Knockout Mouse Models of Differentiation by Transcriptional Analysis a General Survey of Thymocyte

A General Survey of Thymocyte Differentiation by Transcriptional Analysis of Knockout Mouse Models

This information is current as Denis Puthier, Florence Joly, Magali Irla, Murielle Saade, of October 2, 2021. Geneviève Victorero, Béatrice Loriod and Catherine Nguyen J Immunol 2004; 173:6109-6118; ; doi: 10.4049/jimmunol.173.10.6109 http://www.jimmunol.org/content/173/10/6109 Downloaded from

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A General Survey of Thymocyte Differentiation by Transcriptional Analysis of Knockout Mouse Models1

Denis Puthier,2 Florence Joly,2 Magali Irla, Murielle Saade, Genevie`ve Victorero, Be´atrice Loriod, and Catherine Nguyen3

The thymus is the primary site of T cell lymphopoiesis. To undergo proper differentiation, developing T cells follow a well-ordered genetic program that strictly depends on the heterogeneous and highly specialized thymic microenvironment. In this study, we used microarray technology to extensively describe transcriptional events regulating ␣␤ T cell fate. To get an integrated view of these processes, both whole thymi from genetically engineered mice together with purified thymocytes were analyzed. Using mice exhibiting various transcriptional perturbations and developmental blockades, we performed a transcriptional microdissection of the organ. Multiple signatures covering both cortical and medullary stroma as well as various thymocyte maturation intermediates were clearly defined. Beyond the definition of histological and functional signatures (proliferation, rearrangement), we provide the Downloaded from first evidence that such an approach may also highlight the complex cross-talk events that occur between maturing T cells and stroma. Our data constitute a useful integrated resource describing the main gene networks set up during thymocyte development and a first step toward a more systematic transcriptional analysis of genetically modified mice. The Journal of Immunology, 2004, 173: 6109–6118.

ith the systematic sequencing of cDNA libraries and croenvironment that is crucial to control T cell fate. This stroma is the advent of microarray analysis, it has become pos- essentially composed of epithelial cells, dendritic cells, macro- http://www.jimmunol.org/ W sible to describe, at the tissue level, the expression phages, and fibroblasts. It provides developing T cells with essen- pattern of thousands of genes already known or yet to be charac- tial extracellular matrix components, cell surface ligands, and sol- terized. As an example, multitissue experiments using microarrays uble factors. Lymphoid progenitors derived from the bone marrow provided a broad source of information and constituted a first step enter the thymus at the corticomedullary junction, and then migrate in the description of gene expression profiles (1, 2). However, at to the subcapsular region where they undergo multiple cycles of the level of each organ, higher resolution is necessary to specify proliferation and progress to the CD4ϪCD8Ϫ double-negative the histological expression pattern of individual genes. This task is stage (DN)4 through discrete maturation steps: CD44highCD25Ϫ ϩ ϩ currently in progress, but production of informative samples often (DN1), CD44highCD25 (DN2), CD44lowCD25 (DN3), and by guest on October 2, 2021 requires microdissection or purification procedures (3). The exten- CD44lowCD25Ϫ (DN4). During DN to CD4ϩCD8ϩ double-posi- sive development of genetically modified mice in the past few tive (DP) transition, thymocytes relocate to the cortical region. years has led to the generation of numerous models of transcrip- Most DP cells (ϳ97%) die by neglect, because they do not rec- tional perturbations, most of them exhibiting very precise devel- ognize any of the available MHC molecules expressed by thymic opmental blockades and thus quantitative cellular disequilibrium stromal cells. Ultimately, DP thymocytes are subjected to negative (4). Hence, the use of perturbation models for transcriptional anal- selection events occurring both in the cortex and in the medulla, yses may provide us with samples highly enriched in as yet poorly leading to the deletion of autoreactive clones and giving rise to the described maturation intermediates. generation of MHC-restricted CD4 and CD8 single-positive T The thymus is the major site for T lymphocyte maturation. An- ϩ ϩ atomically, it is divided into a subcapsular region, a cortex, where cells (CD4 SP and CD8 SP) (5, 6). Although the molecular path- most of the thymocyte differentiation takes place and a medulla, ways involved in thymocyte ontogeny have been the subject of where newly generated T cells undergo final processes of matura- extensive studies, some of them based on microarray technology tion. Each of these compartments forms a specialized stromal mi- (7–9), our understanding of these mechanisms is still fragmented, and results often remain difficult to situate in an integrated and dynamic view of T cell maturation and T cell-stroma cross talk. Technologies Avanceés pour le Geńome et la Clinique/ERM 206, Parc Scientifique de By taking advantage of the relative enrichment in specific thy- Luminy, Marseille, France mocyte and stromal populations displayed by some genetically en- Received for publication April 26, 2004. Accepted for publication August 17, 2004. gineered mice, microarray analysis should reveal their character- The costs of publication of this article were defrayed in part by the payment of page istic transcriptional signatures. Identification of such signatures charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. may highlight the functional specialization of each cell population 1 D.P. is supported by grants from the Agence Nationale de Recherche sur le Sida. and should allow us to propose a putative role for still poorly This work was supported by Institut National de la Santeét de la Recherche Medicale and Ligue Nationale Contre le Cancer. Microarrays were developed and produced in the microarray platform of Marseille genopole. 2 D.P. and F.J. contributed equally to this work. ϩ ϩ 4 Abbreviations used in this paper: DN, double negative; DP, CD4 CD8 double- 3 Address correspondence and reprint requests to Dr. Catherine Nguyen, Technolo- positive thymocyte; CD4ϩSP, CD4 single-positive thymocyte; CD8ϩSP, CD8 single- gies Avanceés pour le Geńome et la Clinique/ERM 206, Parc Scientifique de Luminy, positive thymocyte; KO, knockout; WT, wild type; DAPI, 4Ј,6Ј-diamidino-2-phe- case 928, 13288 Marseille cedex 09, France. E-mail address: [email protected] nylindole; Ac.CD4ϩP, activated CD4ϩ peripheral T cell; Ac.CD8ϩP, activated CD8ϩ mrs.fr peripheral T cell.

characterized genes. In addition, because T cell and stromal de- cDNA clones, providing internal controls to assess the reproducibility of velopment are interdependent events, the combined analysis of ge- gene expression measurements. netically engineered mouse models affecting one or both compart- Microarray preparation ments should underline such cross-talk events. In the present study, using a 8700 cDNA mouse microarray, we draw up the first PCR amplifications were performed in 96-well microtiter plates using the following primers: 5Ј-CCAGTCACGACGTTGTAAAACGAC-3Ј transcriptional picture of the thymus as a whole by analyzing both and 5Ј-GTGTGGAATTGTGAGCGGATAACAA-3Ј, which were spe- whole thymi from well-defined knockout (KO) mice together with cific of the polylinker sequence of both vectors used. The reactions were purified thymocytes. performed as described (16) by transferring few Escherichia coli from a growth culture with a plastic 96-pin gadget (Genetix, New Milton, U.K.) to 100 ␮l of PCR mix, containing 10 mM Tris-HCl (pH 9), 1.5 ␮ Materials and Methods mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 1.5 M betaine, 250 M Mice dATP, dTTP, dGTP, and dCTP, and5UofTaq polymerase (Promega, Madison, WI). The plates were incubated for 6 min at 94°C, before 38 Mice were housed in a specific pathogen-free animal facility. C57BL/6 cycles of 94°C for 30 s, 65°C for 45 s and 72°C for 210 s, followed by ␣ (wild type (WT)), RAG1°, and TCR ° mice were obtained from the Centre a final elongation phase at 72°C for 10 min. Amplification products ⑀⌬5 ⑀ de Distribution, Typage et Archivage (Orleáns, France). CD3- (CD3 °), were not quantitated, but their quality was systematically checked on LAT°, and RelB° mice were kindly provided by Drs. M. Malissen and P. 1% agarose gels. Eleven percent of the bacterial clones were estimated Naquet (Centre d’Immunologie de Marseille Luming, Marseille, France). to be nonamplified, and 4% showed multiple bands after PCR amplifi- Thymi were isolated from adult mice that were sacrificed between 4 and 6 cation. Unpurified PCR products were then transferred to 384-well mi- wk of age. croplates, before being evaporated, taken up in 40 ␮l of distilled water, and spotted onto nylon membranes (Hybond-Nϩ; Amersham Bio-

