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Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
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Abstract |
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expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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rearrangement (7) and a role in promoting cellular expansion in response to pre-TCR signals (6). Although these studies demonstrate the importance of Notch signaling for normal T cell development and function, the regulation of endogenous Notch signaling during each of these steps in T cell development is still not well understood. In classical models of Notch signaling, regulated proteolysis of the Notch receptor is crucial for generation of a Notch signal, resulting in CSL-mediated transcription of Notch target genes (reviewed in Ref.16). Kuzbanian (Kuz) and TACE are ADAM family proteases that have been described to cleave Notch receptors (17, 18, 19, 20, 21). The putative cleavage site for these proteases, S2, is located extracellularly, very close to the transmembrane domain of Notch. It is thought that upon binding to Notch ligand, the S2 cleavage site becomes exposed to ADAM protease-mediated cleavage. Although it is clear that ADAM family proteases are involved in Notch signaling, the specific roles of Kuz and TACE in Notch cleavage are controversial. Expression of a dominant-negative form of Kuzbanian (dnKuz) in Drosophila and Xenopus resulted in neurogenic phenotypes, consistent with a reduction in Notch signaling (21). Later reports suggested that TACE, not Kuz, was the protease involved in Notch cleavage (17). Kuz knockout mice (22) and TACE knockout mice (23) display very different phenotypes, suggesting that these proteases serve distinct roles. More recent studies have shown that some Notch ligands, Delta-1 and Jagged, also can be cleaved by ADAM proteases (24, 25). Thus, the specific role of ADAM proteases in Notch signaling is an open question.
Conventional Kuz knockout mice are early embryonic lethals (22), preventing their use in the study of thymocyte development. In addition, the related ADAM family protease, TACE, might serve a redundant function with Kuz and obscure a thymic phenotype. To circumvent both problems, we decided to interfere with Kuz activity in thymocytes by generating transgenic (Tg) mice that express dnKuz in the thymus. We show in this study that T cell development is blocked between DN and double-positive (DP) stages of T cell development in dnKuz Tg mice. This block correlates with premature down-regulation of CD25 expression and reduced TCR expression, similar to the effect of Notch-1 deletion (7). Overexpression of Delta-1 provided a nearly complete rescue of thymocyte development in dnKuz Tg mice. Results from mixed bone marrow chimeras show that the effect of dnKuz is cell autonomous, whereas the rescue by Delta-1 is non-cell autonomous. These results fit with the idea that dnKuz impairs Notch signaling in receiving cells when levels of Delta-1 are normal, and that increased Delta-1 expression on sending cells can overcome this defect. Interestingly, although the expression of an activated form of Notch-1 rescued the block in TCR expression, it did not restore DP development in dnKuz Tg mice. These data suggest that Kuz is involved in multiple steps in early thymic development, and that these individual steps may differ in their requirements for components of the Notch pathway.
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Materials and Methods |
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Lck-dnKuz Tg mice were generated by subcloning a murine dnKuz (21) into the p1017 vector (26). Linearized Tg DNA was injected into (CBA x B6)F1 oocytes. Founder mice were backcrossed to the C57BL/6 background. Most data reported in this study were obtained from the fourth backcrossed generation. Animals were analyzed at 3–12 wk of age. Relative transgene copy number was assessed by Southern blotting (data not shown). Unless otherwise indicated, the data reported in this study are from the dnKuz-155 line. Lck-Delta-1 Tg (DL1 Tg) mice were prepared by subcloning a full-length mouse Delta-1 cDNA (27) into the p1017 vector (26) and were injected into (CBA x B6)F1 oocytes. DL1 Tg mice were backcrossed to the C57BL/6 background four times. Overexpression of the DL1 Tg in thymocytes was confirmed by real-time PCR (data not shown). NotchIC-9 (NIC9) (8) Tg mice were bred in our facility. V8.3-SGT Tg mice were a gift from P. Savage (Allison Laboratory, University of California, Berkeley, CA). All mice were housed under conventional conditions and were healthy at the time of analysis. Genotyping of mice was performed by PCR using genomic DNA obtained from ear skin. The primers used were: hHGH 5' primer, 5'-GTC TAT TCC GAC ACC CTC CA-3'; hHGH 3' primer, 5'-GGA TGC CTT CCT CTA GGT CC-3'; dnKuzHGH 5' primer, 5'-GAG GCC CCG AGA GAG TTA TC3–3'; dnKuzHGH3' primer, 5' TGT GCC CAA AGG GAT TTT AG-3'; DLL-1HGH5' primer, 5' TGC ACT ATG GAC AGT TGC TT-3'; V8.3 5' primer, CCA GTA TCT CGA GCG GAT GG-3'; V8.3 3' primer, TGC ACT ACC CCC AGT CCC AC-3'; Notch-1IC forward, 5'-ATG GAC TAC AAA GAC GAT GAC G-3'; and Notch-1IC reverse, 5' CCA TCT GGT CCT CGA ACA TTG.
