Target of the Notch Pathway , a Hes-1 Maintaining the Expression of Early

Home , Homeotic gene

Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway This information is current as of September 29, 2021. Masaki Miyazaki, Hiroshi Kawamoto, Yuko Kato, Manami Itoi, Kazuko Miyazaki, Kyoko Masuda, Satoshi Tashiro, Hiroto Ishihara, Kazuhiko Igarashi, Takashi Amagai, Rieko Kanno and Masamoto Kanno

J Immunol 2005; 174:2507-2516; ; Downloaded from doi: 10.4049/jimmunol.174.5.2507 http://www.jimmunol.org/content/174/5/2507 http://www.jimmunol.org/ References This article cites 54 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/174/5/2507.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway1

Masaki Miyazaki,* Hiroshi Kawamoto,§ Yuko Kato,* Manami Itoi,¶ Kazuko Miyazaki,‡ Kyoko Masuda,ʈ Satoshi Tashiro,† Hiroto Ishihara,* Kazuhiko Igarashi,† Takashi Amagai,¶ Rieko Kanno,* and Masamoto Kanno2*

Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR ␤-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the Downloaded from expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors -isolated from mel-18؊/؊ mice. In addition, mel-18؊/؊ T precursors could not maintain the Hes-1 expression induced by Delta like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors. The Journal of Immunology, 2005, 174: 2507–2516. http://www.jimmunol.org/ he Polycomb group (PcG)3 genes were originally identi- protein complex that also contains M33, BMI-1, RAE-28, fied in Drosophila as a class of regulators responsible for RING1A, and RING1B (3, 4). This mammalian complex is similar T maintaining homeotic gene expression by contributing to to PRC1 in Drosophila, which is able to competitively inhibit the the cellular memory of somite identity throughout cell division. chromatin remodeling complex, SWI/SNF, and interacts with se- PcG genes are conserved from Drosophila to mammals, and their quence-specific, DNA-binding factors (Pipsquesk, Zeste, and protein products have been reported to localize to the nucleus as GAGA) and histone deacetylase (5–7). This PRC1 is shown to multimeric protein complexes. These proteins epigenetically main- collaborate with the ESC-E(Z) complex in regulation of gene ex- tain the repressed state of target genes through the modification of pression through epigenetic modification of chromatin structure (8, by guest on September 29, 2021 chromatin structure. In Drosophila, at least two types of PcG com- 9). Although several studies have recently described the silencing plexes, each with different properties, can be distinguished: Poly- mechanisms of PcG complexes, to our knowledge, it has rarely comb repressive complex 1 (PRC1) and ESC-E(Z) (1, 2). The been reported that the PcG gene is practically required for the mel-18 gene is a mammalian homologue of the Drosophila pos- maintenance of gene expression in the mammalian cell differenti- terior sex combs (Psc) gene, and its product is a member of a PcG ation system. It is well known that PcG genes play a significant role in the regulation of lymphocyte differentiation (10, 11). Mice deficient in Departments of *Immunology and †Biomedical Chemistry, Graduate School of Bio- medical Science, and ‡Department of Developmental Biology, Research Institute for the individual components of the PRC1-like complex, mel-18, Radiation and Medicine, Hiroshima University, Hiroshima, Japan; §Laboratory for bmi-1, rae-28, and m33, display SCID (12–15). Loss of function of Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; ¶Department of Immunology and Microbiology, Meiji University bmi-1 causes a severe block of B cell development (12), and ʈ of Oriental Medicine, Kyoto, Japan; and Department of Immunology and Cell Bi- rae-28 deficiency reduces the generation of pre-B and immature B ology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan cells from fetal liver (FL) hemopoietic progenitors (15). In T cell Received for publication August 6, 2004. Accepted for publication October 31, 2004. development, bmi-1 mutant mice exhibited impaired thymocyte The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance development at an immature stage (16). In mel-18 mutant mice, B with 18 U.S.C. Section 1734 solely to indicate this fact. cell maturation is arrested between the pro- and pre-B cell stages, 1 This work was supported by Grants-in-Aid for Science from the Ministry of Edu- and severe thymic atrophy is also observed (14). In mature resting cation, Culture, Sports, Science, and Technology of Japan. The authors have no fi- B cells, mel-18 negatively regulates B cell receptor-induced pro- nancial conflict of interest. liferation through the down-regulation of the c-Myc/cdc25 cascade 2 Address correspondence and reprint requests to Dr. Masamoto Kanno, Department of Immunology, Graduate School of Biomedical Science, Hiroshima University, (17). Th2 cell differentiation is also impaired in mel-18 mutant 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail address: mkanno@ mice, and mel-18 is involved in the induction of GATA-3 under hiroshima-u.ac.jp Th2-skewed conditions (18). In contrast to the extensive analysis 3 Abbreviations used in this paper: PcG, Polycomb group; DL1, Delta-like-1; DN, double negative; DP, double positive; dpc, days postcoitum; ETP, enhancers of of the roles of mel-18 in both immature and mature B cell devel- Trithorax and Polycomb, early T progenitor; FB, fetal blood; FL, fetal liver; FT, fetal opment as well as in mature T cell function, much remains to be thymus; FTOC, FT organ culture; HOM-C, homeotic gene; HOS, high oxygen submersion; HSC, hemopoietic stem cell; KSL, LinϪScaIϩc-Kitϩ cell; LinϪ, lineage investigated with regard to a potential role for mel-18 in thymocyte marker-negative; PI, propidium iodide; PRC1, Polycomb repressive complex 1; 7Rϩ, development. Therefore, we performed a comprehensive analysis LinϪSca-1ϩc-KitlowIL-7R␣ϩ; SCF, stem cell factor; siRNA, small interfering RNA; SP, single positive; TrxG, Trithorax group; TSt-4/DL1, TSt-4 stroma cell expressing of mel-18 function to clarify its role and the underlying mecha- Delta-like-1. nisms in the regulation of thymocyte development.