Organs and cells sciences, Saclay, France). This step was conducted using a Micro- Downloaded from Grid-II arrayer (Apogent Discoveries, Cambridge, U.K.) equipped with Cells were cultured in RPMI 1640 supplemented with 10% heat-inacti- Ј ␮ a 64-pin biorobotics printhead. A vector probe hybridization (5 -TCA vated FCS, 2 mmol/L glutamine, 100 g/ml streptomycin, 100 U/ml pen- CACAGGAAACAGCTATGAC-3Ј) was performed as previously de- ϫ Ϫ5 Ϫ icillin, and 2 10 M 2-ME. The LMTK ﬁbroblastic cell line and the scribed and showed uniform signal intensities across individual mem- P388D1 macrophage cell line were both obtained from the American Type branes (17). Culture Collection (Manassas, VA). MTE-1D is a thymic epithelial cell line (10). DC2.4 are immortalized dendritic cloned cells derived from bone RNA extraction and cDNA labeling

marrow of C57BL/6 mice (11). 427.1, 1308.1, and 6.1.1 thymic stromal http://www.jimmunol.org/ cell lines were kindly provided by Dr. B. Knowles (The Jackson Labora- Total RNA was isolated using TRIzol reagent (Invitrogen Life Technolo- tory, Bar Harbor, ME) (12). Thymocytes were separated from stroma ac- gies, Carlsbad, CA), and RNA integrity was checked on denaturing agarose cording to standard procedures. CD25ϩ thymocytes were isolated from gels. Reverse transcription was performed as previously described, using 2 RAG1° mice. After labeling with an anti-CD25 Ab, they were purified ␮g of total RNA in the presence of [␣-33P]dCTP (18). A large excess of using magnetic beads (Dynal Biotech, Oslo, Norway) according to manu- oligo(dT) primers was added during cDNA synthesis, and the labeled probe facturer’s guidelines. These samples contain CD44highCD25ϩ (DN2) and was annealed with poly(dA)80 to ensure the complete saturation of poly(A) CD44lowCD25ϩ (DN3) and are referred to as CD25ϩDN. For DP purifi- tails. Hybridizations were conducted for 48 h at 68°C in 500 ␮l of hybrid- cation, thymocytes were sorted using a FACSVantage cell sorter (BD Bio- ization buffer (18). After washing, arrays were exposed to phosphor im- sciences, Mountain View, CA) on the basis of costained profiles with an aging plates, which were scanned using a BAS 5000 (Fuji, Tokyo, Japan) anti-CD4 (RM4-4; BD Pharmingen, San Diego, CA) and anti-CD8 (53-6.7; at a 25-␮m resolution. BD Pharmingen). For SP cell isolation, single-cell suspensions were first by guest on October 2, 2021 incubated with a mixture of Abs composed of an FcR-blocking Ab (2.4G2) Data processing and analysis and either an anti-CD8 (H-59-101-2) or an anti-CD4 (H129.196), for 30 After image acquisition, hybridization signals were quantified using the min at 4°C followed by incubation with sheep anti-rat IgG magnetic beads locally developed Bzscan software (64). All images were carefully in- (Dynal Biotech). The populations recovered were then stained with anti- spected, and spots with overestimated intensities due to neighborhood ef- CD4 (RM4-4) and anti-CD8 (53-6.7) (BD Pharmingen) and FACS sorted. fects were manually excluded. For each of the 66 arrays, background intensity was calculated on the basis of the average of the 400 lowest values Microarray design and subtracted. We defined distinct classes of samples, each of them con- For microarray preparation, the following cDNA libraries (some of which taining all of the replicates of the same sample type (e.g., three replicates were incomplete) were used: the NIA Mouse 15K cDNA clone set, 2NbMT of RAG1° thymi define one class). Of note, all of the cell lines, including (thymus), NbMLN (lymph node), and 3NbMS (spleen). Detailed descrip- dendritic cells, were grouped in the same class. For each sample, the 6000 tions of these cDNA libraries are available at the UniGene database web- highest values were flagged. A cDNA clone was kept for analysis if its site expression values were flagged in all of the samples of at least one class. (www.ncbi.nlm.nih.gov/UniGene/lbrowse2.cgi?TAXIDϭ10090, 2NbMT: Data were filtered (80%), log2 transformed, and centered relative to the Lib.544, 3NbMS: Lib.553, NbMLN: Lib.567, NIA 15K: Lib.8622). All of median for each gene and each array using the Cluster software (19). To the libraries were cloned into pT3T7D-Pac vector, except for the NIA 15K analyze genes with the most fluctuating expression levels across the sam- Mouse cDNA clone set, which was cloned into pSPORT1 vector. The NIA ples, only clones exhibiting a SD of at least 0.35 were kept. Hierarchical Mouse 15K cDNA clone set is a rearrayed and resequenced set of 15,000 clustering (average linkage clustering metric) was applied to the dataset, bacterial clones derived from 11 embryo cDNA libraries (13). It was ob- and results were visualized with the Treeview software (19). All data are tained through the Medical Research Council Geneservice (Cambridge, MIAME compliant and have been submitted to ArrayExpress database (www.ebi.ac.uk/miamexpress; accession no. E-MEXP-96). This article U.K.). 2NbMT, NbMLN, and 3NbMS are sequenced I.M.A.G.E. libraries 5 that were obtained from the Deutsches Ressourcenzentrum fu¨r Genomfor- contains supplemental data (Tables SI, SII, SIII, SIV, and SV). schung Resource Centre (Berlin, Germany). Bacterial clones were anno- tated according to UniGene using the SOURCE database (14) before bioin- Immunofluorescence formatics selection and robotic reorganization. A total of 4300 clones was Primary Abs used were as follows: FITC-anti-CD54/ICAM-1 (BD Phar- selected from the NIA 15K library and had been previously sequence- mingen, San Diego, CA), anti-RANTES (AF478; R&D Systems, Minne- confirmed. However, the reorganized clone set was not subjected to rese- apolis, MN), anti-K5 (AF138; Covance Research, Berkeley, CA), and anti- quencing before being used. The final clone set includes 8750 bacterial CD74 (IN-1). Secondary Abs used were as follows: Alexa-568-goat clones representing ϳ7770 nonredundant UniGene clusters. This clone set anti-rabbit IgG, Alexa-477-goat anti-rat IgG, and Alexa-477-goat anti- encompasses almost all described genes (“known genes”) present in these mouse IgG. Cryosections (8 ␮m) of 8-wk-old WT thymi were generated libraries and has also been supplemented with clones belonging to Uni- from OCT-embedded organs and mounted on glass slides. Sections were Gene clusters that contain at least one RIKEN cDNA clone entry (i.e., with fixed with phosphate buffer containing 4% paraformaldehyde for 1 h before a corresponding putative full-length cDNA) (15). Although the gene con- staining in a humidified chamber. Abs (in 1% BSA, 0.01% Triton, and 0.1 tent of this clone set is quite exhaustive, some key regulators of T cell maturation are still missing (e.g., RAG1, RAG2, ZAP-70). About 10% of the genes included in this clone set are represented by two or more different 5 The on-line version of this article contains supplemental material. The Journal of Immunology 6111

