
-chain mediate this checkpoint, termed selection. After successful pre-TCR signaling, DN thymocytes undergo many rounds of division and multiple phenotypic changes. In addition to genes that encode pre-TCR components, a number of other genes regulate maturation. These genes either affect pre-TCR signaling indirectly or are required for the numerous cellular changes seen during the DN to DP transition (2, 3, 4, 5). The Notch1 signaling pathway has been proposed to play a role during various stages of T cell development. Notch response genes such as Deltex1 are regulated during thymocyte development. Deltex1 is highly expressed in DN, down-regulated in DP cells, and up-regulated in mature CD8 and CD4 single-positive cells (SP) (6). Studies with transgenic mouse models and retrovirally transduced stem cells have shown that expression of the active intracellular portion of Notch1, Notch1IC, can modify the survival, proliferation, and maturation of thymocytes and potentially modulate TCR signaling (6, 7, 8, 9, 10, 11). In addition, pharmacological inhibition of Notch signaling in thymic organ culture systems impairs thymocyte development (12, 13). Specific gene deletion of Notch1 at the earliest lymphoid precursors obviates further development into the T lineage (14, 15).
The Notch1 protein belongs to a family of receptors that upon interaction with ligand releases the intracellular fragment, Notch1IC, via a proteolytic cleavage event. Notch1IC translocates into the nucleus, binds C promoter binding factor 1, and activates transcription (16). Target genes induced by Notch1 in T cell lines include Hes family members, Deltex1, and pre-T (6, 17, 18). Multiple homologues of the Notch1 receptor and a number of different Notch ligands are expressed throughout the normal thymus. The four Notch family members share homology in their intracellular domains, and all have been shown to bind to C promoter binding factor 1. Therefore, intracellular signaling of the family members may converge on similar target genes. For example, overexpression of the active intracellular forms of Notch1, Notch2, and Notch3 is able to up-regulate Hes family members (19, 20, 21).
The mammalian Notch1 receptor was originally identified in a subset of human T cell acute lymphoblastic leukemias (T-ALL), where a chromosomal translocation resulted in a Notch1 truncation, ostensibly Notch1IC, brought under the control of a T cell-specific promoter (22). In mice this has been recapitulated by the expression of intracellular fragments of both human and mouse Notch1 and mouse Notch3 (23, 24, 25). Tumor development with these constructs, even when expressed in all hemopoietic cells, occurs exclusively in immature thymocytes (23, 24). In addition, in vivo mouse mammary tumor virus insertional mutagenesis studies identified intracellular Notch1 constructs as putative collaborators with c-Myc transformation in the majority of the isolated tumors (26). However, the cellular mechanisms that are targeted by Notch1IC signaling during thymomagenesis have not been well characterized.
Notch1 has been clearly implicated in promoting T lineage choice from common lymphoid progenitors, although its role in later development is less clear (15, 27). Inducible gene ablation of Notch1 via Lck-Cre-mediated deletion suggests its role at least through the DN stages (14). However, ablation of Notch1 at the late DN stages via CD4/Cre-mediated gene deletion allows normal thymocyte development (28). On the other hand, overexpression of Notch1IC, pharmacological inhibition of Notch signaling, and the pattern of Notch target gene expression suggest a role at the later stages of DP to SP development (6, 10, 12, 13). In light of the conflicting data on the physiological role of Notch1 during different stages of thymocyte development, we sought to determine at what stages of development is biologically accessible Notch1 receptor expressed on thymocytes and to correlate it with Notch target gene expression. Notch ligand family members Delta and Jagged, have been shown to be expressed throughout the thymus, and all are able to interact with Notch1 (29, 30). This suggests that the surface expression of Notch proteins may play an important role in regulating whether a cell is capable of receiving a Notch signal. We show that specific staining of surface Notch1 is apparent on immature DN thymocytes and is down-regulated as the cells mature to DP and later stages. Using a recombinase-activating gene (RAG)-deficient model and anti-CD3 to provide the pre-TCR signal, we show that enforced expression of Notch1IC augments the DN to DP transition. These data suggest a role for Notch1 signaling in the proliferative expansion and differentiation of DN cells following -selection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human 293 HEK cells were transfected with full-length murine Notch1 (FLN1/293) inserted into the pcDNA3 vector (Stratagene, La Jolla, CA) or with vector alone. Clones were selected in DMEM with 10% BSA and 1 mg/ml G418, and the expression of murine Notch1 was confirmed by Western analysis with Abs to both intracellular and extracellular portions of mNotch1.
