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Transgenic Expression of Numb Inhibits Notch Signaling in Immature Thymocytes But Does Not Alter T Cell Fate Specification¹

Michelle B. French^2,*, Ute Koch^2,, Rachel E. Shaye^*, Melanie A. McGill^*, Sascha E. Dho^*, Cynthia J. Guidos and C. Jane McGlade^3,*

^* Arthur and Sonia Labatt Brain Tumor Research Center and Program in Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The conserved adaptor protein Numb is an intrinsic cell fate determinant that functions by antagonizing Notch-mediated signal transduction. The Notch family of membrane receptors controls cell survival and cell fate determination in a variety of organ systems and species. Recent studies have identified a role for mammalian Notch-1 signals at multiple stages of T lymphocyte development. We have examined the role of mammalian Numb (mNumb) as a Notch regulator and cell fate determinant during T cell development. Transgenic overexpression of mNumb under the control of the Lck proximal promoter reduced expression of several Notch-1 target genes, indicating that mNumb antagonizes Notch-1 signaling in vivo. However, thymocyte development, cell cycle, and survival were unperturbed by mNumb overexpression, even though transgenic Numb was expressed at an early stage in thymocyte development (CD4^-CD8^-CD3^- cells that were CD44⁺CD25⁺ or CD44^-CD25⁺; double-negative 2/3). Moreover, bone marrow from mNumb transgenic mice showed no defects in thymopoiesis in competitive repopulation experiments. Our results suggest that mNumb functions as a Notch-1 antagonist in immature thymocytes, but that suppression of Notch-1 signaling at this stage does not alter / or CD4/CD8 T cell fate specification.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Functionally distinct mature T cells develop in the thymus from multipotent progenitor cells that migrate from the bone marrow. The majority of mature T cells bear the TCR along with either CD4 or CD8 coreceptors, whereas a small population of mature T cells bears the TCR. T cells develop in the thymus from CD4^-CD8^- (double-negative, DN)⁴ cells through an immature CD4⁺CD8⁺ (double-positive, DP) stage into mature CD4⁺ or CD8⁺ (single-positive, SP) cells. Thymocytes commit to the vs lineages very early in T cell development at the CD44^-CD25^- DN (DN4) stage (1). Signals emanating from the pre-TCR and TCR play well-defined roles in T cell development at the DN-DP and DP-SP transitions, respectively.

Members of the Notch family play critical roles in the determination of cell fate and in the maintenance of progenitors during development of a number of different cell types in a variety of different organisms (2, 3). Notch signals are essential for the development of T lineage cells (4, 5, 6), whereas they prevent B cell development (7, 8), suggesting that activation of Notch-1 signaling in multipotential hemopoietic progenitors controls T vs B cell lineage determination. Washburn and colleagues (9) have implicated Notch-1 signals in regulating the choice between the vs T cell lineages during T cell development in the thymus. Activated forms of Notch-1 also promote the survival and maturation of DP thymocytes and influence the DP-SP transition (10, 11, 12, 13). Collectively, these data indicate that Notch-1 may influence the survival and lineage commitment of T cell progenitors at several discrete stages.

Notch activation in Drosophila is regulated by the temporal and spatial distribution of Notch ligands, Delta and Serrate, as well as by the expression of signaling modulators such as Numb (2). Experiments, primarily in Drosophila, indicate that Numb acts upstream of Notch action to inhibit Notch signaling (14, 15, 16, 17). For example, during the development of the sensory organ in Drosophila, loss-of-function mutations in dNumb mimic gain-of-function mutations in Notch, suggesting that Numb inhibits Notch activity (14). Numb may prevent the translocation of the Notch intracellular domain to the nucleus following ligand binding (15, 17). However, the direct effect of Numb on the expression of Notch target genes in vivo has not been reported.

The mechanisms regulating Notch activation in T cell progenitors are not well defined. The Notch ligands, Jagged-1 and Jagged-2, are expressed in the murine (18, 19) and rat (20) thymus, and development of T cells is slightly impaired, but not blocked in mice with a mutation in Jagged-2 (21). Mammalian homologues of dNumb have been identified and are widely expressed in embryos and in adult tissue, including the thymus (22, 23, 24, 25). The function of mammalian Numb (mNumb) remains unclear, but mNumb is thought to influence neurogenesis in vertebrates (17, 22, 26). Mice with a null mutation in the mNumb gene display multiple developmental defects involving the nervous system as well as other cell lineages resulting in embryonic cell death before day E11.5 (26, 27).