ϩ ϩ M Tris buffer (pH 7.4)) were applied to the sections and incubated for 1 h cells (Ac.CD8 P and Ac.CD4 P). Finally, cell lines and dendritic and 30 min at room temperature. Slides were then incubated in blocking cells were also used to delineate genes involved in cellular metab- buffer (3% BSA in 0.01% Triton, 0.1 M Tris buffer, and 5% goat serum) olism. All samples were hybridized to a mouse microarray con- for 10 min before being incubated with Alexa-conjugated-secondary Ab (1:1000 in 1% BSA, 0.01% Triton, and 0.1 M Tris buffer). Between each taining 8750 cDNA selected from embryo, thymus, lymph node, step, slides were washed for 5 min with Tris buffer. Tissues were coun- and spleen libraries. After normalization and data filtering, 4686 terstained with 1 ␮l/ml 4Ј,6Ј-diamidino-2-phenylindole (DAPI) and genes were kept for analysis. Hierarchical clustering was used to mounted with Mowiol fluorescent mounting medium (Calbiochem, Darm- group genes on the basis of their variation of expression over sam- stadt, Germany). Fluorescent images were acquired by Zeiss (Oberkochen, Germany) LSM 510 confocal microscopy. All images were processed after ples. The same method was applied to group the experimental the same exposure time and under the same magnification. samples according to their transcriptional similarity. Results are presented in Fig. 1A. As depicted in Fig. 1B, the hierarchical clus- Results tering procedure gave rise to a highly relevant classification of the Transcriptional microdissection of the murine thymus samples, divided in four major groups. Cell lines and activated To reveal the transcriptional signatures of the cellular compart- peripheral T cells were grouped together due to their metabolic ments that constitute the adult mouse thymus, we used a combi- activity. Indeed, their main transcriptional characteristic relies on nation of whole thymi and of purified cell samples. We assumed the high expression of genes encoding proteins involved in general that comparing the transcriptional status of both WT and KO mice metabolism (Fig. 1A, cluster G, and data not shown). The thymi would help us to reveal specific signatures of over- or underrep- from RAG1°, LAT°, and Cd3⑀° clustered together, whereas a third resented cellular compartments. The RAG1°, LAT°, and CD3-⑀⌬5 group contained all of the purified thymocytes and also naive T

(Cd3⑀°) KO mice were chosen, because they display an early block cells (not shown). The fourth class of samples was composed of Downloaded from in T cell development and consequently a relative enrichment in whole thymi from WT, TCR␣°, and RelB° mice. As shown in Fig. DN T cells and stromal cells (4). Thymi from TCR␣° KO mice that 1A, numerous transcriptional signatures clearly stood out (signa- lack mature T cells and differentiated medullary stromal cells were tures A–G). These signatures were extracted and samples were used to highlight the speciﬁc transcriptional signatures of these reordered according to their types (the signature G corresponding compartments. Mice invalidated for RelB, a member of the NF-␬B to general metabolism will not be discussed herein) (Fig. 1C). A gene expression signature was named by either the cell type in family, were also used, because they are characterized by a disor- http://www.jimmunol.org/ ganized medulla and a lack of medullary dendritic cells (20). In which its component genes were expressed (for example, “DN T addition, RNA from the following sorted thymocyte populations cells” signature) or the biological process in which its component were prepared: CD25ϩ T cells from RAG1° mice (corresponding genes are known to function (for example, the “proliferation” sig- to DN2 and DN3 stages, CD25ϩDN), thymocytes from WT ani- nature). Signatures of T cell maturation stages (DN, DP, and SP) mals (DN, DP, CD4ϩSP, CD8ϩSP), and activated peripheral T as well as functional signatures (proliferation, TCR rearrangement) by guest on October 2, 2021

FIGURE 1. Hierarchical clustering based on gene expression data obtained from 66 RNA samples. A, Each row represents a separate cDNA clone on the microarray, and each column represents a separate mRNA sample. Green, black, and red squares indicate transcript levels lower than, equal to, or greater than the median level, respectively. Gray squares correspond to technically inadequate or missing data. The six different signatures that will be developed thereafter are indicated (A–F). B, The dendrogram generated by the hierarchical clustering procedure (see Fig. 1A) organizes the samples according to their transcriptional similarity and reveals four major groups, presented in different colors. Of note, naive CD4 peripheral T cells cluster with the group of purified thymocytes. C, Supervised representation of the six distinct (A–F) transcriptional signatures. (The samples are not subjected to a classification according to their gene expression similarities but according to their origin.) Abbreviations used are as follows: CD25ϩDN, CD25ϩ cells purified from RAG1° mice; DN, DP, CD4ϩSP, CD8ϩSP, T cell populations sorted from WT animals; CD4ϩP, naive peripheral T cells sorted from WT animals; Ac.CD4ϩP, Ac.CD8ϩP, peripheral CD4 and CD8 T cells activated with coated anti-CD3 and soluble anti-CD28 (1 ␮g/ml). 6112 TRANSCRIPTIONAL MICRODISSECTION OF THE MOUSE THYMUS were defined (Fig. 1C, signatures A–E). For the SP T cell signature Finally, a clear histological signature (F), differentiating the thymic (E), three slightly different signatures from Fig. 1A (E1, E2, E3) stroma from developing thymocytes, was also defined. originally disseminated along the clustering figure were grouped together in Fig. 1C. This signature encompasses genes characteristic of both late DP and SP stages. Indeed, these genes are not, or Proliferation status of developing thymocytes only weakly, expressed in the TCR␣° samples. In addition, it also Close inspection of the genes composing the signature A (Fig. 1B) contains genes more specifically expressed at the SP stage, whose suggests that it corresponds to cell cycle. Bibliographic scanning expression is maintained in peripheral activated T cells. Of note, of cluster A indicates that it is composed of genes that control although we could detect some signatures specific for activated transitions between G1,G2, and S phases, or that monitor entry and peripheral T cells, they will not be considered in the present paper. exit of mitosis: Ccna2, Cdc2, Cdca3/Tome1, and Ube2c (Table I).

Table I. Detailed gene contents of cell cycle-related signature (cluster A)

Functional Associated- Classiﬁcationa Ug.Symbolb Ug.Mmc Ug.Hsd LL.ID (Hs)e MEGf Function

Cell cycle regulator Cdc451 Mm.1248 Hs.114311 8318 MEG1485 Initiation of DNA replication.

Cdc2a Mm.4761 Hs.334562 983 Involved in G1/S and G2/M phase transitions. Downloaded from Ccna2 Mm.4189 Hs.85167 890 Involved in G1/S and G2/M phase transitions. Cdca1 Mm.151315 Hs.234545 83540 Regulatory role in chromosome segregation.

Cdc20 Mm.29931 Hs.82906 991 MEG3165 APC activator during G1 and mitosis. cdca3/Grcc8 Mm.22228 Hs.30114 83461 Regulator of entry and exit of mitosis.

Chromatine structure Hist2 Mm.258496 Hs.348668 126961 Histone Nucleosome structure. H2afx Mm.245931 Hs.147097 3014 Histone DNA repair.

H2afz Mm.916 Hs.119192 3015 MEG2200 Histone Transcriptional control. http://www.jimmunol.org/ Nasp Mm.7516 Hs.446206 4678 H1 histone binding protein. Histone transport. Slbp Mm.4172 Hs.251574 7884 MEG3280 mRNA histone binding.