Ab and cell staining
The anti-Notch1ec reagent is an immunoaffinity-purified rabbit IgG raised against the rat Notch1 epidermal growth factor (EGF) 10-EGF22, residues 381–853 (31) (Upstate Biotechnology, Lake Placid, NY), which cross-reacts with mouse Notch1. Cells were stained using a tertiary protocol with a biotinylated goat anti-rabbit Ab, followed by a streptavidin-conjugated fluorochrome. Purified normal rabbit IgG (Upstate Biotechnology) was also included as a staining control. Fluorochrome-conjugated Abs for CD4, CD8, CD24 (heat shock Ag), CD44, CD25, CD69, B220, CD3 (BD PharMingen (San Diego, CA) and eBioscience (San Diego, CA)) were used to phenotypically characterize and sort cell populations by FACS analysis.
GST fusion protein corresponding to the murine extracellular region recognized by anti-N1ec, residues 381–853 (mN1ec-GST), and GST alone were made as blocking agents and controls for surface staining. Recombinant proteins were produced in BL-21 Gold (DE3) bacteria (Stratagene, La Jolla, CA) and purified using the pGEX2T protocol (American Biosciences, Piscataway, NJ). Anti-N1ec Ab was preincubated with 5 µg/ml of mN1ec-GST or GST for 15 min at room temperature before the FACS staining protocol.
Mice
C57BL/6 (B6) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Notch1IC (N1ic) mice that express the intracellular portion of murine Notch1 under the Lck-proximal thymic promoter (6) were crossed >10 generations onto the B6 background and mated to RAG2-deficient mice on a B6 background (The Jackson Laboratory) to obtain Notch1IC x RAG2-/- and RAG2-/- littermate control mice.
To induce DN to DP development in RAG2-deficient mice, 100 µg of 2C11, anti-CD3
, mAb was delivered in one i.p. injection. Mouse thymi were harvested and analyzed at various time points after injection, as indicated. 2C11 mAb was immunoaffinity purified from ascites and dialyzed against sterile PBS.
Transcript expression analysis
Sorted subsets. Thymocytes were isolated from 4- to 6-wk-old B6 mice and sorted on a FACSVantage (BD Biosciences, San Diego, CA) into phenotypic thymocyte subsets; unsorted thymocytes, DN (CD3-, CD8-, CD4-, B220-), DP (CD8+, CD4+), CD4 (CD4+, CD3+), CD8 (CD8+, CD3+), and DN subsets as previously described (32); unsorted DN (CD3-, CD8-, CD4-, B220-), DN1(DN and CD44+, CD25-), DN2 (DN and CD44+, CD25+), DN3 (DN and CD44-, CD25+), and DN4 (DN and CD44-, CD25-). RNA and cDNA were prepared using RNA STAT 60 (Tel-Test, Friendswood, TX) and SuperScript II (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
The cDNA samples were analyzed in triplicate by real-time PCR with sequence-specific probes on an ABI 7700 sequence detector (PE Applied Biosystems, Foster City, CA). Primers and probes were designed to span exon/intron borders to prevent amplification of genomic sequence: hypoxanthine phosphoribosyltransferase (HPRT): forward, 5'-TGG AAA GAA TGT CTT GAT TGT TGA A; reverse, 5'-AGC TTG CAA CCT TAA CCA TTT TG; probe, 5'-FAM-CAA ACT TTG CTT TCC CTG GTT AAG CAG TAC AGC-TAMRA; Notch1: forward, 5'-GAG ACC AAG AAG TTC CGG TTT G; reverse, 5'-CTT CAC TGT TGC CTG TCT CAA GG; probe, 5'-FAM-CAA TGT TCG AGG ACC AGA TGG CTT CAC A-TAMRA; Deltex1: forward, 5'-TGA GGA TGT GGT TCG GAG GT; reverse, 5'-CCC TCA TAG CCA GAT GCT GTG; probe, 5'-FAM-CGC CTG ATG AGG ACT GTA CCA TTT GCA T-TAMRA; and pre-Ta: forward, 5'-CTG CTT CTG GGC GTC AGG T; reverse, 5'-TGC CTT CCA TCT ACC AGC AGT; probe, 5'-FAM-CCT TTC CGT CTC TGG CTC CAC CCA-TAMRA. In a 50-µl reaction cDNA was incubated with 20 µM forward and reverse primers (Invitrogen), 10 µM 5',6-FAM-3'-TAMRA fluorogenic probe (Biosearch Technologies, Novato, CA), and TaqMan 2x universal PCR Mastermix (PE Applied Biosystems). No template controls and no reverse transcriptase controls were included for each primer/probe set and cDNA set, respectively. Transcript levels were normalized to HPRT levels before determining the relative fold difference in the gene of interest. RT-PCR analysis of the Notch1IC transgene was performed as described previously (6).