The thymus is the major site of T cell differentiation and mat- B220-FITC and -biotin (RA3-6B2); anti-CD19-FITC and -biotin (1D3); uration. Thymocytes can be divided into four main populations: anti-Mac-1-FITC and -biotin (M1/70); anti-Gr-1-FITC and -biotin (RB6- CD4ϪCD8Ϫ double negative (DN), CD4ϩCD8ϩ double positive 8C5); Ter119-biotin; anti-NK1.1-FITC (PK136); anti-CD45.2-FITC (104); ϩ ϩ and anti-CD45.1-PE (A20). Anti-CD127-PE and -biotin (A7R34) and (DP), and CD4 or CD8 single positive (CD4SP or CD8SP) Ter119-FITC were purchased from eBiosciences. Biotinylated Abs were cells. DN cells can be further divided into four subpopulations revealed with streptavidin-CyChrome (BD Pharmingen). To analyze DN (DN1-DN4) according to their CD44 and CD25 expression pat- CD3Ϫ thymocytes, lineage marker-negative (LinϪ)ScaIϩc-Kitϩ cell terns. CD44ϩCD25Ϫ (DN1) thymocytes represent the earliest T (KSL), and common lymphoid progenitors in bone marrow and p-T cell in FL, all cells expressing lineage markers (CD4, CD8, CD3, B220, CD19, progenitors in the thymus. DN1 cells differentiate through a ϩ ϩ Ϫ ϩ Mac-1, Gr-1, Ter119, and NK1.1) were gated out of the analyses. FACS CD44 CD25 (DN2) stage into CD44 CD25 (DN3) cells, in analysis was performed on a FACSCalibur flow cytometer (BD Bio- which the TCR␤ rearrangement takes place. Those with a success- sciences), and data were analyzed using CellQuest software. For cell sort- ful TCR rearrangement at the ␤ locus receive a pre-TCR signal and ing, all cells were stained with biotinylated lineage markers, bound to differentiate through the CD44ϪCD25Ϫ (DN4) stage into DP cells streptavidin-magnetic beads and depleted of lineage-positive cells using MACS separation column (Miltenyi Biotec). The lineage-negative cells (19). During intrathymic T cell development, thymocytes are required were then stained with subsequent Abs as described above. Cells were to expand massively to create a highly diversified TCR repertoire. It subsequently sorted using a FACSVantage SE (BD Biosciences). Dead has been proposed that two independent proliferative phases exist cells were removed from analysis and sorting by staining with propidium during T cell development. One is the period of expansion that occurs iodide (PI; Sigma-Aldrich). Reanalysis of the sorted cells indicated the Ͼ ␤ ␤ purity to be 94% for each cell population. For experiments examining before the TCR gene rearrangement (pre- proliferation), which oc- mRNA expression in sorted cells, 3 ϫ 104 cells from each fraction were curs during the DN1 and DN2 stages (20). The second phase of ex- subjected to real-time PCR. Cells (1 ϫ 103) from 14.5E DN1 and DN2 pansion occurs after TCR␤ gene rearrangement (post-␤ proliferation), fractions were subjected to semiquantitative PCRs. Downloaded from which is stimulated by the pre-TCR signal (21, 22). Several signaling pathways have been shown to be involved in Fetal thymus (FT) organ culture (FTOC) the regulation of proliferation and differentiation of thymocytes. All liquid cultures were performed in RPMI 1640 (Invitrogen Life Tech- Notch signaling has recently drawn wide attention for its role in T nologies) plus 10% FCS for FTOC, 50 ␮M 2-ME (Nacalai Tesque), 1 mM cell development (23). Notch signaling is an evolutionarily con- sodium pyruvate (Invitrogen Life Technologies), 1ϫ nonessential amino acid solution (Invitrogen Life Technologies), and antibiotics. For analysis served pathway that controls multiple cell fate decisions in various http://www.jimmunol.org/ of T cell progenitors, FTOC under high oxygen submersion (HOS) con- tissues throughout ontogeny. Notch-RBP-J signaling is essential in ditions was performed as previously described (34). In brief, sorted cells the cell fate decisions between T and B cell lineages (24–26), ␣␤ from mel-18Ϫ/Ϫ and mel-18ϩ/Ϫ Ly5.2 14.5 days post coitum (dpc), or T cell and ␥␦ T cell lineages (27–29), and Th1 and Th2 cell dif- Ly5.1/5.2 14.5 dpc were plated at one or 25 cells/well onto 96-well, V- ferentiation (30). Furthermore, several studies have indicated that bottom culture plates (Nalge Nunc International) containing deox- Ϫ/Ϫ ϩ/Ϫ Notch-1 signaling regulates the proliferation of early thymocytes yguanosine-treated Ly5.1/5.2(F1) 15.5 dpc, or mel-18 and mel-18 15.5 dpc FT lobes and cultured in medium. Stem cell factor (SCF; 10 in addition to the cell lineage commitment. A stromal cell line ng/ml; R&D Systems) and 50 U/ml IL-7 (Genzyme) were added to the forced to express DL1 can support T cell development to at least medium. Cultures were scored on day 10 or 14, and cells were analyzed by the DP stage as well as the expansion of T progenitors in mono- flow cytometry. layer culture (31). The Hes-1 gene, Notch-1 signaling target gene, by guest on September 29, 2021 was shown to be essential for the expansion of early T progenitor RT-PCR and Southern blot analysis cells, but not for T/B cell lineage commitment. The expression Semiquantitative RT-PCR was performed on cDNAs obtained from 1000 level of Hes-1 is involved in supporting the cellularity of devel- sorted cells of DN1 (CD44ϩCD25Ϫc-Kitϩ) and DN2 (CD44ϩCD25ϩc- ϩ Ϫ Ϫ oping thymocytes (32, 33). Kit ) cell subpopulations in 14.5 dpc mel-18 / or littermate control FT. In this study we found that loss of the PcG gene mel-18 impairs Total RNA was isolated from indicated cells using TRIzol reagent (In- vitrogen Life Technologies) and was reverse transcribed using the Super- the expansion of early T progenitor cells at a stage before rear- Script II RT-PCR system (Invitrogen Life Technologies) with oligo(dT) rangement of the TCR ␤-chain gene in vivo and in vitro. We also primer or random hexamer primer. Real-time PCR was performed using observed that the Hes-1 gene was down-regulated in early T pro- the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) genitors in mel-18Ϫ/Ϫ mice. Based on our analysis, mel-18 appears according to the manufacturer’s instructions. The 50-␮l amplification reaction mixture contained 5 ␮l of cDNA (1/10 dilution), 25 ␮l of TaqMan to be indispensable for the maintenance of the active state of the universal PCR master mix (Applied Biosystems), and 200 nM each of Hes-1 gene in proliferating T progenitors. We propose that mel-18 primers. Each sample was amplified in triplicate using primers and probes contributes to the expansion of early T progenitors through the specific for mel-18. A two-step cycling protocol with combined primer maintenance of cellular memory of these proliferating cells. annealing and elongation at 60°C was used. The TaqMan ribosomal RNA control reagent (Applied Biosystems) was used as the endogenous normal- ization standard. For semiquantitative PCR, cDNAs were amplified using Materials and Methods various oligonucleotide primers chosen on the basis of Oligo 4.0 software Mice (National Biosciences). The primers used for the PCR were as follows: Ϫ Ϫ mel-18 (real-time PCR), 5Ј-GCGACGGGACTTCTATGCA-3Ј and 5Ј- The mel-18 deficient (mel-18 / ) mice were described previously (14), and CGCCTTCGTAGAACTCAATGG-3Ј; mel-18,5Ј-CGGAGAATG animals used in this study were backcrossed to C57BL/6 Ͼ10 times. GAGATGGGGACAAGGAGAAG-3Ј and 5Ј-AAGGTGGAGTGGGG C57BL/6J (B6, Ly5.2) mice were obtained from CREA Japan, and B6 SJL GAAGTAGGATGGGTAG-3Ј; G3PDH,5Ј-GTGAAGGTCGGTGTGAA Ptprca Pep3bBoyJ (B6 Ptprc, Ly5.1) mice were obtained from The Jack- CGGAT-3Ј and 5Ј-CAGAAGGGGCGGAGATGATGAC-3Ј; Notch-1,5Ј- son Laboratory. All mice were kept in accordance with the laboratory CCCAGCAGGTGCAGCCACAG-3Ј and 5Ј-GGTGATCTGGGACGGCA animal science guidelines of Hiroshima University. For timed pregnancies, TGG-3Ј; Hes-1,5Ј-GCCAGTGTCAACACGACACCGG-3Ј and 5Ј-TC the day of vaginal plug was counted as day 0.5. ACCTCGTTCATGCACTCG-3Ј; GATA-3,5Ј-TCGGCCATTCGTACATG Flow cytometric analysis and Abs GAA-3Ј and 5Ј-GAGAGCCGTGGTGGATGGAC-3Ј; LEF-1,5Ј-AACTCT GCGCCACCGATGAG-3Ј and 5Ј-AGAAAAGTGCTCGTCGCTGT-3Ј; Single-cell suspensions from bone marrow, thymus, FL, and fetal blood TCF-1, 5Ј-GCCAGCCTCCACATGGTGTC-3Ј and 5Ј-CGCGTGAGGGAT (FB) were stained with mAbs and second reagents. The following mAbs GGCTGCTG-3Ј; Deltex-1,5Ј-CCACTGCTACCTACCCAACAAT-3Ј and were purchased from BD Pharmingen: anti-CD4-FITC, -PE, -allophyco- 5Ј-AGGCTAGAGGCAAGGCAAAAGG-3Ј; RBP-J,5Ј-CACAGACAAG cyanin, and -biotin (RM4-5); anti-CD8␣-PE, -allophycocyanin, and -biotin GCAGAATACACG-3Ј and 5Ј-TGTAGGTGAAGGTAAGGCTGGT-3Ј; and (53-6.7); anti-CD3⑀-FITC, -allophycocyanin, and -biotin (145-2C11); anti- E2A,5Ј-CATCCATGTCCTGCGAAGCCAC-3Ј and 5Ј-TTCTTGTCCTCT CD44-PE (IM7); anti-CD25-FITC and -allophycocyanin (7D4 and PC61); TCGGCGTCGG-3Ј. PCR amplification was performed using the GeneAmp anti-c-Kit-allophycocyanin (2B8); anti-Sca-1-FITC (D7); anti-CD45R/ PCR system 9700 (Applied Biosystems). The cycle number was 24–34, The Journal of Immunology 2509 because the amplification was found to be within the linear range (data not Results shown). Decreased number of early T progenitors in adult mel-18Ϫ/Ϫ mice Southern hybridization It has been previously reported that the paucity of cell expansion and maturation arrest at the DN stage were observed in adult thy- PCR product was electrophoresed on 2% agarose gel, blotted onto a nylon Ϫ/Ϫ ϫ membrane, and analyzed by Southern blot hybridization with 33P-labeled mocytes from mel-18 mice on a mixed C57BL/6(B6) 129 probes. The signals were processed and quantified by an imaging analyzer background (14). Subsequently, our group and others noticed that (BAS 2000; Fuji Photo Film). The following probes were used: mel-18, the CD4/CD8 profile of thymocytes in mel-18Ϫ/Ϫ mice became 5Ј-ACCTGGCAAAGTTCCTCCGC-3Ј; G3PDH,5Ј-GGTGGACCTGAC ϩ/ϩ Ј Ј comparable to that of mel-18 mice after an extensive backcross CTGCCGTCTAGAAAAAC-3 ; Notch-1,5-ACCCCTTCCTCACCCCA Ͼ TCCCC-3Ј; Hes-1,5Ј-CCGCCGCGCTCAGCACAGACCC-3Ј; GATA-3, to B6 mice ( 10 times; Fig. 1A) (18). Hereafter, experiments were 5Ј-GGTATGCCGCCCGCCTCTGCTG-3Ј; LEF-1,5Ј-GATGCCCAATA performed exclusively with mice on a B6 background. In these TGAACAGCGACCC-3Ј; TCF-1,5Ј-ACCCCCTGTCCCCTTCCTGCGG- 6-wk-old male B6 mel-18Ϫ/Ϫ mice, a severe reduction in total 3Ј; Deltex-1,5Ј-TGCTCATCACCGCCTGGGAACG-3Ј; RBP-J,5Ј-GTC thymocyte number was observed despite the relatively normal CGCCAGCCAGTCCAGGTTC-3Ј; and E2A,5Ј-ATGCCAGCCTCCCC AGCCAGCC-3Ј. CD4/CD8 profile (Fig. 1, A and B). The total thymocyte number in mel-18Ϫ/Ϫ mice was reduced to Ͻ10% of the wild-type number, Immunohistochemistry even in 2-wk-old mice, and was further decreased to ϳ2% of the wild-type number in 10-wk-old mice (Fig. 1B). Next, we investi- Embryos (12.5 dpc) were embedded in OCT compound (Miles) and snap- Ϫ frozen. Freshly cut 5-␮m sections were fixed with acetone at room tem- gated the cellularity of the immature, Lin thymocyte population