Mitotic checkpoint Birc5 Mm.8552 Hs.1578 332 MEG3232 Apoptosis inhibitor expressed during G2 phase. component Bub3 Mm.927 Hs.418533 9184 MEG4512 Mitotic checkpoint component. Ube2c Mm.89830 Hs.93002 11065 Destruction of mitotic cyclins. Cell cycle progression.

Replication Prim1 Mm.2903 Hs.82741 5557 MEG1249 Synthesis of oligoribonucleotide primers on DNA. by guest on October 2, 2021 Top2a Mm.4237 Hs.156346 7153 MEG518 Topoisomerase. Pcna Mm.7141 Hs.78996 5111 MEG2077 Cofactor of DNA polymerase ␦. Smc211/Fin16 Mm.2999 Hs.43910 10592 MEG2321 Mitotic chromosome condensation. DNA repair.

Transcriptional Srebf2 Mm.38016 Hs.108689 6721 Transcription regulator. regulation Zhx1 Mm.37216 Hs.12940 11244 Transcription factor. Hmgb2 Mm.1693 Hs.434953 3148 DNA bending factor.

RNA maturation Sfrs3 Mm.6787 Hs.405144 6428 MEG6285 Splicing factor. Rrm2 Mm.99 Hs.226390 6241 MEG1938 Ribonucleotide reductase M2. Hnrpab Mm.24 Hs.81361 3182 Pre-mRNA processing, mRNA metabolism, and transport. No15a Mm.29363 Hs.376064 10528 MEG418 Pre-rRNA processing. Dutp Mm.276215 Hs.367676 1854 MEG674 Deoxyuridine triphosphatase. DNTP metabolism. Ddx39 Mm.271162 Hs.311609 10212 MEG1184 Nuclear RNA helicase. Silg41 Mm.123859 Hs.30035 6434 MEG6925 Homolog to sfrs10. Splicing factor.

Miscellaneous Ptprv/Esp Mm.4450 375048 Embryonnic stem cell phosphatase. Hpgd Mm.18832 Hs.77348 3248 Hydroxyprostaglandin dehydrogenase 15- (NAD). Topk-pending Mm.24337 Hs.104741 55872 Serine/threonine kinase. Lrpap1 Mm.4560 Hs.75140 4043 MEG2907 Lipoprotein receptor-associated protein. Dnclc1 Mm.56455 Hs.5120 8655 Dynein L chain. Enzymatic activity.

Unknown 2310061F22Rik Mm.37324 Hs.79077 9780 MEG4299 2410017I18Rik Mm.34567 Hs.46680 51001 MEG5810 2810417H13Rik Mm.269025

a Functional classiﬁcation is based upon bibliographic searches. b Mouse UniGene symbols. c Mouse UniGene cluster IDs (release no. 129). d Corresponding human UniGene cluster IDs (release no. 163) based on the HomoloGene database (www.ncbi.nlm.nih.gov/HomoloGene). e Corresponding Human LocusLink IDs based on the HomoloGene database. f Corresponding associated metagenes (Associated-MEG) were retrieved from the Kimlab database (http://cmgm.stanford.edu/ϳkimlab/multiplespecies/Supplement/ stab1.xls). The Journal of Immunology 6113

Many genes (Cdc20, Bub3, and Cdca1) are involved in microtu- highest proliferation rates were observed in activated peripheral T bule-dependent processes that are hallmarks of dividing cells. An- cells and also in WT, RelB°, and TCR␣° thymi. This last obser- other feature of this cluster is the presence of numerous genes vation can be correlated with the presence of both highly dividing encoding components of chromatin structure. It includes three hi- immature single-positive thymocytes and DP cells in these thymi. stones genes, Hist2 and two histones variants, H2afx and H2afz,as Among the most immature thymocyte populations, total DN T well as Nasp and Slbp. Interestingly several genes present in clus- cells, highly enriched in DN4 stage, were found to have the highest ter A actively participate in DNA replication (Cdc45, Prim1, level of proliferation, compared with CD25ϩDN isolated from Top2a, Pcna, and Smc2l1/Fin16). RAG1° thymi. An intermediate level of proliferation was observed To visualize these genes in terms of function, we took advantage in DP T cells, whereas the lowest rate of cycling characterized the of the classification proposed by Stuart et al. (21). They defined SP population, except for CD8ϩSP. Although this could reflect metagenes as sets of orthologous genes conserved between Homo proliferative differences between CD4ϩSP and CD8ϩSP, this more sapiens, Drosophila melanogaster, Caenorhabditis elegans, and likely reveals the presence of a few contaminating and highly di- Saccharomyces cerevisiae. By compiling microarray experiments viding CD8ϩ immature single-positive thymocytes in the cell from these four species, they organized these metagenes into a preparation. gene-coexpression network revealing functional clusters con- Notch1, Smarca4/Brg1, and their targets are highly expressed in served across evolution. Because mouse microarray data have not ϩ been included in their study, we searched for each gene of cluster CD25 DN T cells A, the human orthologue, and then the corresponding metagene. Genes from cluster B were highly expressed in the thymi from Among the 36 genes of cluster A, 19 were found to have an as- mice characterized by an early blockade in T cell differentiation, in Downloaded from sociated metagene (Table I). In the conserved gene coexpression the CD25ϩDN T cells from RAG1° mice, and in DN T cells ob- network, most of them colocalized in an area defined by Stuart et tained from WT animals (Fig. 1B). Expression profiles of these al. (21) as corresponding to cell cycle function (Fig. 2). Dutp and genes in purified T cells are presented in Fig. 3 (only representative Rrm2, two genes known to be involved in RNA metabolism, were genes are depicted). The complete data set including all genes (n ϭ included in this metacluster, suggesting that they have functional 41) is available in Supplemental Table SI. Cluster B was highly links with the cell cycle. Likewise, MEG5810 corresponds to a enriched in genes previously shown to be strongly expressed dur- still-uncharacterized mouse gene (2410017I18Rik) that most likely ing the first thymocyte developmental stages. It includes cell sur- http://www.jimmunol.org/ participates in cell cycle control. Remaining metagenes were face markers such as CD25, CD104/Itgb4, and the Gfra1 (22). mainly found in two regions, highly enriched in genes devoted to general transcription and translation. Two metagenes (MEG4299/ 2310061F22Rik, MEG2907/Lrpap1) were found scattered in the coexpression network. Although we cannot formally exclude their participation in cell cycle-related processes, they may reflect mea- surement errors or clone contaminations. Altogether, this result underscores the capacity of our procedure to group-coexpressed by guest on October 2, 2021 genes in functional networks without any underlying assumption. As expected, the expression levels of all the genes in cluster A fluctuated drastically among the samples. These variations clearly reflected the proliferative status of the biological samples. The

FIGURE 2. Analysis of the cell cycle signature. Visualization of the genes of cluster A in the conserved coexpression network proposed by FIGURE 3. Gene expression programs induced during thymocyte de- Stuart et al. (21). The metagenes corresponding to genes from cluster A velopment. The clusters B, C, D, and E correspond to the major stages of (Table I) were plotted on the conserved coexpression network and visual- thymocyte development (B, double-negative stage; C, gene expression pro- ized using online tools at http://cmgm.stanford.edu/cgi-bin/cgiwrap/kim- gram induced during TCR rearrangement; D, double-positive stage; and E, lab/multiplespecies/visualize.pl. The mouse UniGene symbol correspond- single-positive stage). These clusters are directly derived from Fig. 1C, but ing to each metagene is included. Red circle corresponds to metagenes (i.e., only puriﬁed thymocyte and T cell samples are shown. To clarify, the samples sets of orthologous genes) that are devoted to cell cycle control throughout are ordered according to the developmental scale of thymocyte differentiation. evolution. Green and blue circles correspond to genes with conserved func- For each cluster, only representative genes are depicted. The complete data set tions in general transcription and translation, respectively. is available as Supplemental Tables SI, SII, SIII, and SIV. 6114 TRANSCRIPTIONAL MICRODISSECTION OF THE MOUSE THYMUS