Histology
Analyses of 6- to 8-wk-old B6 mouse thymuses used an indirect enzyme immunohistochemical procedure (33). Briefly, frozen sections of tissue were serially incubated with optimal dilutions of anti-N1ec preincubated with mN1ec-GST or GST as a control. Notch1 was detected by sequential exposure to digoxigenin-conjugated goat anti-rabbit IgG and peroxidase-conjugated Fab of goat anti-digoxigenin Abs. Peroxidase activity was revealed with 3,3'-diaminobenzidine in the presence of hydrogen peroxide.
Cell cycle analysis
Cells were stained with the DNA-binding dye 7-amino actinomycin D to determine DNA content (BD Biosciences). Briefly, cells were stained for cell surface Ags, permeabilized, fixed, and stained with 0.25 µg of 7-amino actinomycin D/1 x 106 cells before FACS analysis. Cells were gated on the appropriate thymic subsets as defined by CD8 and CD4 cells surface expression and were analyzed for the percentage of cells with >2 N DNA content. B6 DP thymocytes, which are mostly in G0, were used to define 2 N gates.
Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation
Thymocytes were pooled from 3- to 5-wk-old Notch1IC x RAG2-/- or RAG2-/- mice. Cells were stimulated with 5 ng/ml PMA at 37°C at 1E7 cells/ml in HBSS with 1% BSA for the indicated times. Cells were lysed in TBS, 1% Triton 0.2 mM Na3VO4, 50 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF. Supernatant from 2.5 x 106 thymocyte equivalents was resolved by SDS-PAGE on a 10% gel and transferred to Optitran nitrocellulose membrane (Schleicher & Schuell, Keene, NH) Immunoblot analysis with anti-phospho-ERK Ab (p44/42 MAPK; Cell Signaling, Beverly, MA) was performed according to the manufacturer’s protocol. The blot was subsequently stripped in 100 mM 2-ME, 2% SDS, and 62.5 mM Tris (pH 6.8) at 50°C for 30 min; reblocked; and immunoblotted for total ERK (ERK2; Santa Cruz Antibodies, Santa Cruz, CA) as a loading control. Proteins were detected by HRP-conjugated secondary Abs (Santa Cruz Antibodies) and ECL according to the manufacturer’s protocol (Amersham Pharmacia Biotech, Piscataway, NJ).
OP9 monolayer cultures
DP and DN3 thymocytes were isolated from 4- to 6-wk old mice. CD8+CD4+CD69low (DP) thymocytes were isolated by FACS sorting. For isolation of DN3 thymocytes, the total DN population was enriched by immunomagnetic separation using biotinylated Abs to CD4 and CD8 (Dynal, Oslo, Norway) according to the manufacturer’s instructions, and the DN3 population was sorted on a FACS vantage. Total DN (CD4-CD8-) thymocytes were negatively gated for lineage markers (CD4, CD8, CD3, B220, Mac1, Gr1, and Ter114), and the DN3 fraction was identified as CD44-CD25+. Sorted thymocyte subsets (1 x 105) were plated on monolayers of OP9 or OP9 cells expressing the Notch ligand, Delta-like 1 (OP9-Dl1) (34), in 24-well plates in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 15% FCS, penicillin/streptomycin, L-glutamine, and 5 ng/ml of rIL-7 and Flt3L (PeproTech, Rocky Hill, NJ). The growth and viability of the thymocytes were assessed by Trypan Blue staining.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To date there has been little characterization of Notch1 surface expression in mammalian systems, including the thymus. We first sought to identify and characterize an Ab that is able to specifically stain surface Notch1. We used a rabbit immunoaffinity-purified IgG against the extracellular portion of rat Notch1, corresponding to EGF10-EGF22 anti-N1ec, for immunohistochemistry and FACS staining (Fig. 1A). Specificity for surface mouse Notch1 was determined by three assays. Firstly, 293 cells stably expressing full-length murine Notch1, FLN1/293, were generated. The transfected cells stained positively using anti-N1ec compared with control cell lines transfected with vector alone (cDNA3/293) and Ab controls (Fig. 1B). Secondly, anti-N1ec was preadsorbed on FLN1/293 or control cDNA3/293 monolayers, and specific staining of Notch1 on the FLN1/293 and thymocytes was compared. Preadsorption on FLN1/293, but not on control cDNA3/293 removed all staining activity (data not shown). Finally, a GST fusion protein containing the murine residues corresponding to the immunogen (i.e., EGF10–22), mN1ec-GST, was able to block staining on FLN1/293 cells and thymocytes when preincubated with anti-N1ec relative to GST controls (Figs. 1C, 2, and 3).
|
|
|
The expression of endogenous Notch1 during thymocyte development has not been well characterized. The majority of published reports have examined mRNA, and these results have been inconsistent. The expression of Notch1 at the cell surface requires several post-transcriptional modifications, including proteolytic processing, assembly in the endoplasmic reticulum, and transport to the cell surface before ligand-induced signaling. To address which cells may be capable of receiving a Notch1 signal, we assayed for the expression of Notch1 in the thymus by immunohistochemistry.