Structurally, mNumb resembles an adapter protein. It has an N-terminal phosphotyrosine-binding (PTB) domain (22, 23, 28), a proline-rich region containing several putative Src-homology 3 binding sites (23), and an Eps 15 homology domain-binding motif at the carboxyl terminus (29). Consistent with the notion that Numb acts as adapter protein, a number of mNumb-binding partners have been identified (29, 30, 31), including Notch-1 (22).

There are four different isoforms of mNumb (p65, p66, p71, and p72), which arise from alternative splicing of mNumb RNA, and are characterized by the presence or absence of an 11-aa insert in the PTB domain and the presence or absence of a 48-aa insert in the proline-rich region. All four isoforms of mNumb are expressed in the thymus, although the p65 and p66 isoforms are predominant (25, 32). This observation, together with the reports that dNumb inhibits Notch activity and that Notch-1 influences T cell development, prompted us to determine whether transgenic (Tg) overexpression of the p66 isoform of mNumb would antagonize Notch signaling in thymocytes and alter T cell development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Tg mice

The complete coding sequence of rat Numb cDNA (nt 196-2007) (23) was cloned into the BamHI site of p1017, which contains the Lck proximal promoter and portions of the human growth hormone gene, including a polyadenylation sequence (33). In the resulting plasmid, the cDNA for Numb was flanked by the mouse Lck proximal promoter at the 5' end and portions of the human growth hormone gene, including introns and a polyadenylation sequence at the 3' end. The rat Numb cDNA that was used encodes the p66 mNumb isoform with the 11-aa insert in the PTB domain, but does not contain the 48-aa insert in the proline-rich region of the molecule (23). The amino acid sequence of the protein encoded is identical to the murine p66 isoform of Numb (25). A SpeI fragment from the plasmid consisting of the entire transgene was purified and injected into (C57BL/6 x SJL)F₂ embyros. Tg⁺ founder mice were identified by PCR amplification of tail DNA using primers that spanned the junction between the Lck proximal promoter and mNumb (5' primer, ATG TCT CCC AGG TAG TCC CC and 3' primer, GTG CAT TCC TCT TGA CTC ATC). Tg⁺ founders were bred to C57BL/6. The majority of the analysis was performed on mice from two lines of founder mice that had been backcrossed three to four times to C57BL/6.

Immunoprecipitation, in vitro binding, and Western blotting

Protein extracts of thymus, spleen, and lymph nodes from Tg⁺ and non-Tg (Tg^-) mice were made by homogenizing the tissues in lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 10% glycerol, 1% Triton X-100, and protease inhibitors (Roche Molecular Biochemicals, Laval, Quebec, Canada)). Lysates were centrifuged at 14,000 rpm at 4°C to pellet insoluble matter. For each immunoprecipitation, 0.5 mg of protein was made up to a volume of 0.5 ml with lysis buffer and incubated for 2–4 h with Ab and protein A-Sepharose beads. Immune complexes bound to protein A-Sepharose were washed four times with lysis buffer and eluted by boiling in SDS sample buffer. Proteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane (Immobilon-P), and immunoblotted with primary Ab for 1–2 h at room temperature. Bound Ab were visualized using HRP-conjugated protein A, followed by ECL. mNumb was immunoprecipitated using affinity-purified anti-Numb-A Ab and blotted with anti-Numb-C Ab. Anti-Numb-A is specific for mNumb and does not recognize the related protein, Numb-like (25). DN cells from Tg⁺ and Tg^- mice were lysed in lysis buffer, and 50 µg of whole cell lysate was separated by SDS-PAGE and analyzed by Western blot with Numb-C Ab. Blots were reprobed with -tubulin Ab (Amersham, Arlington Heights, IL) to confirm equal protein loading.