Importantly, several transcriptional regulators, including Notch1, SIII). Among the transcriptional regulatory genes included in this Smarca4/Brg1, Dtx1/Deltex1, and Hes1/Hry were specifically ex- cluster, Ets2 is already known to be up-regulated at the DP stage pressed in DN T cells. Coregulated expressions of these genes of development (31). The nuclear orphan receptor Rorc/ROR␥ is pointed out some complex transcriptional regulatory networks. an important transcription factor that is tightly regulated during T Dtx1 and Hes1 are two well-established targets of Notch1. cell development: it displays a high expression during the DP stage Smarca4/Brg1 encoding a component of the SWI/SNF chromatin- and is turned off as thymocyte maturation progresses (32). As sug- remodeling complex showed a coordinated expression with Cd25. gested, ROR␥ may reduce cell division (as does cyclin G2/Ccng2) Interestingly, Smarca4/Brg1 is known to be recruited in the Cd25 in developing thymocytes, until they are selected. Strikingly, a core promoter at the CD25ϩDN stages (23). The notion of func- large number of genes involved in TCR-mediated signal transduc- tional links between genes of cluster B was reinforced by the si- tion were induced at the DP stage, as cells become able to receive multaneous expression of several genes involved in calcium-de- signals through their TCR. Three of them are adaptor molecules pendent signaling pathways, as illustrated with the following: that mediate intermolecular interactions. Adaptors are now known Calpain5/Capn5, FK506-bp5, the calcium/calmodulin-dependent to be as crucial for lymphocyte activation as are receptors and eukaryotic elongation factor-2 kinase/Eef2k, and Camk2a/ effectors. Sh2d1a/SAP plays a dual functional role both as an adap- CaMKII, which directly regulates the calcium channel, Cacna1h, tor for FynT and as an inhibitor of SH2 domain-mediated interac- also found in this cluster (24). These data underline the high en- tions (33). Lcp2/Slp76 is an adaptor protein whose deficiency leads richment in this signature of key regulators of CD25ϩDN stages of to a severe block at the DN3 stage (34). Interestingly Sla/SLAP, maturation. They suggest that additional unknown genes may be also present in this cluster, associates with Lcp2/Slp76 and has linked to the Notch1 or Smarca4/Brg1 pathway. These results also been shown to be highly expressed in DP thymocytes. It appears to point out the functional link that may exist between Notch1, play an important role in positive selection by down-regulating the Downloaded from Smarca4/Brg1, and the calcium-signaling pathway. TCR at the cell surface (35). Two genes related to apoptosis phenomena followed the same expression program characteristic of Cluster C corresponds to genes up-regulated during the TCR the DP stage: the product encoded by Arl6ip/Armer is known to rearrangement process protect cells from apoptosis, whereas ASC/TMS-1 encodes a pro- Genes from cluster C show a relatively high expression in all the apoptotic protein containing a pyrin domain (PD) and a caspase- thymi tested, in CD25ϩDN, and in DP T cells (Fig. 1B). In con- recruitment domain (CARD). Altogether, this cluster clearly high- http://www.jimmunol.org/ trast, they were strongly down-regulated in CD4 and CD8 T cells. lights the setting up of some key components of the TCR signaling Five of 55 genes were excluded from cluster C, because they were machinery at the DP T cell stage. not significantly different between immature thymocytes (CD25ϩDN and DP T cells) and mature thymic and peripheral T Cluster E defines the transcriptional status of single-positive cells (CD4ϩSP, CD8ϩSP, Ac.CD4ϩP, and Ac.CD8ϩP) ( p Ͻ 0.05, thymocytes Student’s t test). Close inspection of cluster C showed that it in- Genes from cluster E revealed the terminal maturation status of SP cludes several genes whose expression is known to diminish upon thymocytes subjected to selection events. This signature presents a T cell maturation (Fig. 3 and supplemental data, Table SII). Sev- bias in genes encoding products already known to be involved in by guest on October 2, 2021 eral cell surface markers were found in cluster C (CD24/Hsa, the positive selection of thymocytes (Supplemental Table SIV). CD3⑀, CD3␥, Ly6e, Ly6d, CD26, Cdw127/Il17r, and Sort1/Ntr3). This includes notably cell surface markers, components of the sig- Moreover, numerous nuclear factors, known to regulate TCR ex- nal transduction machinery, transcriptional regulators, and also pression and rearrangements, were highly represented and clus- several members of the cytoskeletal network. Among highly ex- tered in the same expression network. Cutl1 has been shown to pressed cell surface molecules are CD5, CD6, CD52, CD53, bind matrix attachment region sequences, and it has been proposed CCR7, Itgb2, and Itm2a. CD5 and CD53 have been shown to be to be involved in the control of DNA accessibility during V(D)J developmentally regulated and induced by TCR engagement from recombination (25). Sp2 is a zinc finger-containing protein that is the CD4ϩCD8ϩTCRlow cell population stage during selection able to bind the GT box element in TCR V ␣ promoters (26). (36). CD6 is closely related to CD5, and both are physically as- Tcf12/Heb is abundantly detected in thymocytes and is thought to sociated at the immunological synapse. Itm2a is a cell surface- regulate E-box sites present in many T cell-specific gene enhanc- expressed glycoprotein that appears to be transcriptionally up-reg- ers, including TCR-␣ and TCR-␤ (27). Runx1/AML1 has been ulated during positive selection (37). Itgb2 is the ␤-chain of shown to modulate the local accessibility of DNA to the V(D)J LFA-1, the ligand for ICAM1. As for the DP signature, numerous recombinase (28). In the same way, G22p1/Ku70 and Dntt/Tdt are specific signal transducers are found and may regulate the thresh- components of the DNA repair/V(D)J recombination machinery old of TCR-mediated signals. ITK is involved in positive selection, and are crucial for rearrangement and generation of diversity at the presumably by fine-tuning the TCR signals and affecting the effi- V(D)J junction, respectively. Importantly, Terf2/Trf2/TLP acts as ciency with which thymocytes complete their maturation (38). a positive regulator of the Dntt/Tdt promoter (29). The presence of Sh2d2a/Ribp, a T cell adaptor molecule, binds to ITK and regu- Ppp1r1c/Tarpp is in agreement with its recently suggested role in lates TCR-mediated signals (39). The tyrosine phosphatase HCPH/ TCR rearrangement (30). Of note, 16 other still-uncharacterized Shp-1 regulates the strength of TCR-mediated signals in vivo and, genes share the same transcription profile, and some of them may in turn, helps to set the threshold for thymocyte selection (40). be functionally related to rearrangement events. Among transcriptional regulatory factors expressed by developing thymocytes, Tox is a member of DNA binding HMG box protein Cluster D is mainly composed of genes highly expressed in WT, that is up-regulated during positive selection of DP thymocytes ␣ RelB°, and TCR ° mice, and corresponds to DP T cell (41). Egr1 is another transcriptional regulator of interest, because signature it promotes positive selection of both CD4 and CD8 SP cells (42). As shown in Fig. 3, genes from cluster D are exclusively induced The Kruppel-like zinc finger transcription factor (Klf2/Lklf) is in- at high levels at the DP stage. DP thymocytes were characterized duced during the maturation of SP thymocytes and rapidly down- by the expression of genes encoding cell surface proteins such as regulated after lymphocyte activation. It has been proposed to Cd4, Cd8a, CCR9, Cd97, and Blr1/Cxcr5 (Supplemental Table maintain the selected thymocytes in a quiescent state enhancing The Journal of Immunology 6115 their survival (43). The large representation of genes involved in terized or poorly described genes may be of particular interest for the cytoskeleton architecture is another striking feature of this further studies. Strikingly, a hierarchical clustering analysis re- cluster. Actin cytoskeletal rearrangements play an essential role in stricted to whole thymi samples indicated that this set of genes was shaping the cell response to extracellular signals providing struc- able to distinguish between WT and TCR␣° and RelB° thymi (Fig. tural frameworks for the spatial reorganization of signaling effec- 4A). As minor variations in the high expression values observed in tors. Components of the cytoskeleton network are found (tubulin RAG1°, Cd3⑀°, and LAT° mice strongly influenced gene cluster- ␣4 (tuba4), myosin (Myh9)) in addition to actin assembly regula- ing, these samples were excluded from analysis. This allows us to tors (thymosin ␤10 (Tmsb10), profilin 1 (Pfn1), Vasp, and calpo- define a new signature containing genes that display a low expres- nin 2). Interestingly, ITK also regulates TCR/CD3-induced actin- sion level in RelB° and TCR␣° mice as compared with WT mice dependent cytoskeletal events (44). Of the 69 genes defining this (Fig. 4B, cluster F.1). This signature revealed a high enrichment of SP signature, 15 encode still-uncharacterized proteins that may be genes already known to be highly or specifically expressed in the crucial regulators of the final steps of thymocyte maturation. medullary stromal cells (Fig. 4C). Among the most obvious ones are the two cytokeratin genes, Krt2–5 and Krt1–17 (shown in H. Stromal genes specifically devoted to thymocyte education sapiens), regulators or components of the Ag presentation machin- Genes from cluster F belong to the stroma signature: they are ery, C2ta/CIITA and some of its target genes (Invariant chain/Ii, highly expressed in thymi from RAG1°, Cd3⑀°, and LAT° mice, H2-Aa, H2-Eb1, H2-Oa) (45), the cell surface marker ICAM1, whereas relatively weakly expressed in all of the purified T cell CXCL10 (shown in H. sapiens), and the CXCL16 chemokine. To populations tested (Fig. 1B). In addition, these genes are not highly clearly establish the capability of our procedure to group genes expressed in all of the thymic epithelial or fibroblastic cell lines specific to the medullary stroma compartment, immunofluorescent Downloaded from tested, suggesting that they are likely to correspond to more spe- stainings for some of them were performed. As previously re- cific genes that may be devoted to the specialized task of thymo- ported, Krt2–5/K5 and the invariant chain Ii/CLIP/CD74 are al- cyte education. This signature contains 96 genes, and several of most exclusively expressed in the medulla with some scattered them have already been described in the thymic stroma (Supple- positive cells in the cortex (Fig. 5, A and B) (46, 47). Concerning mental Table SV). This signature includes notably 1) the cell sur- ICAM1/CD54, its expression is particularly strong in the medulla, face markers CD83, Ly75/CD205, ALCAM/CD166, VCAM1, and although faint staining is also observed throughout the cortex as ICAM1/CD54, 2) chemokines, TECK/CCL25, SDF1/CXCL12, Lepique et al. (48) recently reported (Fig. 5C). Ccl5/RANTES is http://www.jimmunol.org/ Cxcl16, CCL5/RANTES, and CXCL10/IP-10, 3) nuclear factors, already known to be expressed in the thymus, although its precise Whn/Foxn1 and Pax1, 4) proteins known to be involved in anti- expression pattern is not yet documented. Of particular interest and genic presentation Ii/CLIP/CD74, H2-Eb1, H2-Aa, H2-Oa, Ca- as underlined by its transcriptional profile, high levels of Ccl5/ thepsin L/Ctsl, and CIITA, 5) intermediate filaments, Krt2–8/K8, RANTES chemokine immunostaining were also found in the med- Krt1–18/K18, Krt2–5/K5, Krt1–17/K17, and finally 6) gene prod- ullary areas (Fig. 5D). Finally, the weak expression of the genes ucts whose function remains to be elucidated, SPATIAL, TSCOT, from cluster F.1 in RelB° and TCR␣° thymi demonstrates that thymus LIM protein (Tlmp-pending), and PRSS16. Beyond this cross-talk events with SP thymocytes are required for their