Using anti-N1ec to stain C57BL/6 thymus sections, we found that specific Notch1ec staining is localized primarily to the outer cortex and subcapsular zone of the thymus (Fig. 2). This staining is blocked in the presence of mN1ec-GST fusion protein. The expression appears to be intercellular and suggests cell surface expression of Notch1. In the medulla, where mature SP thymocytes are thought to reside before export, Notch1 staining is less apparent (Fig. 2). This pattern of staining is consistent with expression by early thymocytes. T cell progenitors are thought to enter the thymus at the cortical-medullary junction and migrate to the subcapsular zone during DN development. Specifically at the subcapsular zone late DN populations, DN3 and DN4 thymocytes, are present (35).
To identify the population of thymocytes expressing surface Notch1, we used anti-N1ec in FACS analysis of cells isolated from normal C57BL/6 mice. We detected Notch1 surface expression in the majority of DNs and on a subset of DP and CD8 SP cells (Fig. 3B). Staining of CD8 SP cells was surprising given the lack of staining in the thymus medulla (Fig. 2). This apparent discrepancy was resolved when we analyzed Notch 1 surface expression on immature CD8 SPs (ISPs), which express high levels of CD24 and are an intermediate step between DN and DP stage, vs staining on mature, CD24low, CD3highCD8 SP thymocytes. The data in Fig. 3C show that only the immature, pre-DP CD8+ T cells stain at the surface for Notch 1.
We further examined Notch1 surface expression on the DN subpopulations by costaining for CD44 and CD25. We found surface expression of Notch1 up-regulated at the DN2 stage, when cells become fully committed to the T lineage, and remained throughout the DN and ISP compartments until down-regulated at the DP stage (Fig. 3, B and C, and Fig. 4B) (36). The early expression in DN thymocytes was further confirmed by real-time cDNA analysis of DN, DP, and mature CD4 and CD8 SP subsets (Fig. 3D). We found that the Notch1 transcript is highly expressed in the DN subset relative to the levels in DP and SP subsets.
|
, are regulated to the same extent (6). This may be due to additional control by other transcriptional regulators and other Notch family members. These data suggest a role for Notch1 and activation of the Notch signaling pathway during immature thymocyte development, specifically during the late DN transition. This is consistent with a previous report that intracellular Notch1 expression is highest in DN thymocytes (38). Notch1 activation during the DN-to-DP transition increases thymic cellularity and cell cycling
Given the expression pattern of Notch1 at the cell surface and the evidence for activation of the pathway, as indicated by target gene expression, we investigated the function of Notch1 during early thymocyte development. During the DN to DP transition there are several distinguishing hallmarks of thymocyte development. Upon functional rearrangement of the TCR, a pre-TCR signal is delivered, allowing transition from the DN3 to the DP stage and a coincident proliferative expansion. We asked what role the expression of an active Notch1 transgene would play during this developmental stage.
The lack of RAG recombinase machinery prevents TCR rearrangement and thus precludes -selection. However, dysregulation of other genes that affect survival and cell cycle, TCR signaling, or transcriptional activation, such as p53, Lck, -catenin, and FADD, permit development to the DP stage independent of pre-TCR signaling. (39, 40, 41, 42) To determine whether Notch1 activation alone would cause DN to DP development in the absence of a pre-TCR signal, we analyzed thymi from mice expressing an active form of Notch1, Notch1IC, under the thymus-specific Lck-proximal promoter on a RAG2-deficient background, Notch1IC x RAG2-/-. Activated Notch1IC is unable to overcome the RAG developmental block, as thymus cellularity is similar to RAG2-/- controls, and the cells remain at the DN3 stage (Fig. 5A). To check that the Notch1IC transgene is expressed at this early stage, transgene-specific primers were used in semiquantitative RT-PCR assay to show the presence of the NotchIC transcript. As expected from the Lck-proximal promoter, Notch1IC is expressed in the RAG2-deficient background, although at lower levels than in the RAG-sufficient thymocytes, which are predominately DP cells (Fig. 5B). This result is consistent with the failure of Notch1IC retrovirally introduced into RAG-deficient hemopoietic stem cells to develop into DP cells (43). Additionally, the Notch1IC x RAG2-/- mice or retrovirally transduced hemopoietic stem cells do not generate thymomas normally associated with enforced Notch1IC expression, suggesting a downstream pathway relative to the pre-TCR signal for Notch1IC signaling in thymomagenesis.