Immunohistochemistry

Thymic tissue from Tg⁺ mice was rapidly removed, flash frozen to -80°C, and sectioned using a cryostat modular cryosection apparatus. The 5-µm thymic sections were mounted on positively charged slides (Fisher Scientific, Mississauga, Canada) and stored at -20°C. A dual immunofluorescent staining procedure was used to demonstrate Numb membrane colocalization with the AP-2 protein. Thymic sections were fixed via incubation at room temperature with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 30 min. Following the incubation, sections were washed three times for 15 min each with 1x PBS. To permeabilize the tissue, slides were incubated with a solution of 1x PBS containing 0.2% Triton X-100 at room temperature for 30 min. The slides were washed three times for 10 min each with 1x PBS. To prevent nonspecific binding of the secondary Ab used in the detection of Numb and AP-2, slides were incubated with a solution of 1x PBS containing 5% goat serum (The Jackson Laboratory, Bar Harbor, ME) at 37°C for 30 min. Slides were then washed as described above. To detect the Numb protein, slides were treated with a 1/10 dilution of a 1 mg/ml stock solution of rabbit anti-Numb polyclonal anti-Numb-C Ab (23) for 24 h at 4°C. The sections were washed three times for 10 min each and treated with a 1/300 dilution of a stock 2 mg/ml goat anti-rabbit Ab conjugated to AlexaFlour 488 (Molecular Probes, Eugene, OR) for 2 h at room temperature. Slides were washed and incubated with a 1/100 dilution of a stock 1 mg/ml mouse anti-AP-2 mAb (Affinity BioReagents, Golden, CO) for 24 h at 4°C. Slides were washed and treated with a 1/300 dilution of a stock 2 mg/ml goat anti-mouse Ab conjugated to CY3 (Molecular Probes) for 2 h at room temperature. Slides were coverslipped (Baxter Diagnostics, McGraw Park, IL) with fluorescent mounting media (DAKO Diagnostics, Carpinteria, CA) and analyzed using a confocal microscope.

RT-PCR analysis of endogenous Numb, Tg Numb, and Notch-1 target genes in Tg^- and Tg⁺ thymus

RNA from thymi from Tg⁺ and Tg^-littermates that were 3- to 4-wk-old was prepared using the TRIzol system (Life Technologies, Grand Island, NY). Superscript II reverse transcriptase (Invitrogen, San Diego, CA) was used to prepare cDNA, which was then normalized for equivalent template amounts by serial dilution and amplification using primers specific to -actin (5'-GTCGTACCACAGGCATTGTGATGG-3' and 5'-GGTGGTACATGGGTCCGTAACG-3'). RT-PCR analysis was conducted as described previously (11). Briefly, normalized cDNA amplified for 35 cycles (95°C, 1 min; 60°C, 30 s; 72°C, 1 min) using primers specific for all isoforms of endogenous mNumb (5'-CTACGGCAAAGCTTCAGGAGA-3' and 5'-TCAGCAACTTTTCACTAATCC-3') and Tg mNumb (5'-AGAAGTGTCAAAGAGTGTGCG-3' and 5'-CAG GCTTTTTGACAACGCTAT-3'). To analyze specific Notch-1 target genes, normalized cDNA was serially diluted 3-fold and amplified for 35–60 cycles (95°C, 1 min; 55°C, 1 min; 72°C, 1 min) using Ifi-D3 (5'-ACTTCCTCTGTGTTAGAGGCTGC-3' and 5'-AAAGCTGTCATTTAGAGGTG-3'), Hes-1 (5'-GCCAGTGTCAACACGACACCGG-3' and 5'-TCACCTCGTTCATGCACTCG-3'), and preT (5'-TGGCTGCAACTGGGTCATGCTTC-3' and 5'-GGCTCAGAGGGGTGGGTAAGATC-3') primers. PCR products were separated by electrophoresis through a 0.8% agarose gel.

Abs and flow cytometry

Single-cell suspensions were prepared from thymus, spleen, lymph node, and bone marrow; stained (2 x 10⁶ cells/sample) with various Ab; and analyzed by flow cytometry. The following mAb, which were generated in the laboratory unless otherwise indicated, were used: anti-CD4 PE; anti-CD4 FITC (GK1.5); anti-CD4 biotin (YTS191.1); anti-CD8 APC; anti-CD8 FITC (53-6.7); anti-CD8 biotin (YTS169.4); anti-B220 FITC (6B2); anti-B220 biotin; anti-B220 APC (Cedarlane, Hornby, Ontario, Canada); anti-CD3 biotin (YCD3); anti-TCR FITC; anti-TCR biotin (H57-597); anti-TCR biotin (GL3; BD PharMingen, San Diego, CA); anti-CD25 FITC (7D4; BD PharMingen); anti-CD44 PE (IM781); and anti-IgM FITC (R6-60.2; BD PharMingen). Biotinylated Ab were detected either with streptavidin-PE or streptavidin-Cy5PE. Where required, Ab directed against CD45.1 and/or CD45.2 (FITC or PE conjugated; BD PharMingen) were included. All FACS analysis was performed on a FACSCalibur flow cytometer with CellQuest software (BD Biosciences, Mountain View, CA). Dead cells were excluded by gating for forward scatter and propidium iodide (PI) exclusion. For each sample, 50,000–125,000 cells were analyzed. Cell cycle analysis was performed by PI staining of ethanol-fixed, RNase-treated cells, followed by flow cytometric analysis of DNA content, as previously described (34).