quite exhaustive listing of T cell maturation regulators, uncharac- induction. by guest on October 2, 2021

FIGURE 4. Genes deﬁning the thymic stroma signature. A, Cluster F is composed of 96 genes that discriminate RAG1°, Cd3⑀°, and LAT° from RelB°, TCR␣°, and WT thymi. Corresponding gene names are given in Supplemental Table SV. B, Exclusion of RAG1°, Cd3⑀°, and LAT° thymi leads to the identiﬁcation of F1 signature. C, The F1 signature is composed of 19 genes expressed in medullary stromal cells. 6116 TRANSCRIPTIONAL MICRODISSECTION OF THE MOUSE THYMUS

does not display canonical CBF1/Su(H) binding sites (49). Be- cause Smarca4/Brg1 is recruited in the promoter region of Cd25 (23), this supports the notion that multiple pathways overlap in this cluster. The transcriptional analysis of Notch1 and Smarca4/Brg1 conditional targeting would be of great interest to resolve such a complexity and to help us to temporally organize these transcriptional processes. Importantly, the recent findings of Raya et al. (50), establishing a calcium-dependant activation of the Notch1 pathway, is clearly in agreement with the simultaneous expression of numerous calcium-related genes together with Notch1. Further- more, the high expression level of Hes1 and Dtx1/Deltex1 in RAG1°, CD3⑀°, and LAT° T cells implies that, in these thymi, the microenvironment is able to deliver a Notch1 signal. Although an active Notch1 pathway has been clearly established in early DN thymocytes (51), this underlines the capacity of our approach to detect receptor-ligand activation signals and cellular cross talks. Consequently, in a more systematic analysis of genetically modified mice, this procedure could point out stromal defects in trig- gering developmental signals. As underlined by the proliferation signature, some of the clus- Downloaded from ters identified include numerous genes involved in specific functions. We revealed a cluster containing important genes involved in TCR expression and V(D)J recombination processes. The co- regulation of genes whose role is still unclear such as the transcription factor Runx1/AML1 strongly suggests that they are part of this functional pathway. Runx1 is highly expressed in cortical http://www.jimmunol.org/ thymocytes (52). It has been suggested to be crucial for cell fate determination during DN to DP transition and to be involved in FIGURE 5. Immunofluorescence analysis confirms the high medullary proliferation events (53). Nevertheless, it has been previously re- expression of four genes from cluster F1 in WT C57BL/6 mice (ϫ200). ported that TCR-␣ and TCR-␤ genes harbor AML1 binding sites Cryostat sections of whole thymi were stained with anti-K5/Krt2–5 (A), in their enhancer regions (54). Our data support the notion that it anti-Ii/Cd74 (B), anti-Icam1/Cd54 (C), and anti-Ccl5/RANTES (D). All could be involved in TCR rearrangement processes or in the reg- sections were counterstained using DAPI to distinguish between medullary ulation of TCR gene expression. More generally, such functional and cortical zones. From left to right, images correspond to Ab staining clusters may give substantial clues to infer putative functions for (left), Ab vs DAPI staining (middle), and DAPI staining (right). Abbrevi- by guest on October 2, 2021 ations: c, cortex; m, medulla; cmj, corticomedullary junction. poorly described genes. One other interesting aspect of this work resides in the visualization of the initiation of the thymocyte signaling machinery. For Discussion example, we could define genes, related to a calcium signaling As systematic characterization of the expression profiles of genes pathway and specifically expressed at the DN stage (Capn5, across tissues are currently in progress, data are needed to improve FK506-bp5, Eef2k, Camk2a/CaMKII, and Cacna1h). Calcium our knowledge concerning their precise expression pattern and plays important roles in thymocytes, controlling differentiation, their putative function (3). In the thymus, notably, the role of nu- proliferation, and apoptosis, depending on the intensity and the merous genes has yet to be elucidated. The use of whole thymi duration of signaling events. Our result is to be linked to the recent along with purified samples constitutes the major originality of our findings suggesting that DN thymocytes exhibit a greater ability to approach. Indeed, analyzing whole organs from genetically engi- support capacitative Ca2ϩ entry than do DP thymocytes (55). This neered mice allows us to visualize the transcriptional perturbations calcium-related property may contribute to the ligand-independent in targeted populations and their effects on cross-talk events with signaling by pre-TCR complexes. In DN thymocytes, the potent neighboring cells. However, purified cell samples are required to Ca2ϩ mobilization induced by the pre-TCR may also lead to the resolve the complexity of some of the signatures identified with transcriptional induction of survival molecules (56). DP and SP total organs. As an illustration, purified CD25ϩDN were necessary clusters, as well, are highly represented by molecular components to separate the thymocyte and stromal components of the RAG1°-, of the signal transduction machinery. Because thymocytes are able Cd3⑀°-, and LAT°-derived signatures. It also proved to be useful to receive signals through their TCR, they may induce a transcrip- to delineate specific signatures of poorly represented cell popula- tional program leading to the assembly of a specialized transduc- tions. For example, the differences that exist, at the T cell level, tion system composed of numerous molecular sensors at the cell between TCR␣° and WT mice were more clearly underlined by the surface, of coactivators, and of downstream transducers such as sorted SP samples. Sh2d1a/SAP, Slp76, Sla/SLAP, ITK, Sh2d2a/Ribp, and SHP-1. In Interestingly, this procedure enables us to visualize the coordi- addition, the SP stage is marked by the expression of proteins nate expression of components of regulatory networks. As an ex- involved in cytoskeletal dynamics that may be required to improve ample, the DN specific signature contains several transcriptional the efficiency of TCR-mediated signals. In this regard, systematic regulators, as well as some of their identified targets. Indeed, once transcriptional analysis of mouse models for each of these com- activated, Notch1 is able to convert the transcription factor CBF1/ ponents may lead to the dissection of TCR-mediated signal trans- Su(H) from a repressor to an activator state. Hes1 and Dtx1/Deltex1 duction pathways and to a better understanding of ␣␤ T cell se- are two well-known targets of CBF1. Notch1 has also been pro- lection processes. Such an approach may help to unravel posed as a Cd25 regulator, although the promoter region of Cd25 conflicting results in this area. The Journal of Immunology 6117