|
-selection and results in proliferation and developmental progression from the DN to the DP stage (44). To determine whether constitutive activation of the Notch1 pathway can influence early thymocyte development after -selection, we injected anti-CD3 into Notch1IC x RAG2-/- mice and RAG2-/- littermate controls. Two days after anti-CD3 treatment, Notch1IC x RAG2-/- mice show an increased rate of progression from DN to DP and increased thymic cellularity compared with their RAG-/- littermates similarly injected with anti-CD3 (Fig. 6, A and B). The greatest difference is seen in the ISP CD8+ compartment, with a 3-fold increase compared with RAG2-/- treated with anti-CD3 (Fig. 6C). The time course after anti-CD3 treatment shows an increase in cellularity as early as 2 days until 7 days after injection. In addition, the kinetics of development, as measured by the percentage of cells that have left the DN compartment, appear to be increased in the Notch1IC x RAG2-/- relative to controls from day 2 to day 4 (Fig. 6A and data not shown). Time points after 14 days showed similar cellularity at 
300 x 106 thymocytes, presumably due the depletion of anti-CD3 Ab over time or complete filling of the thymic compartment (data not shown).
|
Pre-TCR signals are not affected by Notch1 activation
To determine whether the increase in cellularity and the fraction of cycling cells in anti-CD3-treated Notch1IC x RAG2-/- mice is due to an effect of Notch1 activation on pre-TCR signaling, we analyzed three components of the pre-TCR signaling cascade. Because the pre-T gene is a target of Notch1 activation, we analyzed the expression of pre-T and surface CD3 levels to determine whether the difference in response could be attributed to differences in the levels of the TCR components. We saw no increase in basal levels of surface CD3 by FACS analysis in Notch1IC x RAG2-/- mice compared with littermate controls (data not shown). Similarly, using real-time PCR to measure mRNA transcripts, we found no significant increase in pre-T expression in Notch1IC x RAG2-/- thymocytes (Fig. 7A). We also performed a kinetic analysis of ERK phosphorylation in response to PMA stimulation to determine whether proximal phosphorylation events of the TCR pathway were modulated by an active Notch1 signal. This kinetic analysis allowed us to determine whether changes in the absolute levels or in the duration of signals were affected by enforced activation of Notch1. We found that ERK phosphorylation is induced and sustained after stimulation with the same kinetics in Notch1IC x RAG2-/- and control RAG2-/- thymocytes (Fig. 7B).
|
These results indicate that both proximal and distal points of the pre-TCR signaling cascade are unaffected by concurrent activation of the Notch1 pathway, suggesting that Notch1IC affects proliferation by augmenting a parallel pathway to the pre-TCR cascade or via other downstream effectors of the pre-TCR cascade outside the ERK signaling branch.
Delta-like-1-dependent proliferation of DN in vitro
To examine the role of Notch signaling in a more physiological setting, i.e., without an active NotchIC transgene, we turned to the recent description of T lymphopoiesis in vitro driven by a stromal cell line expressing the Notch ligand, Dl1 (34). The OP9 stromal cell line can support B cell development from fetal liver or bone marrow precursors, and Schmidt and Zuniga-Pflucker (34) showed that following expression of full-length Dl1 in OP9, T, but not B, cell development ensued. To determine whether Notch-Notch ligand signaling would enhance the expansion of DN cells from normal mice, we FACS-purified DN3 and DP cells from normal B6 thymus, plated them on monolayers of control OP9 and OP9-Dl1, and followed their progress by cell counts. The results presented in Fig. 8 complement our previous conclusions on the role of Notch signaling at the DN to DP transition. Thus, DN3 cells plated on OP9-Dl1 expanded dramatically, while following plating on control OP9 monolayers, they only maintained the starting number. Also in line with our Notch1 expression analysis, CD69low DP thymocytes showed no difference in response when plated on control or Dl1-expressing OP9 monolayers.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our staining results are in general agreement with previous work by Hasserjian et al. (38). In that work, intracellular staining of BALB/c mouse thymocytes using an Ab against the cytoplasmic portion of human Notch1 suggested that all thymus subsets express Notch1, compared with the staining with an anti-GST-negative control. They reported highest levels in DN subsets and lower levels in DP and SP subsets.
We have previously shown that Notch target genes are up-regulated in both CD4 and CD8 SP compared with the levels in their DP precursors (6, 7). Thus, it remains possible that other Notch family members are important for DP to SP development. This possibility is supported by data showing that low levels of presenilin inhibitors can block SP development in fetal thymic organ cultures (12, 13). In these studies the inhibitors block 
-secretase/presenilin-dependent cleavage, a step shared by all Notch family members for ligand-dependent signaling of the Notch receptors. Finally, multiple ligands that interact with all the Notch receptors are expressed throughout the thymus (data not shown), allowing the potential for ligand-dependent Notch signaling at a variety of developmental stages. These findings suggest that the effect of Notch1IC on SP development may be interpreted as activating other Notch pathways or target genes that may be physiologically important during SP development.