Purification of DN2/3 thymocytes and peripheral T cells

DN2/3 thymocytes from Tg⁺ and Tg^- mice were isolated and purified as follows. Thymocytes expressing CD4, CD8, and/or CD3 surface receptors were removed using purified Abs directed against CD4, CD8, and CD3, and sheep anti-rat IgG magnetic beads (Dynal, Great Neck, NY). CD44⁺CD25⁺ (DN2) and CD44^-CD25⁺ (DN3) cells were then isolated (99% pure) by cell sorting. Peripheral lymph node cells were enriched for T cells (peripheral T cells) by magnetic bead depletion of cells that express B220, Mac-1, and Gr-1 cell surface receptors.

Generation of mixed bone marrow chimeras

Mixed bone marrow chimeras were generated using an adaptation of the engraftment model (35). T cell-depleted bone marrow was prepared from Ly-5.2⁺ mNumb Tg⁺ or Tg^- littermates and Ly-5.1⁺ C57BL/6 mice (B6.SJL-ptprc^a/BoAiTac; Taconic Farms, Germantown, NY). T cells were depleted from bone marrow by magnetic depletion of Thy-1.2-expressing cells. A total of 5 x 10⁶ cells was injected i.v. into RAG-2^-/- mice (B10.D2/nSnJTac-Rag2^tm1 N10; Taconic Farms) that had been gamma-irradiated with a lethal dose (1000 cGy, Gammacell 137C source; Atomic Energy, Ottawa, Canada) 4–18 h before injection. Chimeras were analyzed 4–5 wk following reconstitution.

Cell culture

Thymocytes (3 x 10⁶ cells/ml) from Tg⁺ or Tg^- mice were cultured in RPMI 1640 supplemented with 12.5% FBS, 25 mM HEPES (pH 7), 50 µM 2-ME, and 2 mM L-glutamine for 24 or 40 h. Cell cycle analysis and the degree of apoptosis were determined by FACS analysis of the DNA content, as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we have shown that misexpression of the p66 isoform of mNumb is sufficient to produce cell fate transformations in the Drosophila peripheral nervous system (23). p66 mNumb is the predominant isoform expressed in the mouse thymus (25), and therefore we chose to examine the effects of p66 mNumb overexpression on T cell development. We generated Tg mice with the p66 mNumb expressed under the control of the Lck proximal promoter. This promoter becomes active coincident with T cell commitment in CD44^-CD25⁺ DN (DN3) thymocytes, and remains active during the DP thymocyte stage (36). Accordingly, we assessed the affect of mNumb overexpression on vs TCR⁺ T cell and CD4⁺ vs CD8⁺ T cell development. Four lines of Tg mice were generated. Most of the experiments were done using the two founder lines, A and B, which had the highest levels of thymic mNumb expression. Similar results were obtained with each line; therefore, the results have been combined.

Immunoprecipitation and Western blot experiments demonstrated that mNumb protein was dramatically overexpressed in thymocytes (Fig. 1A). This was evident at the early DN stage of thymic development, since Numb was detected by Western blot in Tg⁺ DN, but not in Tg^- DN cells (Fig. 1B). mNumb was also elevated in the spleen and lymph node cells of Tg⁺ mice in comparison with their Tg^- littermates (Fig. 1A). Using anti-Numb antisera recognizing different regions of the Numb protein, we confirmed that the transgenically expressed mNumb protein is full length (data not shown). Confocal microscopy revealed that Tg mNumb was localized to thymic cell membranes and colocalized with AP-2, a marker of clathrin-coated pits and early endosomes (Fig. 1, C–E). This is in agreement with previous studies showing that mNumb proteins interact with components of the early endocytic machinery and localize to endocytic vesicles (37) (S. E. Dho, C. A. Smith, and C. J. McGlade, manuscript in preparation). To further examine the timing of Tg mNumb and endogenous mNumb expression during thymocyte development, we performed RT-PCR on RNA extracted from DN2/3 and DP subpopulations of thymocytes and from mature peripheral T cells isolated from 4-wk-old mice. Two primer pairs, one specific for endogenous mNumb and the other specific for Tg mNumb, were used in the reaction. Both endogenous mNumb and Tg mNumb mRNA was detected in all of the subpopulations of cells (Fig. 1F). Therefore, Tg mNumb protein was expressed during thymocyte development before - and -chain rearrangement and before CD4/CD8 lineage commitment, and it localized correctly within the cells.