Western blotting
Thymocytes (106) were lysed in 1% Triton X-100 with protease inhibitors (Roche) for 20 min on ice, then centrifuged at top speed for 15 min at 4°C. SDS-reducing sample loading buffer was added to the lysate and boiled for 5 min. Samples were loaded onto a 10% SDS-PAGE separating gel with 5% stacking gel. Eight microliters of Rainbow Marker (Amersham Biosciences) was also loaded in one lane. Gels were run at a constant 25 mA, then transferred to ECL nitrocellulose at 200 mA for 2 h. The membranes were incubated with 0.2% Tween 20-TBS-10% milk overnight at 4°C, washed, incubated with peptide affinity-purified anti-Kuz C-terminal peptide-specific Ab (1/1000; produced for us by Zymed Laboratories) for 30 min at room temperature, washed, and incubated with goat anti-rabbit IgG-HRP (1/5000) for 30 min at room temperature, and washed. ECL reagent was added to the membrane for 1 min, and then the membrane was exposed to ECL Hyperfilm (Amersham Biosciences).
Flow cytometry
Thymi were harvested and crushed with the base of a 5-cc syringe. Bone marrow cells were flushed from the bones using a 25-cc needle. Cells were resuspended in medium 199–2% FCS medium. RBC were lysed with ammonium-chloride-potassium lysis buffer, and cell counts were obtained using trypan blue exclusion. Cells were pelleted and resuspended in FACS buffer, and 2.4G2 supernatant was added to all cells before staining. CD4-FITC, 
TCR-FITC, CD8-FITC, CD8-PeCy5, CD25-PE, CD3-FITC, CD4-PETR, CD8-PETR, CD44-CyChrome, CD44-PeCy5, CD45 (clone 30-F11)-PeCy5, CD45.1-PE, CD45.2-FITC, CD45.1-FITC, Thy1.2-PE, CD3-biotin, CD19-biotin, CD11b-biotin, GR1-biotin, DX5-biotin, V8.3-FITC, purified anti-TCR, streptavidin-FITC, streptavidin-CyChrome, streptavidin-613, and streptavidin-PETR were purchased from BD Pharmingen, eBioscience, Caltag Laboratories, and Invitrogen Life Technologies. For intracellular staining, extracellular staining was performed first, then cells were fixed and permeabilized using the BD Pharmingen kit. TCR-FITC was added to cells and incubated for 30 min, washed with 1x Perm/Wash buffer (BD Pharmingen), then resuspended in FACS buffer. FACS was performed with an ELITE FACS machine (Beckman Coulter). Data analysis was performed with FlowJo software (TreeStar).
Cell sorting of lineage-negative thymocytes
Lineage-negative thymocytes were enriched from whole thymocytes using anti-CD8 magnetic beads and collecting CD8-negative cells using AutoMACS (Miltenyi Biotec). MACS-enriched CD8-negative thymocytes were then stained with anti-CD3-biotin, anti-CD4-FITC, and anti-CD8-FITC, anti-CD11b-biotin, DX5-biotin, and/or Gr-1-biotin plus streptavidin-FITC. FITC-negative thymocytes were collected using a MoFlow cell sorter. Sorted populations were 95–100% pure.
Semiquantitative RT-PCR
Sorted Lin– thymocytes were pelleted and resuspended in TRIzol. RNA was isolated using standard protocols, and cDNA was generated using SuperScript II reverse transcriptase (Invitrogen Life Technologies). For PCR, the input amount of cDNA was normalized between controls and dnKuz samples using -actin. The following primers were used: -actin forward, 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3'; -actin reverse, 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'; and pre-T, Deltex and Hes-1 primers (previously reported) (28, 29). PCR products were Southern blotted, probed with gene-specific 32P-labeled oligonucleotide probes, and exposed on PhosphorImager screens (BD Biosciences).