In a recent study Wolfer et al. (14) reported that deletion of Notch1 at the DN2 stage via the expression of Lck-Cre resulted in an impairment in the rate of VDJ recombination at the TCR locus. They also noted an accumulation of aberrant DN4 thymocytes that lacked a functionally rearranged TCR-chain. To explain the latter observation they hypothesized that signaling through Notch1 is required to eliminate DN thymocytes that have failed TCR selection (14). In line with this, our results show that Notch1 is highly expressed at the cell surface from the DN2 to ISP stage, and the levels of Deltex1 mRNA are highest at the DN3 stage. Breeding the Notch1IC transgenic mice to RAG2-/- mice allowed us to ask what effect constitutive Notch signaling would have on early thymocytes. We found no evidence that enforced Notch signaling increased the turnover rate or apoptosis of RAG2-/- thymocytes that do not express a functional -chain. Staining RAG2-/- controls vs Notch1IC x RAG2-/- thymocytes for DNA content or for apoptotic cells showed no difference between the two (data not shown).
Enforced Notch1 signaling also did not alter the phenotype of the RAG2-/- thymus; cellularity was unchanged, and most cells remained arrested at the DN3 stage (Fig. 5). This establishes a hierarchy of Notch1 signaling relative to pre-TCR signaling. For example, constitutive activation of Lck and -catenin in RAG-deficient mice results in DN to DP transition, suggesting that development in these mice is pre-TCR-independent and that Lck and -catenin are proposed downstream targets after initiation of the pre-TCR signal (41). Conversely, the DN3 block seen in Notch1IC x RAG2-/- suggests that Notch1 signaling at this stage is upstream or separate from the pre-TCR signaling cascade. Previously, two groups using Notch1IC and Notch3IC have shown that rapid leukemia development requires pre-TCR signaling. From these experiments it is unclear whether NotchIC requires a specific stage development, i.e., after -selection, to induce transformation or whether NotchIC plays a role in affecting pre-TCR signaling, which leads to transformation. The developmental timeline in our experiments evaluates developmental and cellular changes from a few hours to a few days after -selection, i.e., proximal to the pre-TCR signal (25, 43).
Mimicking pre-TCR signaling by injection of anti-CD3 Ab allowed us to examine whether the Notch1IC transgene would have an effect on the kinetics of the DN to DP transition that follows -selection. Notch1IC increases the rate of development of DN3 cells to the ISP and then the DP compartment. Thus, in addition to the role for Notch1 signaling in driving TCR rearrangement (14), we propose that Notch signaling may play a positive role following pre-TCR signaling. One explanation for the enhanced development of Notch1IC x RAG2-/- thymocytes following anti-CD3 injection could have been that the DN3 cells in these mice express a higher level of CD3 and would therefore receive a stronger signal from the anti-CD3 Ab. Relevant to this is the fact that pre-T has been shown to be a target of Notch signaling in thymomas and thymocytes (7). Even in the absence of a TCR-chain, it is conceivable that increased levels of pre-T could up-regulate the surface expression of CD3. Our expression analysis, however, showed no heightened expression of pre-T nor any increase in detectable levels of surface CD3 (data not shown) in the Notch1IC transgenic thymocytes. Furthermore, by studying the degree and kinetics of ERK phosphorylation and CD69 up-regulation in stimulated thymocytes from RAG2-/- and Notch1IC x RAG2-/- mice, we found no evidence that enforced Notch signaling modulated the pre-TCR signaling cascade. Finally, the comparison of the in vitro growth of DN3 thymocytes from control B6 mice on OP9 vs OP9-Dl1 stromal cells strongly supports a role for Notch signals in the DN to DP expansion (Fig. 8).
The effect of Notch1IC on proliferation may shed light on the cellular mechanisms that Notch1IC targets as an oncogene in the maintenance of thymoma growth and tumorigenesis (47). Recently, Aster’s group (48) has shown that pharmacological inhibition of the Notch pathway in Notch1IC-induced T-ALL lines results in cell cycle arrest and induction of apoptosis. This suggests that constitutive Notch1 activation is required for the growth potential of Notch1IC-induced thymomas in addition to any effects Notch1IC may have on differentiation of the tumor.