View larger version (75K): [in this window] [in a new window]   FIGURE 1. mNumb is overexpressed in Tg+ mice. A, Overexpression of mNumb protein in the thymus, spleen, and lymph nodes of Tg+ mice. mNumb was immunoprecipitated from equal amounts of lysate (0.5 mg of protein per immunoprecipitation) from thymus, spleen, and lymph nodes of two lines of Tg+ mice (Tg+A and Tg+B) and their Tg- littermates using anti-Numb-A Ab and Western blotted with anti-Numb-C Ab (23 ). Arrowhead, mNumb. B, Overexpression of mNumb protein in Tg+ DN thymocytes. DN thymocytes purified by magnetic bead depletion (97% purity) from Tg+ and Tg- littermates were lysed, and equivalent amounts of total protein were separated by SDS-PAGE and Western blotted with anti-Numb-C Ab. Blots were reprobed with anti-tubulin Ab. C–E, Transgenically expressed mNumb is membrane associated and colocalizes with AP-2. Thymic sections from Tg+ (A) animals were fixed 4% paraformaldehyde and permeabilized in a solution of 1x PBS containing 0.2% Triton X-100. Slides were preblocked in PBS containing 5% goat serum and coimmunostained with mouse anti-AP-2 mAb, followed by goat anti-mouse Ab conjugated to CY3 (red) and affinity-purified rabbit anti-Numb-C polyclonal Ab (23 ), followed by goat anti-rabbit Ab conjugated to AlexaFlour 488 (green). Images showing AP-2 staining (C), Numb staining (D), and the merged images (E) were obtained using confocal microscopy. F, Expression of endogenous and Tg mNumb during T cell development. RNA extracted from depleted and FACS-sorted populations of cells from CD44+CD25+ and CD44-CD25+ (DN2/3) and DP thymocytes, and peripheral T cells (PT) from 3- to 4-wk-old Tg+ and Tg- mice were used to prepare cDNA. cDNA pools were normalized for equivalent template amounts by amplification using primers specific to -actin. PCR amplifications were performed using primers specific for endogenous mNumb and Tg mNumb.

preT

preT

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Expression pattern of Hes-1, preT, and Ifi-D3 is altered by Tg mNumb expression. A, RNA extracted from the thymi of 3-wk-old Tg+ and Tg- littermates was used to prepare cDNA. cDNA pools were normalized for equivalent template amounts by serial dilution and amplification using primers specific to -actin. PCR amplification of Notch-1 target genes was performed using primers specific for Ifi-D3, Hes-1, and preT. PCR products were separated by electrophoresis through a 0.8% agarose gel. Lanes 1–4, Serial dilution of Tg- thymic cDNA; lanes 5–8, serial dilution of Tg+ thymic cDNA. Representative results obtained from three independent pairs of Tg+ and Tg- and littermates are shown. B, RNA was prepared from DN2/3 and DP thymocytes and peripheral T cells (PT) from 3- to 4-wk-old Tg+ and Tg- mice and used to make cDNA. The cDNA was amplified with Ifi-D3 and -actin-specific primers, and PCR products were separated as above.

View larger version (56K): [in this window] [in a new window]   FIGURE 3. Phenotypic profile of thymocytes from mNumb Tg+ and Tg- mice. A, Phenotypic profile of total thymocytes. Two-parameter 7% probability contour plots depict surface expression of CD4 vs CD8 on thymocytes from representative 4-wk-old Tg+ and Tg- mice. The percentages of DN, DP, CD4 SP, and CD8 SP in each sample are indicated. The histograms depict expression of TCR on total thymocytes. B, Phenotypic profile of immature DN thymocytes. Two-parameter 7% probability contour plots depict the expression of CD44 vs CD25 (top panels) and CD44 vs B220 (bottom panels) in DN thymocytes of Tg+ and Tg- mice. DN thymocytes were identified by lack of staining with Ab specific for CD4, CD8, CD3, and TCR. The numbers within the quadrants represent the percentage of live cells gated by PI exclusion.