perature for 2 min. Sections were incubated in rabbit anti-keratin Ab (wide subdivided by CD44/CD25 (DN1-DN4) expression criteria. The Downloaded from spectrum screening; DakoCytomation) or rabbit anti-Ikaros Ab (54), then severe reduction of the absolute cell number (to Ͻ10% of normal) incubated in HRP-conjugated goat anti-rabbit IgG (Vector Laboratories) at was apparent at the most immature stage, DN1. No maturation arrest Ј room temperature. Peroxidase activity was developed with 0.1% 3,3 -dia- among DN subpopulations was observed (Fig. 1, A and B). From minobenzidine and 0.02% H2O2 in PBS. Sections were then counterstained with hematoxylin. these results, we speculated that the severe reduction of the cell number might begin at the DN1 stage or earlier in mel-18Ϫ/Ϫ mice. TUNEL assay and immunofluorescence staining Recent reports have revealed that the DN1 stage comprises at http://www.jimmunol.org/ high ␣neg/low high Freshly cut 5-␮m sections were fixed with acetone at room temperature for least two subpopulations, c-Kit IL-7R (c-Kit ) and c- neg/low ϩ high 2 min. Sections were incubated in rabbit anti-keratin Ab for 2 h, then Kit IL-7R␣ , and that only cells within the c-Kit pool incubated in goat anti-rabbit IgG-Texas Red conjugate (Molecular Probes) have T-lineage potential in adult mice (37). Additionally, our for1hatroom temperature. TUNEL reaction was performed on sections preliminary observations suggested that c-Kithigh DN1, but not using an In Situ Cell Death Detection kit, Fluorescein (Roche), according c-Kitneg/low DN1, cells retained T-lineage reconstitution potential to the manufacturer’s protocol. Briefly, sections were fixed in 4% parafor- maldehyde in PBS for 10 min at room temperature, incubated in 0.1% (data not shown), and a recent study supported our data (38). Triton X-100/0.1% sodium citrate for 2 min at 4°C, then incubated in Therefore, we examined whether these c-Kithigh cells were affected TUNEL reaction mixture for1hat37°C. by a lack of mel-18. The absolute cell number of c-Kithigh cells in Ϫ/Ϫ ϩ/ϩ mel-18 mice was reduced to 3% of that observed in mel-18 by guest on September 29, 2021 Cell cycle analysis mice (1,200 cells and 40,600 cells/thymus, respectively; Fig. 1C). The amount of nuclear DNA was determined by PI staining as follows. Next, we investigated whether these cellular defects occurred DN1 and DN2 14.5E FT cells were fixed in 50% ethanol at 4°C for 30 min after migration into the thymus or at a prethymic progenitor stage and incubated in PBS containing 1 mg/ml RNase (Sigma-Aldrich) at 37°C in adult bone marrow. We analyzed the absolute number of he- for 20 min. The cells were washed in PBS, resuspended in PBS containing 100 ␮g/ml PI, and analyzed by a FACSCalibur (BD Biosciences). mopoietic progenitors, defined as KSL cells, and so-called common lymphoid progenitors, defined as LinϪSca-1ϩc-KitlowIL- In vitro survival or proliferation assay 7R␣ϩ (7Rϩ) cells (39) in bone marrow. There was no significant difference between the numbers of 7Rϩ and KSL cells in mel- The in vitro survival assay was conducted as previously described (35). Sorted ϩ/ϩ Ϫ/Ϫ cells were cultured in 96-well, flat-bottom plates in a volume of 0.15 ml of 18 and mel-18 mice (Fig. 1D). RPMI 1640 with 10% FCS and 2-ME. SCF (100 ng/ml) and IL-7 (150 U/ml) The expression level of mel-18 was assessed in normal cell pop- were added to the medium. After 0 or 20 h, cells were harvested, and the ulations by real-time PCR (Fig. 1E). The level of mel-18 mRNA numbers of surviving T cell precursors were counted by flow cytometry. was higher in the DN subset than in the DP and SP subsets. Within the DN population, the mel-18 expression level was highest in T cell development on Tst-4/DL1 DN1 and was gradually decreased in the stages leading up to the Tst-4, a thymus-derived fibroblast cell line (36), was transfected with ret- more differentiated DN4. The level in DN1 was higher than that in rovirus containing Delta-like-1 (DL1) gene. DL1 expressing TSt-4 effi- ϩ Ϫ ϩ KSL and 7R cells in bone marrow. The level of mel-18 mRNA ciently support the differentiation of T cells from Lin c-Kit progenitors high of FL and bone marrow in a monolayer culture, whereas they suppress B in the c-Kit subset was comparable to that in the entire popu- cell generation (H. Kawamoto, unpublished observation). IL-7Rϩ cells in lation of DN1 cells (data not shown). Therefore, these data show 12.5 dpc mel-18Ϫ/Ϫ or control FL were sorted and cocultured with Tst-4/ mel-18 ϩ ϩ that the gene is expressed at a higher level in DN1 thymo- DL1 cell in FTOC medium. After day 3 or 6 of culture, Ly5 /Thy1.2 cytes, including c-Kithigh cells, than any at other stage during the cells were sorted. developmental process from HSC to mature T cells in adult mice. Small interfering RNA (siRNA) preparation and transfection Based on our data, we speculate that the thymic atrophy in adult mel-18Ϫ/Ϫ mice is probably caused by defects in the earliest c- Dicer siRNA for mel-18 or GFP were generated using the Dicer siRNA Kithigh DN1 cells, although it remains to be clarified whether mi- Generation Kit (Gene Therapy Systems). In brief, cDNA construct for Ϫ/Ϫ mel-18 with T7 promoters at the 717 and 3Ј ends were made by PCR and gration into the thymus is affected in adult mel-18 mice. subjected to in vitro transcription to produce dsRNA. Dicer siRNAs were produced by digestion of dsRNA with recombinant Dicer enzyme. KKC cells (1 ϫ 105) were transfected with 125 ng of dicer-siRNA for either Impairment of early thymocyte development in mel-18Ϫ/Ϫ fetus mel-18 or GFP using Lipofectamine 2000 (Invitrogen Life Technologies). After 48 h, cells were harvested and total RNA was extracted and subjected We also examined whether early T cell development was impaired Ϫ Ϫ to semiquantitative RT-PCR analysis. in the mel-18 / fetus. In the FT at 12.5 dpc, when thymocytes are 2510 mel-18 REGULATES EXPANSION OF EARLY T PROGENITORS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Phenotypic characterization of mel-18Ϫ/Ϫ thymocytes and expression level of mel-18 during T cell development. A, A representative flow cytometric analysis of CD4 vs CD8 on total thymocytes, and analysis of CD44 vs CD25 on LinϪ DN thymocytes from 6-wk-old male wild-type control littermate (mel-18ϩ/ϩ) and mel-18Ϫ/Ϫ mice. Percentages of CD4 SP, CD8 SP, DP, DN, and DN1–4 cells are indicated. B, Absolute cell numbers were calculated for total thymocytes and the thymocyte subpopulations described in A. The bars represent the mean Ϯ SD. The cell number of total thymocytes from adult mel-18ϩ/ϩ, mel-18ϩ/Ϫ, and mel-18Ϫ/Ϫ mice, 2–10 wk old, are plotted. C, Total thymocytes from 6-wk-old mel-18ϩ/ϩ and mel-18Ϫ/Ϫ mice were stained for lineage markers, CD44, CD25, c-Kit, and IL-7R␣, and 5 ϫ 106 cells were analyzed. The percentage of total thymocytes and the absolute number of cells within the indicated region are shown. D, Bone marrow cells from femurs and tibiae were stained for lineage markers, Sca-1, c-Kit, and IL-7R␣. A representative flow cytometric analysis of c-Kit vs IL-7R␣ on gated LinϪSca-1ϩ bone marrow cells is shown. Total cell numbers of KSL and IL-7Rϩ from femurs and tibiae were calculated and presented in graphic histograms. The bars represent the mean Ϯ SD. E, Real-time PCR analysis of mel-18 expression in thymocyte and bone marrow cell fractions described in A and D from adult male B6. Expression in DP was defined as 1.