Preparation of mixed bone marrow chimeras
Bone marrow cells were harvested from the femurs and tibiae of WT B6 (Ly5.2/5.2 or Ly5.2/5.1), dnKuz (Ly5.1/5.2), DL1 Tg (Ly5.2/5.2), and dnKuz/DL1 double-Tg (Ly5.2/5.2) mice. WT B6 (Ly5.1/Ly5.1) recipient mice were lethally irradiated with 1000 rad from a cesium source. At least 4 h after irradiation, 15 x 106 bone marrow cells from WT, dnKuz Tg, DL1 Tg, or dnKuz/DL1 mice alone (single donor chimeras) or a mixture of WT plus dnKuz, WT plus dnKuz/DL1, or DL1 Tg plus dnKuz Tg (total of 15 x 106) bone marrow cells were transferred to these hosts via tail vein injection. Animals were given antibiotic-containing water and were housed in sterile microisolator cages. Animals were killed 6 wk after bone marrow transplant. FACS analysis of thymocytes and bone marrow cells was performed.
Real-time PCR
RNA was isolated using standard protocols and was treated with RNase-free DNase I (Roche) to remove any possible genomic DNA contamination. RT was performed using the TaqMan RT kit (Applied Biosystems) according to the manufacturer’s suggestions. Real-time PCR was performed using the ABI TaqMan 5700 thermal cycler default cycling protocol. SYBR Green PCR Master Mix (2x; Applied Biosystems) was used. The following primers were used: Delta-1 forward, CAC TAT GGA CAG TTG CTT TGA AGA GT; and Delta-1 reverse, TGG CTC ATA GTA ATC CAA GAT AGA CG. GAPDH primer/probe sets were obtained from Applied Biosystems. PCRs were performed in triplicate, and the ratio of Delta-1 to GAPDH expression was calculated and averaged. The mean ratio across samples was then calculated and compared with the reference sample.
Statistical analysis
Mann-Whitney tests to test the difference in medians were used (InStat 3; GraphPad). Differences were considered statistically significant at p < 0.05.
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Results |
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To generate dnKuz Tg mice, a cDNA encoding a dominant negative form of Kuzbanian (21) was subcloned into the p1017 vector, which contains the Lck-proximal promoter (26). Six independent founders containing different transgene copy numbers were obtained and backcrossed to C57BL/6 mice. The dnKuz protein was expressed in all founders (Fig. 1 A and our unpublished observations). A clear effect of the dnKuz transgene on thymus size was evident in the higher copy lines (Fig. 1B). The thymic cellularity in the highest copy dnKuz Tg mice (dnKuz-155; hereafter simply called dnKuz Tg) ranged between 10 and 40% of non-Tg littermate controls.
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cells in the thymus were significantly reduced in dnKuz mice (data not shown). To further investigate where the developmental block might be occurring, lineage-negative (Lin–; CD3–CD4–CD8–CD11b– DX5–Gr1–) thymocytes were stained for CD25 and CD44 expression, delineating the DN1-DN4 stages (Fig. 2A). Calculation of the absolute number of DN1 cells showed a slight reduction in dnKuz Tg mice compared with controls, but this difference was not statistically significant (Fig. 2B). However, the absolute number of DN2 cells was significantly reduced in dnKuz Tg mice compared with non-Tg controls (Fig. 2B). Therefore, thymocyte development in dnKuz Tg mice may be blocked as early as the DN2 stage.
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In addition to this aberrant CD25 expression, TCR expression was affected in dnKuz Tg mice. Fewer intracellular TCR-positive (icTCR+) cells were present in the DN3 and DN4 stages compared with non-Tg controls (Fig. 2C). In both non-Tg and dnKuz Tg mice, the icTCR+ cells were larger than icTCR– cells (Fig. 2C). This suggests that the icTCR+ thymocytes in both non-Tg and dnKuz Tg mice passed selection and proliferated normally. To confirm that the lack of TCR expression was contributing to the DN to DP block in dnKuz Tg mice, the mice were crossed with V8.3 SGT Tg mice (a gift from P. Savage), which express a rearranged V8.3 transgene under control of the natural TCR regulatory elements (32). Coexpression of the rearranged TCR gene and the dnKuz transgene resulted in a reduced DN to DP ratio (Fig. 2, D and E). The total thymocyte number in dnKuz/V8.3 Tg mice was slightly higher than that in dnKuz littermates (Fig. 2E), although this difference was not statistically significant (p = 0.528). These results imply that reduced TCR expression at the DN stage is partly responsible for the reduced number of DP thymocytes seen in dnKuz Tg mice.