View larger version (55K): [in this window] [in a new window]   FIGURE 4. Phenotypic profile of splenocytes and lymphocytes from mNumb Tg+ and Tg- mice. A, The histograms depict expression of B220 on splenocytes (top panels). Two-parameter 7% probability contour plots show surface expression of CD4 vs CD8 on splenocytes (bottom panels). B, Staining profile of lymphocytes using two-parameter 7% probability contour plots. The numbers within the quadrants represent the percentage of live cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Cell cycle analysis of total, SP/DP, and DN thymocytes from mNumb Tg+ and Tg- mice. DNA content of thymocytes from Tg+ and Tg- mice was determined by PI staining and flow cytometric analysis. The percentages of cells in G0 and G1 or in S and G2-M phases of the cell cycle in total, CD4/CD8 SP and DP, or DN thymocytes are shown. DN cells were analyzed by gating on FITC-negative thymocytes stained with anti-CD4 and anti-CD8 mAbs. The results are representative of three independent experiments.

The percentages of Tg⁺ or Tg^- cells contributing to the thymus, spleen, and lymph nodes were similar in recipient mice that received mixtures of Tg⁺ and wild-type Ly-5.1⁺ bone marrow or those that received control mixtures of Tg^- and wild-type Ly-5.1⁺ bone marrow (Table II). In addition, the proportions of Tg⁺ and wild-type Ly-5.1⁺ cells contributing to DN, DP, CD4⁺, and CD8⁺ SP and TCR⁺ subsets in these tissues were similar. There was a small increase in the proportion of Tg⁺ B cells (B220⁺) and a small decrease in the proportion of Tg⁺ T cells (CD4⁺ SP or CD8⁺ SP) in the spleen and lymph nodes of the recipient mice reconstituted with mixtures of Tg⁺ and wild-type Ly-5.1⁺ bone marrow cells (Fig. 6). However, these differences were also observed in the control recipients that were injected with mixtures of Tg^- and wild-type Ly-5.1⁺ cells. Therefore, it is unlikely that these differences reflect an effect of Tg mNumb overexpression.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Transgenic overexpression of mNumb does not alter lymphocyte development. Mixtures of T cell-depleted bone marrow cells from Ly-5.2+ mNumb Tg+ mice (left panel) or Tg- littermates (right panel) and Ly-5.1+ wild-type mice were injected into irradiated RAG-2-/- mice. The thymus (A), spleen (B), and lymph nodes (C) of the recipient mice were analyzed by FACS 4–5 wk after reconstitution. The number of cells in a specific subset is represented as a percentage of the number of Ly-5.2+ or Ly-5.1+ cells in each tissue. Each dot represents the value from one recipient mouse, and the bar represents the average.

In previous work by others, Tg expression of a constitutively active form of Notch-1 also promoted survival of thymocytes in culture (10). Therefore, we determined whether thymocytes with Tg overexpression of mNumb would undergo apoptosis more readily in culture. We observed similar amounts of cell death in cultures of Tg⁺ and Tg^- thymocytes (Table III), suggesting the Tg mNumb does not increase apoptosis in vitro.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We analyzed two different lines of Tg mice to determine the effect of mNumb overexpression on endogenous Notch-1 activity and on T cell development. High levels of functional p66 mNumb protein were expressed in thymocytes of Tg mice, including DN cells. Overexpression of mNumb resulted in reduced expression of three genes, Hes-1, preT, and Ifi-D3, previously shown to be up-regulated by activated Notch (11), providing evidence that Numb antagonizes endogenous Notch-1 signaling. This reduction was evident as early as the DN2/3 stage of development coincident with commitment to the T cell lineage. Despite this evidence for a direct effect of Numb on Notch-1 signaling, we were unable to detect any changes in / or CD4/CD8 lineage commitment or thymocyte cell cycle or survival. Moreover, the lymphopoietic activity of bone marrow from mNumb Tg⁺ mice was equivalent to that of Tg^- bone marrrow in competitive repopulation experiments.