In contrast to most of the microarray-based studies of thymocyte analysis of the human and mouse transcriptomes. Proc. Natl. Acad. Sci. USA development, we considered thymocytes and microenvironment as 99:4465. 2. Bono, H., K. Yagi, T. Kasukawa, I. Nikaido, N. Tominaga, R. Miki, Y. Mizuno, two compartments displaying an indissociable and coordinated de- Y. Tomaru, H. Goto, H. Nitanda, et al. 2003. Systematic expression profiling of velopment. Of 96 genes enclosed in the stromal signature, several the mouse transcriptome using RIKEN cDNA microarrays. Genome Res. have already been described as playing key roles in thymus de- 13:1318. 3. Higgins, J. P., L. Wang, N. Kambham, K. Montgomery, V. Mason, velopment (e.g., Whn/Foxn1, Pax1). In the same way, this cluster S. U. Vogelmann, K. V. Lemley, P. O. Brown, J. D. Brooks, and M. Van De Rijn. also contained specific genes that may be finely regulated. Spatial 2003. Gene expression in the normal adult human kidney assessed by comple- expression is thought to depend on the three-dimensional organi- mentary DNA microarray. Mol. Biol. Cell 15:649. 4. Fischer, A., and B. Malissen. 1998. Natural and engineered disorders of lympho- zation of the thymus, and Tscot expression is lost in epithelial cell cyte development. Science 280:237. lines and may require extracellular signals to be maintained (57). 5. Kishimoto, H., and J. Sprent. 1999. Several different cell surface molecules con- The cluster corresponding to medullary stromal cells contains trol negative selection of medullary thymocytes. J. Exp. Med. 190:65. 6. Ghendler, Y., R. E. Hussey, T. Witte, E. Mizoguchi, L. K. Clayton, A. K. Bhan, genes highly expressed in WT as compared with RelB° and S. Koyasu, H. C. Chang, and E. L. Reinherz. 1997. Double-positive T cell re- TCR␣° thymi. As expected, it includes several genes whose ex- ceptor(high) thymocytes are resistant to peptide/major histocompatibility com- pression is known to depend on the RelB/NF-␬B pathway in dif- plex ligand-induced negative selection. Eur. J. Immunol. 27:2279. 7. DeRyckere, D., D. L. Mann, and J. DeGregori. 2003. Characterization of tran- ferent cellular models (RANTES/Ccl5, Cxcl10/IP-10, Icam1/ scriptional regulation during negative selection in vivo. J. Immunol. 171:802. CD54, MHC class II). More surprisingly, like Cd54/Icam1 and the 8. Hoffmann, R., L. Bruno, T. Seidl, A. Rolink, and F. Melchers. 2003. Rules for MHC components, the great majority of the genes belonging to gene usage inferred from a comparison of large-scale gene expression profiles of ␥ T and B lymphocyte development. J. Immunol. 170:1339. this signature have been shown to be regulated by IFN- (Ifit-3, 9. Schmitz, I., L. K. Clayton, and E. L. Reinherz. 2003. Gene expression analysis of Cxcl10/IP-10, Krt1–17/K17, and Oasl2) (10, 58–62). Altogether, thymocyte selection in vivo. Int. Immunol. 15:1237. these genes exhibit features of tightly regulated genes that reflect 10. Naquet, P., H. Lepesant, A. Luxembourg, P. Brekelmans, C. Devaux, and Downloaded from ϩ M. Pierres. 1989. Establishment and characterization of mouse thymic epithelial the activated phenotype of UEA1 medullary thymic stromal cell lines. Thymus 13:217. cells, known to be absent in RelB° mice. Their expression is de- 11. Shen, Z., G. Reznikoff, G. Dranoff, and K. L. Rock. 1997. Cloned dendritic cells pendent on SP-derived signals, because they are not expressed in can present exogenous antigens on both MHC class I and class II molecules. ␣ J. Immunol. 158:2723. TCR ° thymi. Therefore, this cluster pinpoints crucial cross-talk 12. Faas, S. J., J. L. Rothstein, B. L. Kreider, G. Rovera, and B. B. Knowles. 1993. events that exist in the medulla. Lymphotoxin (LT␣1␤2) and Phenotypically diverse mouse thymic stromal cell lines which induce prolifera- LIGHT are known to be produced by SP and to deliver activation tion and differentiation of hematopoietic cells. Eur. J. Immunol. 23:1201. http://www.jimmunol.org/ ␤ 13. Tanaka, T. S., T. Kunath, W. L. Kimber, S. A. Jaradat, C. A. Stagg, M. Usuda, signals to medullary stromal cells through the LT receptor. They T. Yokota, H. Niwa, J. Rossant, and M. S. Ko. 2002. Gene expression profiling are presumably required for the transcriptional induction of these of embryo-derived stem cells reveals candidate genes associated with pluripo- genes, although a putative role of IFN-␥ in medullary stromal cell tency and lineage specificity. Genome Res. 12:1921. 14. Diehn, M., G. Sherlock, G. Binkley, H. Jin, J. C. Matese, T. Hernandez-Boussard, activation cannot be excluded. C. A. Rees, J. M. Cherry, D. Botstein, P. O. Brown, and A. A. Alizadeh. 2003. Our analysis both at the RNA and protein levels additionally SOURCE: a unified genomic resource of functional annotations, ontologies, and shows for the first time that RANTES/Ccl5 expression is restricted gene expression data. Nucleic Acids Res. 31:219. 