Expression of Notch target genes in dnKuz Tg mice
If Notch signaling were inhibited by dnKuz, we might expect the expression of Notch target genes to be reduced in dnKuz Tg thymocytes. To test this, the expression of Deltex-1, Hes-1, and pre-T, three reported Notch target genes (33, 34, 35), was examined by semiquantitative RT-PCR. Lineage-negative DN thymocytes were isolated from dnKuz Tg and non-Tg littermates, and RNA was isolated from these sorted populations. The expression of pre-T in dnKuz Tg mice was similar to that in controls (Fig. 3). In contrast, the expression of Deltex-1 and, to a lesser extent, Hes-1, was reduced in dnKuz Tg mice. These results are consistent with reduced Notch signaling in DN thymocytes in dnKuz Tg mice.
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In many respects, the block in T cell development observed in dnKuz Tg mice is similar to that observed in conditional Notch-1 knockout mice, in which Cre is driven by the Lck promoter (7). This similarity suggested that Notch signaling was impaired in the thymocytes of dnKuz Tg mice. Kuzbanian has been implicated in the cleavage of both Notch and its ligand, Delta-1 (24, 36, 37). Regardless of whether dnKuz impairs the cleavage of Notch or Delta, we would expect that overexpression of Delta might rescue the resulting Notch signaling defect by increasing the number of receptor-ligand interactions and therefore compensating for the reduced efficiency of Notch signaling. To test this idea, dnKuz Tg mice were crossed with mice that overexpress full-length Delta-1 in thymocytes under control of the Lck promoter (Fig. 4). Delta-1 Tg (DL-1Tg) mice express 13 times more Delta-1 mRNA in the thymus compared with WT thymus (data not shown) and do not display any differences in thymocyte cellularity compared with WT controls (Fig. 4C). Importantly, the DN to DP block was rescued in dnKuz/DL-1 double-Tg mice, as indicated by the DN to DP ratio (Fig. 4A, top panels, and Fig. 4B) and the overall thymic cellularity (Fig. 4C). In addition, the CD25/CD44 profiles of DN thymocytes and the expression of icTCR in dnKuz/DL1 mice were similar to those in DL1 controls (Fig. 4A, middle and lower panels, and Fig. 4D). These data indicate that overexpression of Delta-1 can substantially rescue the developmental block induced by dnKuz in the thymus.
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In the classical model of Notch signaling, Notch ligands expressed on sending cells are thought to induce Notch signaling in the receiving cells (38). Kuzbanian might be involved in the cleavage of both Notch receptors (19, 20, 21) and the Notch ligand, Delta-1 (24, 25, 36, 37). Therefore, if dnKuz affects Delta-1 expression or function, the effect of dnKuz on thymocyte development might be cell nonautonomous. In contrast, if dnKuz affects the cleavage of Notch receptors, the effect of dnKuz would be cell autonomous. To examine these possibilities, we prepared mixed (WT + dnKuz) bone marrow chimeras. Thymocyte development in the mixed chimera was compared with that in chimeras in which WT or dnKuz bone marrow was transferred alone. Thymocytes derived from the WT donor developed normally in both the WT
WT and the (WT+dnKuz)WT chimeras (Fig. 5A, first and third rows). In contrast, thymocytes derived from the dnKuz donors comprised only 5% of the total thymocytes in the (WT+dnKuz) mixed chimeras (Fig. 5B, top graph) despite the fact that equal numbers of donor cells were injected, and bone marrow engraftments by WT-derived and dnKuz-derived bone marrow were similar (our unpublished observations). Interestingly, the DN to DP block was more pronounced in the dnKuz thymocytes developing in the mixed chimera compared with chimeras receiving only dnKuz bone marrow (Fig. 5A, second and fourth rows, and Fig. 5B, bottom graph) and intact dnKuz Tg mice (Fig. 1C). Some possible explanations for this enhanced block will be addressed in Discussion. These experiments demonstrate that the effect of the dnKuz transgene is cell autonomous and indicate that the dnKuz transgene impairs the ability of thymocytes to receive a Notch signal.