exclusively composed of DN1 cells (Fig. 2B), we found no sig- mostly T cell lineage-restricted progenitors, and those cells iso- nificant difference in the total number of thymocytes between mel- lated from 13.5 dpc FB also represented T cell progenitors (40, ϩ Ϫ Ϫ Ϫ 18 / and mel-18 / mice; however, at 13.5 dpc, the number of 41). In 12.5 dpc FL and FB, the percentage of IL-7Rϩ cells in Ϫ Ϫ thymocytes in the mel-18 / fetus was reduced to about half; fur- mel-18Ϫ/Ϫ fetuses was the much same as that in mel-18ϩ/Ϫ fetuses thermore, from 14.5 dpc to birth, the number of thymocytes was (data not shown). Furthermore, at 13.5 dpc, in which the cell num- ϩ/Ϫ reduced to less than one-third of the number in the mel-18 fetus ber in mel-18Ϫ/Ϫ FT was decreased to about half that in mel-18ϩ/Ϫ (Fig. 2A). However, we observed a small decrease in the percent- FT, there was no significant difference in the percentage of IL-7Rϩ age of DN2 cells, but no differentiation arrest in CD44/25 profile cells in FB between the two genotypes (Fig. 2C). To rule out the B of the FT at 12.5–14.5 dpc (Fig. 2 ). These observations suggested second possibility, the impairment of migration, the distribution of that a lack of mel-18 also affected the development of early T hemopoietic progenitors and thymic epithelial cells was investi- progenitors in the fetus. gated in sagittal sections of whole embryos at 12.5 dpc by immu- To confirm this, we had to rule out the following possibilities: nohistochemistry. Hemopoietic progenitors and thymic epithelial first, a decreased number of prethymic progenitors in FL or FB, and second, an obstructed migration of these progenitors into the cells were determined by staining with an anti-IKAROS or an anti- FT. We performed flow cytometric analysis to detect the number keratin Ab, respectively. Hemopoietic progenitors normally begin im- of LinϪc-Kithigh cells (hemopoietic progenitors) and LinϪc- migration into the thymic mesenchymal layer at 11.5 dpc and enter KithighIL-7Rϩ (IL-7Rϩ) cells in FL and FB. We have previously the epithelial region at 12.5 dpc (42). IKAROS-positive cells that shown that IL-7Rϩ cells in both 12.5 dpc FL and FB contained reside in the mesenchymal layer surrounding the epithelial region The Journal of Immunology 2511