Physiological Notch signaling as well as oncogenic Notch1IC signaling may be important in up-regulating genes that promote proliferation, such as the bHLH gene, Hes1. Hes1 is up-regulated in DN subsets, and deletion of Hes1 results in an autonomous defect in the ability of immature thymocytes to proliferate (49, 50). In vivo Hes1 deficiency blocks development at the DN2 and DN3 stages, which correlates with Notch1 pathway activity (Fig. 4C). Although the molecular mechanism for Hes1 involvement in proliferation is not well characterized, there is evidence that Hes1 inhibits E protein activity by binding E47 and preventing transcriptional repression. In line with this, the loss of E47 activity results in early DN proliferation and tumorigenesis (51). We hypothesize that enforced expression of Notch1IC results in an increase in Hes1-dependent proliferation at the DN3 stage. Additionally, Notch1IC may augment thymocyte proliferation by inhibiting E protein activity in a manner independent of Hes1 (52). It will be a challenge for the future to determine the molecular mechanism by which Notch1IC induces the development and perpetuates the growth of T-ALL.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Michael J. Bevan, Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195-7370. E-mail address: mbevan{at}u.washington.edu
3 Abbreviations used in this paper: DN, double negative; DP, double positive; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; HPRT, hypoxanthine phosphoribosyltransferase; ISP, immature single positive; RAG, recombinase-activating gene; SP, single positive; T-ALL, T cell acute lymphoblastic leukemia.
Received for publication February 14, 2003. Accepted for publication July 1, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene in development of but not 
T cells. Nature 375:795.[Medline]
-secretase activity modulates thymocyte development. Proc. Natl. Acad. Sci. USA 98:9312.-Secretase inhibitors repress thymocyte development. Proc. Natl. Acad. Sci. USA 98:7487. rearrangement and allows pre-TCR-independent survival of early lineage thymocytes. Immunity 16:869.[Medline]
and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis. Proc. Natl. Acad. Sci. USA 99:3788.-catenin bypasses pre-TCR signaling and TCR selection in thymocyte development. Nat. Immunol. 2:863.[Medline]
-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon. Proc. Natl. Acad. Sci. USA 90:11401.This article has been cited by other articles:
|
|
 |
|
  Y. Yashiro-Ohtani, Y. He, T. Ohtani, M. E. Jones, O. Shestova, L. Xu, T. C. Fang, M. Y. Chiang, A. M. Intlekofer, S. C. Blacklow, et al. Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A Genes & Dev., July 15, 2009; 23(14): 1665 - 1676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dumont, A. Corsoni-Tadrzak, S. Ruf, J. de Boer, A. Williams, M. Turner, D. Kioussis, and V. L. J. Tybulewicz Rac GTPases play critical roles in early T-cell development Blood, April 23, 2009; 113(17): 3990 - 3998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Van de Walle, G. De Smet, M. De Smedt, B. Vandekerckhove, G. Leclercq, J. Plum, and T. Taghon An early decrease in Notch activation is required for human TCR-{alpha}{beta} lineage differentiation at the expense of TCR-{gamma}{delta} T cells Blood, March 26, 2009; 113(13): 2988 - 2998. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kleinmann, A.-S. Geimer Le Lay, M. Sellars, P. Kastner, and S. Chan Ikaros Represses the Transcriptional Response to Notch Signaling in T-Cell Development Mol. Cell. Biol., December 15, 2008; 28(24): 7465 - 7475. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chari and S. Winandy Ikaros Regulates Notch Target Gene Expression in Developing Thymocytes J. Immunol., November 1, 2008; 181(9): 6265 - 6274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Martin, P. S. Woll, Z. Ni, J. C. Zuniga-Pflucker, and D. S. Kaufman Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells Blood, October 1, 2008; 112(7): 2730 - 2737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Varnum-Finney, M. H. Dallas, K. Kato, and I. D. Bernstein Notch target Hes5 ensures appropriate Notch induced T- versus B-cell choices in the thymus Blood, March 1, 2008; 111(5): 2615 - 2620. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-F. Wang, M. Aoki, Y. Nakashima, Y. Shinozuka, H. Tanaka, M. Taniwaki, M. Hattori, and N. Minato Development of Notch-dependent T-cell leukemia by deregulated Rap1 signaling Blood, March 1, 2008; 111(5): 2878 - 2886. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Michie, A. C. Chan, M. Ciofani, M. Carleton, J. M. Lefebvre, Y. He, D. M. Allman, D. L. Wiest, J. C. Zuniga-Pflucker, and D. J. Izon Constitutive Notch signalling promotes CD4 CD8 thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals Int. Immunol., December 1, 2007; 19(12): 1421 - 1430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Gothert, R. L. Brake, M. Smeets, U. Duhrsen, C. G. Begley, and D. J. Izon NOTCH1 pathway activation is an early hallmark of SCL T leukemogenesis Blood, November 15, 2007; 110(10): 3753 - 3762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Laky and B.J. Fowlkes Presenilins regulate {alpha}{beta} T cell development by modulating TCR signaling J. Exp. Med., September 3, 2007; 204(9): 2115 - 2129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Serwold, K. Hochedlinger, M. A. Inlay, R. Jaenisch, and I. L. Weissman Early TCR Expression and Aberrant T Cell Development in Mice with Endogenous Prerearranged T Cell Receptor Genes J. Immunol., July 15, 2007; 179(2): 928 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Tanaka, T. Era, S.-i. Nishikawa, and S. Kawamata Forced expression of Nanog in hematopoietic stem cells results in a {gamma}{delta}T-cell disorder Blood, July 1, 2007; 110(1): 107 - 115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Heinzel, C. Benz, V. C. Martins, I. D. Haidl, and C. C. Bleul Bone Marrow-Derived Hemopoietic Precursors Commit to the T Cell Lineage Only after Arrival in the Thymic Microenvironment J. Immunol., January 15, 2007; 178(2): 858 - 868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Lehar and M. J. Bevan T Cells Develop Normally in the Absence of both Deltex1 and Deltex2 Mol. Cell. Biol., October 15, 2006; 26(20): 7358 - 7371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Guidos Synergy between the pre-T cell receptor and Notch: cementing the {alpha}{beta} lineage choice J. Exp. Med., October 2, 2006; 203(10): 2233 - 2237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Sun, S. M. Lehar, and M. J. Bevan Augmented IL-7 Signaling during Viral Infection Drives Greater Expansion of Effector T Cells but Does Not Enhance Memory J. Immunol., October 1, 2006; 177(7): 4458 - 4463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Patra, T. Drewes, S. Engelmann, S. Chuvpilo, H. Kishi, T. Hunig, E. Serfling, and U. H. Bommhardt PKB Rescues Calcineurin/NFAT-Induced Arrest of Rag Expression and Pre-T Cell Differentiation J. Immunol., October 1, 2006; 177(7): 4567 - 4576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Schito, O. N. Demidov, S. Saito, J. D. Ashwell, and E. Appella Wip1 Phosphatase-Deficient Mice Exhibit Defective T Cell Maturation Due To Sustained p53 Activation. J. Immunol., April 15, 2006; 176(8): 4818 - 4825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Selvaraj, D. Bunick, C. Finnigan-Bunick, R. W. Johnson, H. Wang, L. Liu, and P. S. Cooke Gene Expression Profiling of 17{beta}-Estradiol and Genistein Effects on Mouse Thymus Toxicol. Sci., September 1, 2005; 87(1): 97 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. T. Petrie and P. W. Kincade Many roads, one destination for T cell progenitors J. Exp. Med., July 5, 2005; 202(1): 11 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Goetz, I. R. Harmon, J. J. O'Neil, M. A. Burchill, T. M. Johanns, and M. A. Farrar Restricted STAT5 Activation Dictates Appropriate Thymic B versus T Cell Lineage Commitment J. Immunol., June 15, 2005; 174(12): 7753 - 7763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kawazu, T. Asai, M. Ichikawa, G. Yamamoto, T. Saito, S. Goyama, K. Mitani, K. Miyazono, S. Chiba, S. Ogawa, et al. Functional Domains of Runx1 Are Differentially Required for CD4 Repression, TCR{beta} Expression, and CD4/8 Double-Negative to CD4/8 Double-Positive Transition in Thymocyte Development J. Immunol., March 15, 2005; 174(6): 3526 - 3533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Lehar, J. Dooley, A. G. Farr, and M. J. Bevan Notch ligands Delta1 and Jagged1 transmit distinct signals to T-cell precursors Blood, February 15, 2005; 105(4): 1440 - 1447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tabrizifard, A. Olaru, J. Plotkin, M. Fallahi-Sichani, F. Livak, and H. T. Petrie Analysis of Transcription Factor Expression during Discrete Stages of Postnatal Thymocyte Differentiation J. Immunol., July 15, 2004; 173(2): 1094 - 1102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ciofani, T. M. Schmitt, A. Ciofani, A. M. Michie, N. Cuburu, A. Aublin, J. L. Maryanski, and J. C. Zuniga-Pflucker Obligatory Role for Cooperative Signaling by Pre-TCR and Notch during Thymocyte Differentiation J. Immunol., May 1, 2004; 172(9): 5230 - 5239. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). C, FLN1/293 cells stained with anti-N1ec (black line), GST (gray line), and mN1ec-GST fusion protein (dashed line). Cells were stained without primary anti-N1ec Ab (
; CD-CD8-CD44-CD25+) or DP (
and 
; CD4+CD8+CD60low) thymocytes were plated on monolayers of OP9 (