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The observation that dnKuz mutation is cell autonomous (Fig. 5) together with the rescue in development seen by overexpression of Delta-1 on dnKuz thymocytes (Fig. 4) imply that Delta-1 might rescue thymocyte development by increasing Kuz/Delta-1 interactions between thymocytes (i.e., in trans). To test this prediction, we prepared mixed (WT + dnKuz/DL1) bone marrow chimeras, in which the Delta-1 and dnKuz transgenes were expressed on the same thymocyte population (i.e., in cis-), and mixed (DL1 + dnKuz) bone marrow chimeras, in which Delta-1 and dnKuz transgenes were expressed on different thymocyte populations (i.e., in trans). In the (WT + dnKuz/DL1) mixed chimeras, dnKuz/DL1-derived thymocytes comprised only 1.5% of the total thymocytes and displayed a DN to DP ratio of 0.91 (Fig. 5B). In contrast, in the (DL1 + dnKuz) mixed chimeras, dnKuz-derived thymocytes comprised 15% of the total thymocytes and displayed a DN to DP ratio of 0.1 (Fig. 5B). Although dnKuz thymocytes from (DL1 + dnKuz) mixed chimeras still showed some developmental impairment, the block was much less severe than that seen in control mixed chimeras (i.e., (WT + dnKuz) and (WT + dnKuz/DL1) mixed chimeras). Taken together, these data indicate that dnKuz expression in thymocytes impairs Notch signaling, and that overexpression of Delta-1 on neighboring thymocytes can partially compensate for this impairment.
An activated form of Notch-1 rescues TCR expression, but does not promote the DN to DP transition in dnKuz Tg mice
If Kuzbanian acts to cleave Notch receptors and promotes Notch signaling, we would expect that introduction of a cleaved, ligand-independent, activated form of Notch would rescue thymocyte development in dnKuz Tg mice. The dnKuz Tg mice were crossed with NIC9 Tg mice, which express an intracellular, activated form of Notch-1 (Notch-1IC) under control of the Lck promoter (8, 39). A higher expression of intracellular TCR expression was observed in dnKuz/NIC9 mice compared with dnKuz Tg mice (Fig. 6A). However, despite this increased TCR expression, the block between the DN to DP stages was still present in dnKuz/NIC9 double-Tg mice (Fig. 6B), and thymocyte cellularity between dnKuz and dnKuz/NIC9 Tg mice was not significantly different (Fig. 6C). These data can be interpreted in light of earlier studies showing that multiple Notch-dependent steps are involved in the DN to DP transition. In an early step, Notch promotes TCR expression to allow for the formation of the pre-TCR, and in a later step promotes cellular expansion in response to pre-TCR signaling (6). Our data suggest that although dnKuz blocks both these steps, activated Notch-1 can substitute for the first Notch-dependent step, but not the second. The possible implications of this will be discussed below.
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Discussion |
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The block in thymocyte development in dnKuz Tg mice is similar in many ways to that in the Lck-conditional Notch-1 knockout (Notch-1–/–) mouse (7). Both dnKuz and Notch-1–/– mice display blocks in the DN to DP transition, and this block correlates with a defect in TCR expression at the DN3 stage. In addition, CD25 appears to be prematurely down-regulated in DN thymocytes from dnKuz and Notch-1–/– mice (7). This is consistent with the up-regulation of CD25 seen in thymocytes and mature T cells in response to activated Notch (9, 13) and suggests that CD25 expression is regulated by Notch-1.
A connection between Kuzbanian and Notch signaling during thymocyte development is also suggested by the expression of some Notch target genes. The expression of Deltex-1 and, to a lesser extent, Hes-1 was reduced in dnKuz Tg mice, consistent with an inhibition of Notch signaling. The modest effect of the dnKuz mutation on Hes-1 expression may be a reflection of Notch-independent expression of this gene as well as the transient nature of Hes-1 induction in response to Notch signaling (42, 43). These considerations may also explain the observation that Hes-1 expression was not significantly altered in RBP-J
mutant mice, in which CSL-dependent Notch signaling is abolished (44). In addition, although pre-T was suggested to be a Notch target gene based on in vitro reporter assays by Reizis and Leder (34), analysis of conditional Notch-1 knockout mice did not reveal any difference in its expression (7). Similarly, we did not observe any change in the expression of pre-T in dnKuz Tg mice. Therefore, pre-T may not be a major target of Notch in vivo, and it is likely that other mechanisms are involved in the regulation of its expression.