FIGURE 2. Analysis of fetal thymocytes and prethymic lymphoid progenitors in mel-18Ϫ/Ϫ mice. A, Cell number of total thymocytes from a control littermate (mel-18ϩ/Ϫ) and a mel- 18Ϫ/Ϫ fetus. The cell numbers of total thymocytes from 12.5 dpc to neonatal mice are plotted. There were no differences in cell number or FACS profiles between mel-18ϩ/ϩ and mel-18ϩ/Ϫ fetuses in this period (data not shown). B, Representative flow cytometric analysis of CD44 vs CD25 on fetal thymocytes from mel-18ϩ/Ϫ and mel-18Ϫ/Ϫ fetus in 12.5, 13.5, and 14.5 dpc fetuses. The percentages of DN1-DN4 cells are indicated. C, Fetal blood cells in 13.5 dpc fetuses were stained for lineage markers (CD3, B220, CD19, Gr-1, Ter119, Thy1.2, and NK1.1), c-Kit, and IL- Downloaded from 7R␣. Representative flow cytometric analyses are shown. Two experiments with independent litters were performed with similar results. D, Distributions of epithelial and hemopoietic progenitor cells in the thymus anlage. Serial frozen sections of 12.5 dpc mel-18ϩ/Ϫ and http://www.jimmunol.org/ mel-18Ϫ/Ϫ embryos were incubated with anti-keratin or anti-IKAROS Abs. The lines indicate the border between the epithelial region and the mesenchymal layer of the thymus anlage. Scale bar, 50 ␮m. were regarded as immigrating progenitors. Taken together with the with a dGuo-treated FT lobe of B6 mice (Ly5.1/5.2 F1) at one cell data presented in Fig. 2, A, C, and D, the number of immigrating per lobe under HOS conditions. After 14 days, the thymocytes by guest on September 29, 2021 progenitors in mel-18Ϫ/Ϫ mice was comparable to that in mel-18ϩ/Ϫ generated in the culture were collected and analyzed by flow cy- mice (Fig. 2D). These results indicate that the mel-18 deficiency af- tometry to determine the potential of the initial progenitor cell for fected neither prethymic T progenitors nor their migration into the generating T cells. The frequency of reconstituted T progenitors Ϫ Ϫ thymus anlage. from mel-18 / DN1 cells was reduced to 8 of 30 (26.7%), in contrast to the 17 of 30 (56.7%) observed for mel-18ϩ/Ϫ. Further- Ϫ/Ϫ Impaired expansion of T progenitors in the mel-18 fetus at a more, the number of cells generated from a single mel-18Ϫ/Ϫ DN1 stage before TCR ␤-chain gene rearrangement cell was about half that generated by a mel-18ϩ/Ϫ DN1 cell (Fig. The above results raised the question of whether the reduced cell 3B). These results suggest that the reduced cell number of fetal number of mel-18Ϫ/Ϫ FT was caused by impaired expansion of thymocytes was due to a decreased number of early T progenitors Ϫ/Ϫ early thymocytes or an inability on the part of the thymic envi- and the impaired expansion of the T progenitors in mel-18 ronment to support thymocyte development. To assess the poten- mice. Taken together with the results shown in Fig. 2A, in which Ϫ/Ϫ tial of the thymic lobe to support T cell development, we per- thymocyte number in mel-18 FT was obviously reduced at 14.5 formed a FTOC. DN1 cells (CD44ϩCD25Ϫc-Kithigh; Ly5.1/5.2 dpc, we concluded that mel-18 deficiency impairs the pre-␤ pro- F1) were transferred into each lobe of mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ liferation of early T progenitors. (Ly5.2) FTs that had been treated with dGuo. After a 10-day cul- To confirm this, we established breeding mice, which lacked Ϫ/Ϫ Ϫ/Ϫ ture period, the number of donor-derived thymocytes, Ly5.1-pos- both the Rag1 and mel-18 genes (Rag1 mel-18 ). Rag1 is itive cells, was counted and analyzed. No significant difference essential for TCR gene rearrangement, and thymocytes from Rag1 was observed in cell number or in the CD4/CD8 expression profile mutant mice arrest at the DN3 stage. Therefore, backcrossing to of the recovered Ly5.1ϩ cells (data not shown), indicating that the Rag1 mutant mice makes it possible to investigate pre-␤ prolifer- Ϫ/Ϫ mel-18Ϫ/Ϫ thymic lobe sustained the capacity to support immature ation specifically. The number of thymocytes in the Rag1 mel- Ϫ/Ϫ thymocyte development. Next, 14.5 dpc FT lobes from either mel- 18 fetus was reduced to less than one-third that observed in Ϫ/Ϫ ϩ/ϩ Ϫ/Ϫ ϩ/Ϫ 18ϩ/Ϫ or mel-18Ϫ/Ϫ mice were cultured for 3 and 6 days. As either the Rag1 mel-18 or the Rag1 mel-18 fetus at shown in Fig. 3A, we found that the early expansion of immature 17.5 dpc (Fig. 3C). These results clearly demonstrate that mel-18 thymocytes was impaired in mel-18Ϫ/Ϫ FT, specifically on day 3, is required for the expansion of early T progenitors before TCR whereas there appeared to be normal maturation of T cell devel- ␤-chain gene rearrangement. opment, as defined by the CD4/CD8 profiles (data not shown). Ϫ/Ϫ To acquire a more detailed understanding of the impaired ex- Decreased cell number in mel-18 FT is due to increased cell pansion, we examined the T cell reconstitution frequency of DN1 death in DN1/DN2 cells cells at the single-cell level using the progenitor assay. DN1 cells It was previously reported that the defect in T cell development in (Ly5.2) from 14.5 dpc mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ FT were cultured mel-18Ϫ/Ϫ mice was due to the impaired proliferation of thymocytes 2512 mel-18 REGULATES EXPANSION OF EARLY T PROGENITORS Downloaded from http://www.jimmunol.org/

FIGURE 3. Impaired expansion of fetal DN1 cells in mel-18Ϫ/Ϫ mice. A, A total of 14.5 dpc FT lobes from either mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ fetus was cultured for 3 and 6 days in organ. Indicated are viable cell numbers per lobe. B, Single CD44ϩCD25Ϫc-Kitϩ (DN1) FT cells from 14.5 dpc mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ fetus (Ly5.2) were picked up under microscopic visualization and were seeded into wells containing a dGuo-treated FT lobe (Ly5.1/5.2 F1) in the presence of IL-7 and SCF. After 14 days of culture under HOS conditions, cells in each well were analyzed by ﬂow cytometry.