In support of our results, it was recently reported that Notch-1 signaling is not required after T cell commitment for thymocyte survival, differentiation, or fate specification in mice with a conditional, loss-of-function mutation of Notch-1 (43). There is the possibility that other Notch genes may have acted redundantly in place of Notch-1 in that study, since downstream consequences of the conditional Notch-1 deletion were not directly measured (43). However, results from our analyses of Numb Tg⁺ mice, in which Notch signaling is down-regulated through Numb overexpression, support the conclusion that Notch-1 signaling is not required in thymocytes for / or CD4/CD8 fate specification.

In contrast, previous studies on the role of Notch-1 signaling in T cell development, in which a truncated, constitutively active form of Notch-1 was overexpressed in thymocytes, led to the conclusion that Notch-1 signaling plays a critical role in / and CD4/CD8 lineage decisions during T cell development (12, 13). This form of Notch is known to be oncogenic in several cell types, including T cells, and may have produced a nonphysiologic effect (44, 45, 46). It is also possible that activated Notch-1 may have altered the activity or expression of proteins that are normally influenced by other Notch genes. The role of these other Notch family members was not addressed in experiments in which Notch-1 was constitutively deleted (43), and the effect of mNumb on the activity of other Notch genes is not known.

Reduction of Notch-1 levels in bone marrow cells results in an enhancement in the development of TCR⁺ T cells, which can be observed in competitive reconstitution experiments with mixtures of Notch-1 heterozygous mutant and normal bone marrow (9). We used a similar strategy to study the effect of Tg overexpression of mNumb on the TCR vs TCR lineage commitment. Transgenic mNumb was readily expressed in DN2/3 thymocytes, and the expression of the Notch target gene, Ifi-D3, was significantly reduced in this subpopulation. Thus, an effect of mNumb on lineage commitment, if present, should have been observed at this developmental stage; however, we found that mNumb Tg⁺ and Tg^- cells contributed equally to the different cell lineages in repopulation experiments at the different time points analyzed. Therefore, Tg overexpression of mNumb and down-regulation of Notch signaling at this stage of development do not mimic the effect of reduced Notch-1 levels in competitive repopulation experiments. It is possible that the inhibition of Notch by Numb overexpression in our experiments was insufficient or perhaps the reduction in Notch-1 may be required earlier, since in the experiments with heterozygous Notch-1 mutants (9), Notch-1 expression would be reduced at all stages of development.

In mammals, there are four isoforms of mNumb, and several lines of evidence imply that the Numb isoforms may have distinct cellular functions (25, 32). It is currently unknown whether the Numb isoforms differ in their capacity to antagonize Notch-1, or whether they interact with other Notch family members. In this study, we have shown that the p66 isoform of Numb is able to suppress expression of Notch-1 target genes in vivo. We cannot exclude the possibility that a different isoform of Numb may have a different effect on Notch-1 signaling or on other Notch genes expressed in the thymus, which would alter T cell development. However, we are aware of unpublished results in which the p65 isoform of mNumb was transgenically expressed using the same promoter, and like our results, no alterations in T cell development were observed (Y.-R. Zou and D. R. Littman, personal communication).

Conditional targeting of Notch-1 much earlier in development has revealed an essential role in specification of T vs B lineage precursors (4, 7, 8). Therefore, during the course of lymphocyte development, there is an apparent transition between Notch-1 dependence and independence. As such, a role for Numb in lymphocyte development will likely be revealed by modulating its levels at earlier stages of hemopoiesis. Our results, in keeping with recently published work (43), support the conclusion that in immature thymocytes, Notch-1 signaling is not essential for / or CD4/CD8 T cell lineage commitment or survival.

	Footnotes

 
¹ This work was supported by grants from the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society (to C.J.M.) and the Medical Research Council of Canada (to C.J.G.). U.K. is supported by a NCIC postdoctoral fellowship award, C.J.G. is a Medical Research Council of Canada Scientist, and C.J.M. is a Research Scientist of the NCIC, supported with funds from the Canadian Cancer Society.

² M.B.F. and U.K. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. C. Jane McGlade, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. E-mail address: jmcglade{at}sickkids.on.ca

⁴ Abbreviations used in this paper: DN, double negative; dNumb, Drosophila numb; DP, double positive; mNumb, mammalian Numb; PI, propidium iodide; PTB, phosphotyrosine binding; SP, single positive; Tg, transgenic.

Received for publication May 11, 2001. Accepted for publication January 22, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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