There are, however, some slight differences between the Notch-1–/– and dnKuz mice. DN3 thymocytes from Notch-1–/– mice were reported to have decreased rearrangement of the TCR gene, suggesting that reduced TCR expression in these cells results from an impairment of gene rearrangements. In contrast, we did not observe a consistent decrease in TCR gene rearrangements in dnKuz Tg mice (our unpublished observations). Although this may reflect a difference in phenotype, it is also possible that this difference is due to limitations of this assay, which in our hands is not sufficiently quantitative to reliably detect differences of <10-fold. Thus, it is unclear whether the primary effect of the dnKuz mutation is on rearrangement or expression of the TCR gene. However, our observation that expression of a TCR transgene in dnKuz mice rescues thymocyte development (Fig. 2, D and E) strongly suggests that a defect in TCR expression is contributing to the block.
Another difference between the Notch-1–/– and dnKuz mice is found in the thymocyte populations that are affected by the different mutations. Specifically, in the Notch-1–/– mice, no differences in the absolute numbers of DN thymocytes or T cells were observed (7), whereas in dnKuz mice, both these populations were reduced (Figs. 1 and 2 and data not shown). This discrepancy might be explained by an earlier inhibition of Notch signaling in the dnKuz mice compared with the Notch-1–/– mice. For example, in dnKuz mice, the dnKuz transgene might be expressed as early as the DN1 stage (30), and the dnKuz could begin inhibiting Notch signaling as soon as it is expressed. In the Lck-Notch-1–/– mice, in contrast, deletion of the Notch gene is not complete until the DN3 stage (7). At this stage and beyond, common defects in thymocyte development are observed in both dnKuz and Notch-1–/– mice.
Although our results indicate that dnKuz impairs Notch signaling in the receiving cell, we also see clear evidence for an impact of neighboring thymocytes on the phenotype of the mutation. In particular, the effect of the dnKuz mutation is exaggerated when dnKuz thymocytes develop in the presence of wild-type thymocytes. This is reflected in the greater block seen in (WT plus dnKuz) mixed chimera compared with intact dnKuz mice and in the partial rescue seen in (DL1 plus dnKuz) mixed chimeras compared with intact dnKuz/DL1 Tg mice. This enhanced block in development in the mixed chimeras might reflect a general inability of the dnKuz-derived thymocytes to compete with WT-derived thymocytes in the thymus. For example, a decrease in overall proliferative ability might contribute to the reduced competitive fitness of the dnKuz-derived thymocytes. A more interesting possibility is that the enhanced block in development might be due to a lateral signaling mechanism. During lateral signaling, Notch ligand expression is up-regulated in cells that receive lower Notch signals (38, 45). If a lateral signaling mechanism operates during thymic development, reduced Notch signaling in dnKuz-expressing cells could cause these cells to up-regulate ligand expression. Increased levels of Notch ligand could partially relieve the block to Notch signaling in intact dnKuz Tg mice, a situation in which all thymocytes express dnKuz. In mixed chimeras, neighboring WT thymocytes would express normal levels of Notch ligand. This normal level of Notch ligand would be insufficient to bypass the block in Notch signaling the dnKuz-expressing thymocytes, resulting in a more severe developmental block. Although we did not observe any impact of the dnKuz transgene on the levels of Delta1 mRNA (Fig. 7 ), additional detailed analysis of the expression levels of Delta-1 and other Notch ligands is necessary to investigate the existence of lateral signaling mechanisms controlling Notch in thymocytes.
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We propose a model to explain our results, in which Kuz promotes Notch signaling in thymocytes by increasing their sensitivity to ligand expressed on a neighboring cell. The mixed bone marrow chimera experiments presented in Fig. 5 show that the effect of dnKuz is cell autonomous, yet the rescue by Delta-1 is non-cell autonomous. This suggests that dnKuz impairs Notch signaling in thymocytes, such that Delta-1 expression in the thymus becomes limiting, and overexpression of Delta-1 on thymocytes can compensate for this impairment. The requirement for Kuz activity in the receiving cell fits with a model in which Kuz cleaves Notch in response to Delta binding, helping to initiate Notch signaling and leading to the transcription of Notch target genes and TCR rearrangement and/or expression (Fig. 8 A). Overexpression of Delta-1 would overcome the effect of dnKuz, perhaps by providing a strong agonist signal through Notch, and thus overriding the block in Notch processing. The cell-autonomous nature of dnKuz mutation is also consistent with an alternative model in which Delta-1 is the substrate for Kuz, but Kuz activity is restricted to Delta on an adjacent (sending) cell that is engaged in active Notch signaling with the Kuz-expressing cell (the receiving cell; Fig. 8B). Kuz-mediated cleavage of Delta-1 on an adjacent thymocytes might promote transendocytosis, a mechanism that is important for the promotion of Notch signaling (48, 49). In this model, overexpression of Delta-1 in trans would overcome the effect of dnKuz by providing more substrate for endogenous Kuz.