Ly5.1-positive cells were gated out (data not shown). The numbers of T by guest on September 29, 2021 progenitors per 14.5 dpc FT CD44ϩCD25Ϫc-Kitϩ cells are scored among 30 cells (reconstitution frequency). The numbers of cell recoveries in reconstituted clones are plotted (single DN1 cell proliferation). Bars denote the mean value (mean, 12,073 and 6,730, respectively). C, Absolute cell numbers were calculated for total thymocytes from Rag1Ϫ/Ϫ mel-18ϩ/ϩ, Rag1Ϫ/Ϫ mel-18ϩ/Ϫ,orRag1Ϫ/Ϫ mel-18Ϫ/Ϫ fetus at 17.5 dpc. Shown is a Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ Ϫ 17.5 dpc fetal thymus from Rag1 / mel-18 / and Rag1 / mel-18 / FIGURE 4. Dysfunction of DN1 cells in mel-18Ϫ/Ϫ fetus is possibly fetus. Scale bar, 1 mm. caused by increased susceptibility to cell death. A, Cell cycle analysis of sorted DN1(CD44ϩCD25Ϫc-Kitϩ) and DN2(CD44ϩCD25ϩc-Kitϩ) cells from 14.5 dpc mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ fetus. Three independent experi- in response to IL-7, although IL-7 and IL-7R expression and the JAK/ ments were performed with similar results. B, Serial frozen sections of 12.5 ϩ/Ϫ Ϫ/Ϫ STAT signaling pathway were found to be normal (14). These stud- dpc mel-18 or mel-18 fetus were stained with anti-keratin (red) and subsequently processed for TUNEL assay (green). The lines indicate the ies, however, were primarily focused on the total thymocyte popula- border between the epithelial region and the mesenchymal layer of the tion and not specifically on early T progenitors. We chose to ϩ/Ϫ thymus anlagen. The numbers of TUNEL-positive cells within the epithe- investigate the cell cycle parameters of T progenitors in mel-18 lial region were counted on every two serial sections (5-␮m thick) through- Ϫ/Ϫ and mel-18 FT in two different ways. First, DN1/DN2 cells from out the anlagen. The total numbers of TUNEL-positive cells from the sec- 13.5 dpc FT were stained with PI, and cell cycle profiles were ana- tions of a single anlage of mel-18Ϫ/Ϫ mice were almost twice those of ϩ/Ϫ lyzed by flow cytometry. The proportion of cells in S-G2-M phase in control littermates (mel-18 ; total, 155 and 84, respectively). The rep- mel-18Ϫ/Ϫ DN1 cells was comparable to that in mel-18ϩ/Ϫ DN1 cells resentative results of two experiments (two mel-18ϩ/Ϫ and three mel-18Ϫ/Ϫ (34.9 Ϯ 3.4 and 35.3 Ϯ 0.4%, respectively). Similar results were fetuses) are shown. Scale bar, 50 ␮m. C, In vitro survival assay. Sorted ϩ/Ϫ Ϫ/Ϫ observed for DN2 cells (Fig. 4A). Second, to assess the number of DN1 and DN2 cells FT cells from a 14.5 dpc mel-18 or mel-18 fetus cells containing newly synthesized DNA, 14.5 dpc fetuses were pulse- were cultured with IL-7 or SCF. After 0 or 20 h, the numbers of viable cells were counted by flow cytometry. Three independent experiments were per- labeled with BrdU in vivo. DN1/DN2 cells were sorted and double- formed with similar results. stained with an anti-BrdU Ab and Hoechst 33342. Consistent with the cell cycle profiles, we could not find any difference in the proportion of BrdU-positive cells between mel-18ϩ/Ϫ and mel-18Ϫ/Ϫ DN1/DN2 apoptotic cells in 12.5 dpc FT. As shown in Fig. 4B, we observed cells (both ϳ40%; data not shown). Therefore, we considered that the a 2-fold increase in the number of TUNEL-positive cells in mel- reduction in fetal thymocytes did not correlate with a change in mi- 18Ϫ/Ϫ FT compared with that in mel-18ϩ/Ϫ FT (see Fig. 4B). This totic activity in mel-18Ϫ/Ϫ mice. increase in the number of TUNEL-positive cells could explain the Consequently, to explain the impaired expansion of early T pro- data shown in Fig. 3B; that is, early T progenitors in mel-18Ϫ/Ϫ FT genitors in mel-18Ϫ/Ϫ FT, we performed TUNEL staining to detect were prone to cell death during the process of pre-␤ proliferation, The Journal of Immunology 2513 and this resulted in a low frequency of T progenitors within the has been reported that OP-9 cells ectopically expressing DL1 ac- DN1 cell population and impaired expansion from a single DN1 quired a capacity for T cell differentiation in monolayer culture cell. We suggest that the reduction of fetal thymocytes is due to an (31). Therefore, we used a similar system, with a thymus-derived increased susceptibility to cell death or an insufficient presence of fibroblast cell line, TSt-4, transfected with the DL1 gene. IL-7Rϩ survival signals in early T progenitors in mel-18Ϫ/Ϫ mice. cells (LinϪc-KitϩIl-7Rϩ) in 14.5 dpc FL were cultured on TSt-4 Because IL-7R signaling was known to be required for cell sur- stroma cells expressing DL1 (TSt-4/DL1). We found that TSt-4/ vival during immature T cell development, especially from the DL1 cells suppressed B cell generation while inducing T cell dif- DN1 to the DN3 stage (43, 44), we next examined the possibility ferentiation (data not shown). When IL-7Rϩ cells from 12.5 dpc that the increased cell death in mel-18Ϫ/Ϫ FT was due to an im- mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ FL were sorted and cocultured with TSt- pairment of IL-7R signaling. In an in vitro survival assay, DN1/2 4/DL1 cells for 5 days, the number of cells generated from IL-7Rϩ cells in mel-18Ϫ/Ϫ FT displayed a normal response to SCF and cells in mel-18Ϫ/Ϫ FL was less than one-third that in mel-18ϩ/Ϫ IL-7, suggesting that the increased cell death of T progenitors in cells (data not shown). Using this system, we examined the precise mel-18Ϫ/Ϫ FT is not correlated with IL-7 or SCF signaling time course of Hes-1 expression. After 3 and 6 days of culture, T (Fig. 4C). precursors (Thy1.2ϩ cells) were sorted, and the expression of the Hes-1 gene was analyzed by semiquantitative RT-PCR Southern Mel-18 deficiency affects the expression of Hes-1, a Notch-1- blot analysis. Indeed, on day 6, the expression of Hes-1 in mel- signaling target gene 18Ϫ/Ϫ Thy1.2 cells was decreased by 20% compared with the ex- ϩ Ϫ To identify underlying mechanisms involved in this dysfunction of pression in mel-18 / cells, whereas Hes-1 expression in mel- Ϫ Ϫ Ϫ Ϫ early T progenitors in mel-18 / ] FT, we analyzed the expression 18 / cells on day 3 was comparable to that in the control (Fig. Downloaded from of several genes that are involved in early thymocyte development. 6A). From these results, we advanced the hypothesis that mel-18 As shown in Fig. 5, mel-18 deficiency had no effect on the ex- was engaged in maintaining the active transcriptional state of the pression of many transcription factors, including GATA-3, TCF-1, Hes-1 gene once induced by DL1. To investigate this hypothesis, LEF-1, and E2A. we first surveyed the expression of Hes-1 and mel-18 in several Surprisingly, the expression of the Hes-1 gene, one of the target DN thymocyte cell lines. KKC cells, a DN1 thymocyte cell line genes of the Notch-1 signal, was remarkably decreased in mel- (45), displayed Hes-1 expression in the absence of the Notch-1 http://www.jimmunol.org/ 18Ϫ/Ϫ DN1 and DN2 cells compared with mel-18ϩ/Ϫ cells. In contrast, there were no significant differences in the expression of Notch-1, RBP-J,orDeltex-1. These results suggested that mel-18 might be involved in Notch-1 signaling, especially in the regulation of Hes-1 expression, in early T progenitors. Moreover, we investigated the expression of Hes-1 in DN1/DN2 cells in adult mel-18Ϫ/Ϫ mice, and indeed, we observed down-regulation of the Hes-1 gene similar to that observed in FT (data not shown). by guest on September 29, 2021 Mel-18 plays a role in the maintenance of Hes-1 gene expression To confirm that mel-18 is involved in Hes-1 expression induced by Notch-1 signal, we set up an in vitro coculture system for studying T cell development with DL1-expressing stroma cell. Recently, it

FIGURE 6. Mel-18 is associated with maintenance of Hes-1 gene expression. A, Sorted IL-7Rϩ cells in 12.5 dpc mel-18ϩ/Ϫ or mel-18Ϫ/Ϫ FL were cocultured with TSt-4/DL1 for 3 and 6 days. Expression of the Hes-1 gene was assessed by semiquantitative RT-PCR Southern blot analysis using cDNAs isolated from sorted T precursor cells (Ly5ϩThy1.2ϩ). Ar- bitrary densitometric ratios of Hes-1/G3PDH are shown. The expression in mel-18ϩ/Ϫ cells was defined as 1.0 in each panel. B, KKC cells express Hes-1, Notch-1, and mel-18 mRNA in the absence of Notch-1 ligands (data not shown). KKC cells were transfected with dicer-siRNA for either mel-18 or GFP. mRNA levels of mel-18, Hes-1, and G3PDH were deter- FIGURE 5. The expression of Hes-1, a target gene of Notch-1 signal, is mined by semiquantitative RT-PCR and Southern blot analysis with 4-fold decreased in mel-18Ϫ/Ϫ DN1 and DN2 cells. Semiquantitative RT-PCR serial dilution of template cDNA. The data with asterisks correspond to was performed with primers specific for each gene on cDNAs obtained each other. The relative ratio of mel-18/G3PDH was calculated with cells from sorted DN1 and DN2 cells in 14.5 dpc mel-18Ϫ/Ϫ or littermate control transfected with mel-18 siRNA or GFP siRNA. The expression level of FT. After RT-PCR, the products were subjected to Southern blot analysis. mel-18 was decreased to 0.02 by mel-18 siRNA compared with that in As an internal control, the expression of G3PDH was investigated. The siRNA for GFP. Arbitrary densitometric ratios of Hes-1/G3PDH are in- results were representative of two independent litters. dicated. The ratio in siRNA for GFP was defined as 1.0. 2514 mel-18 REGULATES EXPANSION OF EARLY T PROGENITORS

FIGURE 7. Schematic illustration of mel-18 function in the pre-␤ rearrangement proliferation of early T progenitors through the maintenance of Hes-1 gene expression.

ligand, and the mel-18 gene was also highly expressed in this cell starts from DN1 cells, and this proliferation is also essential for the Downloaded from line (data not shown). Because the Hes-1 gene seemed to be main- formation of diversified TCR ␤-chains (20, 41). The Rag1-mel-18 tained in an active state in KKC cells, we investigated, using this double-knockout (Fig. 3C) provides clear evidence that the lack of cell line, whether the reduction of mel-18 expression affected the mel-18 impairs the pre-␤ proliferation of early T progenitors in FT, expression level of the Hes-1 gene. KKC cells were transfected indicating that the PcG/mel-18 complex plays a role in developing with a dicer-siRNA for either mel-18 or GFP. The expression of the diversified immune system by supporting the pre-␤ prolifera- mel-18 was reduced to ϳ2% by the siRNA for mel-18 compared tion of early T progenitors. with the expression level observed upon application of the siRNA http://www.jimmunol.org/ for GFP (Fig. 6B). Under these conditions, the expression level of Ϫ Ϫ the Hes-1 gene was decreased to 5% by the siRNA for mel-18. Down-regulation of Hes-1 in mel-18 / T progenitors Based on these results, we propose that mel-18 is indispensable for We observed that expression of the Hes-1 gene was dramatically maintaining the active state of the Hes-1 gene once it is established decreased in mel-18Ϫ/Ϫ DN1 and DN2 cells, although other by the Notch-1 signal. Notch-1 signal-related genes were expressed normally (Fig. 5). In Fig. 7, we provide a model explaining how mel-18 regulates Hes-1 is a basic helix-loop-helix transcriptional factor and func- ⌻ ␤ the expansion of early T progenitors before CR rearrangement tions as a negative or positive regulator of cell growth and differ- by maintaining Hes-1 gene expression. entiation in various systems (49). In T cell development, prethymic by guest on September 29, 2021 T cell progenitors in FL express a relatively low level of Hes-1, Discussion even though they express Notch-1 receptor. After migration into Mel-18-dominant PcG complex plays a role in the pre-␤ the thymus, T progenitors interact with thymic stromal cells and expansion of T progenitors receive a signal through the Notch-1 receptor, and the Hes-1 gene It has been previously shown that expression levels of individual is up-regulated by this signal (50) (Fig. 6B). We note that a mel-18 PcG genes varied during hemopoiesis (10, 46). Mutant mice lack- deficiency affects early T progenitors after migration into the thy- ing each constituent of a PRC1-like complex tended to display a mus, not at the prethymic stage (Figs. 1 and 2, B and C), demon- characteristic defect at the stage of hemopoiesis in which the par- strating that the consequences of mel-18 deficiency correlate with ticular gene under investigation was highly expressed. For in- the expression pattern of the Hes-1 gene. Interestingly, the pheno- Ϫ Ϫ stance, the expression of mel-18 is high in immature DN thymo- type of fetal thymocytes from Hes-1 / mice is strikingly similar Ϫ Ϫ cytes, especially in DN1 cells, and the lack of mel-18 affects DN1 to that of mel-18 / mice (Figs. 2 and 3). Prethymic T progenitors Ϫ Ϫ cells in the adult thymus (Fig. 1). In addition, Bmi-1 and Rae-28 in FL and FB were not affected in Hes-1 / mice (K. Masuda and are essential for the self-renewal activity of adult or fetal HSCs, H. Kawamoto, unpublished observations). Transfer of Hes-1-null respectively, in which the expression of each gene is particularly FL cells into Rag2-null host mice failed to generate mature T cells high (46–48). These observations suggest that the subunit stoichi- in the thymus, and the expansion of DN cells before TCR gene ometry of the PRC1-like complex changes during hemopoiesis, rearrangement was severely affected (32). In the FTOC system, Ϫ Ϫ and that a stage-specific PRC1-like complex can determine a dis- Hes-1 / fetal thymocytes were found at a reduced number de- tinct spectrum of target genes. Consequently, this complex plays a spite the almost normal development to the DP and SP stages (33). specific role at each developmental stage. We believe that the mel- The level of Hes-1 expression is profoundly involved in the cel- 18-dominant PRC-1-like complex (PcG/mel-18) may be responsi- lularity of developing thymocytes, especially for the expansion of ble for the expansion of early T progenitors. early T progenitors. In conclusion, we suggest that down-regula- In T cell development, the expansion of T progenitors that have tion of the Hes-1 gene is a major cause of the impaired expansion migrated into the thymus is required for clonal diversification of of early T progenitors in mel-18Ϫ/Ϫ FT. TCR, and this diversification profoundly contributes to the recog- Because we found a normal proportion of cycling cells and an nition of a wide variety of Ags. It has been proposed that there are increased number of TUNEL-positive cells in 12.5 dpc mel-18Ϫ/Ϫ two independent expansion phases of T progenitors. The so-called FT (Fig. 4, A and B), we believe that the reduced cell number in post-␤ proliferation takes place immediately after TCR␤ rear- mel-18Ϫ/Ϫ FT resulted from the increased cell death of early T rangement, which makes it possible to generate diverse TCR progenitors. We also found that bcl-2 could partially rescue (50– ␣-chains from a single TCR ␤-chain (21, 22). In contrast, the pre-␤ 80%) thymocyte cell number in the absence of mel-18 from the proliferation, which is predicted to be on the order of 1000-fold, analysis of bcl-2 Tg/mel-18Ϫ/Ϫ mice (data not shown). The Journal of Immunology 2515