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expression at the DN3 stage and to allow proliferation and survival of thymocytes that pass selection (6). Our results indicate that dnKuz inhibits both of these Notch-dependent steps, but that Notch-1-IC can restore the first, but not the second, step. Although the explanation for the failure of Notch-1-IC to restore DP development in dnKuz mice is not clear, several possible explanations could be considered. One could imagine that the timing and/or levels of Notch signaling at the DN stage need to be precisely controlled, and that presence of high levels of Notch activity throughout this stage interfere with a process required for normal DN to DP transition. It is also worth noting that although Notch-1-IC provides a potent Notch signal, it may not trigger all the same downstream effects triggered by endogenous Notch. Indeed, some of the effects of Notch-1-IC could be mediated by increased signaling through endogenous Notch and might thus still require Notch proteolysis. The up-regulation of endogenous Notch in response to activated Notch is consistent with this idea (8, 50). Notch signaling can be regulated in a variety of ways, can trigger a diverse set of cellular responses, and can be deployed at successive developmental stages. These complexities pose a major challenge to a full understanding of the role of Notch signaling in T cell development. Our results provide the first indication that ADAM family proteases regulate the sensitivity of DN thymocytes to Notch signaling and, as such, represent an important piece to this complex puzzle.
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Acknowledgments |
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8.3 SGT Tg mice, Hector Nolla for expert technical assistance, and Gerry Weinmaster, Bill Sha, and members of the Robey laboratory for helpful discussions and their critical review of the manuscript. |
Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants 1F32GM64975 (to J.O.M.) and 5RO1AI42033 (to E.A.R.). 
2 Current address: Room 780, Center for Neurological Diseases, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115.
3 Address correspondence and reprint requests to Dr. Ellen A. Robey, Department of Molecular and Cell Biology, 471 Life Sciences Addition, University of California, Berkeley, CA 94720. E-mail address: erobey{at}berkeley.edu
4 Abbreviations used in this paper: ADAM, a disintegrin and a metalloprotease; CSL, CBF1, suppressor of hairless Lagl; DL1, Delta-1; DN, double negative (CD4–CD8–); dnKuz, dominant-negative Kuz; DP, double positive (CD4+CD8+); hHGH, human growth hormone; icTCR, intracellular TCR-chain; ISP, immature single positive; Kuz, Kuzbanian; NIC9, NotchIC-9; Notch-1-IC, intracellular activated form of Notch-1; PETR: Phycoerythrin-Texas Red; SP, single positive; TACE, TNF -converting enzyme; Tg, transgenic.
Received for publication September 16, 2004. Accepted for publication March 18, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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  G. van Tetering, P. van Diest, I. Verlaan, E. van der Wall, R. Kopan, and M. Vooijs Metalloprotease ADAM10 Is Required for Notch1 Site 2 Cleavage J. Biol. Chem., November 6, 2009; 284(45): 31018 - 31027. [Abstract] [Full Text] [PDF]
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 E. C. Bozkulak and G. Weinmaster Selective Use of ADAM10 and ADAM17 in Activation of Notch1 Signaling Mol. Cell. Biol., November 1, 2009; 29(21): 5679 - 5695. [Abstract] [Full Text] [PDF]
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 L. Tian, X. Wu, C. Chi, M. Han, T. Xu, and Y. Zhuang ADAM10 is essential for proteolytic activation of Notch during thymocyte development Int. Immunol., September 1, 2008; 20(9): 1181 - 1187. [Abstract] [Full Text] [PDF]
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 N. Li, K. Boyd, P. J. Dempsey, and D. A. A. Vignali Non-Cell Autonomous Expression of TNF-{alpha}-Converting Enzyme ADAM17 Is Required for Normal Lymphocyte Development J. Immunol., April 1, 2007; 178(7): 4214 - 4221. [Abstract] [Full Text] [PDF]
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 G. Weinmaster and R. Kopan A garden of Notch-ly delights Development, September 1, 2006; 133(17): 3277 - 3282. [Abstract] [Full Text] [PDF]
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