Notch signaling is known to be associated with cell death as in control cells. For the later phase, we also observed that the well as differentiation and proliferation, although the molecular expression of Hes-1 is down-regulated to a remarkably low level mechanisms through which Notch activation affects cell death re- when the amount of mel-18 mRNA was reduced by siRNA in the main to be elucidated (51). In Hes-1Ϫ/Ϫ mice, the expansion of the KKC cell line, which expresses the Hes-1 gene in the absence of earliest thymocytes was severely impaired, but it was not deter- Notch ligands (Fig. 6B). These results suggest that mel-18 plays a mined whether Hes-1 deﬁciency affected cell proliferation or in- key role in maintenance of the active state of the Hes-1 gene in creased susceptibility to cell death (32). During normal thymocyte proliferating T progenitors. development, immature thymocytes are themselves vulnerable to To maintain gene expression levels in proliferating cell popu- cell death. It is, therefore, likely that the down-regulation of Hes-1 lations, daughter cells must inherit a cellular memory of the state expression could lead to cell death, even though the cells have the of gene expression from the mother cell after cell division. As potential to proliferate. However, it is not yet known whether the shown in Fig. 7, prethymic T progenitors migrate into the thymus, down-regulation of the Hes-1 gene directly causes thymocyte cell then they interact with stroma cells to receive several signals, in- death in mel-18Ϫ/Ϫ FT or if other mechanisms are involved. cluding a Notch-1 signal. Prompted by these signals, T progenitors We also investigated whether mechanisms other than the Notch start to proliferate massively and differentiate from DN1 to DN2 signaling pathway were involved in the dysfunction of early T cells to create a diversity of TCR ␤-chains (pre-␤ proliferation). In progenitors in mel-18Ϫ/Ϫ FT. In a previous report the reduced mel-18Ϫ/Ϫ FT, early T progenitors could not proliferate appropri- number of mel-18Ϫ/Ϫ thymocytes was attributed to their lack of ately and were susceptible to cell death, probably due to their responsiveness to IL-7, based on the observation that total thymo- inability to maintain the Hes-1 expression level after each cell Ϫ Ϫ cyte of adult mel-18 / mice displayed a low proliferative re- division. Downloaded from sponse to PMA plus IL-7 (14). In addition, it is well known that In Drosophila melanogaster, the expression of HOM-C genes is IL-7 and SCF play a key role in the proliferation and survival of initially induced by gap and pair-rule genes, and these genes are immature thymocytes (43, 44, 52). Therefore, we thought it pos- transiently expressed during early embryogenesis. Subsequently, sible that an impaired response to IL-7 resulted in the cell death in the Trithorax group (TrxG) and PcG genes are necessary to main- mel-18Ϫ/Ϫ fetal T progenitors. However, when we focused on fetal tain proper expression of HOM-C throughout development. TrxG

DN1 and DN2 cells, these cells proliferated normally in response proteins are generally responsible for preserving the active state, http://www.jimmunol.org/ to IL-7 or SCF (Fig. 4C), indicating that the dysfunction of T whereas PcG proteins maintain a silenced state. However, recent progenitors in mel-18Ϫ/Ϫ FT is not associated with IL-7 signal. observations revealed that some members of the PcG and TrxG Several lineage-specific genes (GATA-3, Tcf-1, Lef-1, and E2A), groups exhibit the dual property of maintaining both the activated which have been shown to be essential for early thymocyte devel- and inactivated states of homeotic gene expression. They are iden- opment (53), were expressed normally in mel-18Ϫ/Ϫ DN1/DN2 tified as enhancers of Trithorax and Polycomb (ETP) (11), al- cells (Fig. 5). It was reported that the induction of GATA-3 was though it remains to be determined whether ETP maintains both affected under Th2-skewed conditions in mel-18Ϫ/Ϫ naive CD4 T states of gene expression directly or indirectly. Interestingly, cells (18); however, we observed normal expression of GATA-3 in mel-18 has been classified as a member of the mammalian ETP. In mutant DN1/DN2 cells. Therefore, GATA-3 was not a target of this paper we demonstrate that mel-18, as an ETP, could, in prin- by guest on September 29, 2021 Mel-18 in the T progenitor population. Furthermore, we suspected ciple, play a positive role in Hes-1 transcriptional maintenance in that ink4a, another known downstream target gene of the PcG proliferating early T progenitors (ETPs), i.e., ETP controls expan- complex, was involved in the dysfunction of mel-18Ϫ/Ϫ early T sion of ETPs. However, it is at this time unclear whether mel-18 progenitors. However, we were not able to detect the expression of acts directly on Hes-1 gene expression. the ink4a gene in either mel-18Ϫ/Ϫ or control DN1/DN2 cells, as In summary, we demonstrated that the PcG gene mel-18 is in- examined by RT-PCR Southern hybridization procedure (data not dispensable for the expansion of adult and fetal early T progeni- shown). Therefore, it is not likely that the defects in T progenitors tors. The PcG/mel-18 protein complex appears to regulate the ex- in mel-18Ϫ/Ϫ mice are correlated with the expression of the pression of Hes-1, a target gene of the Notch-1 signaling pathway, ink4a gene. in these progenitors. We propose that PcG/mel-18 plays a crucial role in pre-␤ proliferation, which is required for the formation of PcG/mel-18 contributes to the maintenance of Hes-1 gene a diversified TCR ␤-chain, through the maintenance of Hes-1 gene expression in proliferating T progenitors expression. Our study indicates that PcG/mel-18, in principle, In the TSt-4/DL1 coculture system, we noted that after 6 days, the works as a cellular memory system in pre-␤ proliferation, thereby expression level of Hes-1 in mel-18Ϫ/Ϫ T precursors was partic- supporting the development of the diversified immune system. ularly decreased compared with that in mel-18ϩ/Ϫ cells, whereas Ϫ/Ϫ after day 3 the expression of Hes-1 was not affected in mel-18 Acknowledgments T precursors (Fig. 6A). In parallel, we noticed that the difference in ϩ/Ϫ Ϫ/Ϫ We thank Kenji Tanigaki and Tasuku Honjo for providing the vectors used Hes-1 expression between mel-18 and mel-18 cells wid- in the luciferase assays, Kiyokazu Kakugawa for providing the TSt-4/DL1 ened as differentiation proceeded from DN1 to DN2 (Fig. 5). From cell line, Akihiko Muto and Tomoo Ueno for technical assistance, and these results, we hypothesize a biphasic regulation of Hes-1 ex- Yoshihiro Takihara, Yosuke Takahama, and Yoshimoto Katsura for help- pression. During the early phase, the Hes-1 gene is transactivated ful discussions and advice. by Notch-1 signaling. In the later phase, the Hes-1 expression level is maintained during the expansion of early T progenitors. Regard- Disclosures ing the early phase, we examined the possibility of involvement of The authors have no financial conflict of interest. mel-18 in transactivation of the Hes-1 gene by Notch-1 signals. However, we did not find that mel-18 influenced the Hes-1 pro- moter activities mediated by Notch-1-IC in a luciferase assay (data References not shown). This finding corresponds well with the results from the 1. van Lohuizen, M. 1999. The trithorax-group and Polycomb-group chromatin modifiers: implications for disease. Curr. Opin. Genet. Dev. 9:355. TSt-4/DL1 assay, in which the Hes-1 expression level in mel- Cell Ϫ Ϫ 2. Orlando, V. 2003. Polycomb, epigenomes, and control of cell identity. 18 / T precursors after 3 days of culture was comparable to that 112:599. 2516 mel-18 REGULATES EXPANSION OF EARLY T PROGENITORS

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