15. Hayashizaki, Y. 2003. RIKEN mouse genome encyclopedia. Mech. Ageing Dev. to the medullary stroma. Despite the fact that RANTES has been 124:93. shown to be induced in T lymphocytes under some circumstances, 16. Diehl, F., B. Beckmann, N. Kellner, N. C. Hauser, S. Diehl, and J. D. Hoheisel. by guest on October 2, 2021 its expression in the thymus is predominantly found in the med- 2002. Manufacturing DNA microarrays from unpurified PCR products. Nucleic Acids Res. 30:e79. ullary stromal cells. In the same way, our data indicate that, as for 17. Tanaka, T. S., S. A. Jaradat, M. K. Lim, G. J. Kargul, X. Wang, M. J. Grahovac, human (63), Cxcl10 is expressed in medullary stromal cells. The S. Pantano, Y. Sano, Y. Piao, R. Nagaraja, et al. 2000. Genome-wide expression low level of expression of Cxcl16, Cxcl10/IP-10, and RANTES/ profiling of mid-gestation placenta and embryo using a 15,000 mouse develop- ␣ mental cDNA microarray. Proc. Natl. Acad. Sci. USA 97:9127. Ccl5 in TCR ° mice underscores the mandatory presence of SP 18. Verdeil, G., D. Puthier, C. Nguyen, A. M. Schmitt-Verhulst, and thymocytes for the induction of an efficient chemokine network in N. Auphan-Anezin. 2002. Gene profiling approach to establish the molecular the medulla. bases for partial versus full activation of naive CD8 T lymphocytes. Ann. NY Acad. Sci. 975:68. The present work underlines the benefit of using models of per- 19. Eisen, M. B., P. T. Spellman, P. O. Brown, and D. Botstein. 1998. Cluster anal- turbed development to describe complex transcriptional processes ysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA in the mouse. The combined analysis of a still-limited number of 95:14863. 20. Naquet, P., M. Naspetti, and R. Boyd. 1999. Development, organization and models of transcriptional perturbation is sufficient to summarize function of the thymic medulla in normal, immunodeficient or autoimmune mice. the major phenomena related to thymus function in an integrated Semin. Immunol. 11:47. way. The identification of functionally relevant signatures will 21. Stuart, J. M., E. Segal, D. Koller, and S. K. Kim. 2003. A gene-coexpression network for global discovery of conserved genetic modules. Science 302:249. bring functional clues to uncharacterized transcripts coexpressed 22. Kondo, S., H. Kishi, Y. Tokimitsu, and A. Muraguchi. 2003. Possible involve- with well-known genes. Most importantly, the systematic analysis ment of glial cell line-derived neurotrophic factor and its receptor, GFR␣1, in of KO models should be a very promising method to precisely and survival and maturation of thymocytes. Eur. J. Immunol. 33:2233. 23. Yeh, J. H., S. Spicuglia, S. Kumar, A. Sanchez-Sevilla, P. Ferrier, and J. Imbert. extensively microdissect the complex events occurring during T 2002. Control of IL-2R␣ gene expression: structural changes within the proximal cell development. enhancer/core promoter during T-cell development. Nucleic Acids Res. 30:1944. 24. Welsby, P. J., H. Wang, J. T. Wolfe, R. J. Colbran, M. L. Johnson, and P. Q. Barrett. 2003. A mechanism for the direct regulation of T-type calcium Acknowledgments channels by Ca2ϩ/calmodulin-dependent kinase II. J. Neurosci. 23:10116. 25. Goebel, P., A. Montalbano, N. Ayers, E. Kompfner, L. Dickinson, C. F. Webb, We are grateful to P. Naquet, N. Auphan, and B. Malissen for critical and A. J. Feeney. 2002. High frequency of matrix attachment regions and cut-like reading of the manuscript, and to R. Houlgatte for helpful discussions. We protein x/CCAAT-displacement protein and B cell regulator of IgH transcription thank A. Carrier for providing RNA from purified CD25ϩ DN T cells, binding sites flanking Ig V region genes. J. Immunol. 169:2477. A. Bourgeois for technical help, and N. Brun and M. Barad for cell sorting. 26. Kingsley, C., and A. Winoto. 1992. Cloning of GT box-binding proteins: a novel We also thank Dr. B. B. Knowles for kindly giving us thymic stromal cell Sp1 multigene family regulating T-cell receptor gene expression. Mol. Cell. Biol. 12:4251. lines. 27. Barndt, R., M. F. Dai, and Y. Zhuang. 1999. A novel role for HEB downstream or parallel to the pre-TCR signaling pathway during ␣␤ thymopoiesis. J. Immu- nol. 163:3331. References 28. Hernandez-Munain, C., and M. S. Krangel. 1995. c-Myb and core-binding factor/ 1. Su, A. I., M. P. Cooke, K. A. Ching, Y. Hakak, J. R. Walker, T. Wiltshire, PEBP2 display functional synergy but bind independently to adjacent sites in the A. P. Orth, R. G. Vega, L. M. Sapinoso, A. Moqrich, et al. 2002. Large-scale T-cell receptor ␦ enhancer. Mol. Cell. Biol. 15:3090. 6118 TRANSCRIPTIONAL MICRODISSECTION OF THE MOUSE THYMUS

29. Ohbayashi, T., M. Shimada, T. Nakadai, T. Wada, H. Handa, and T. Tamura. 47. Farr, A., P. C. DeRoos, S. Eastman, and A. Y. Rudensky. 1996. Differential 2003. Vertebrate TBP-like protein (TLP/TRF2/TLF) stimulates TATA-less ter- expression of CLIP:MHC class II and conventional endogenous peptide:MHC minal deoxynucleotidyl transferase promoters in a transient reporter assay, and class II complexes by thymic epithelial cells and peripheral antigen-presenting TFIIA-binding capacity of TLP is required for this function. Nucleic Acids Res. cells. Eur. J. Immunol. 26:3185. 31:2127. 48. Lepique, A. P., S. Palencia, H. Irjala, and H. T. Petrie. 2003. Characterization of 30. Kisielow, J., A. C. Nairn, and K. Karjalainen. 2001. TARPP, a novel protein that vascular adhesion molecules that may facilitate progenitor homing in the post- accompanies TCR gene rearrangement and thymocyte education. Eur. J. Immu- natal mouse thymus. Clin. Dev. Immunol. 10:27. nol. 31:1141. 49. Adler, S. H., E. Chiffoleau, L. Xu, N. M. Dalton, J. M. Burg, A. D. Wells, 31. Anderson, M. K., G. Hernandez-Hoyos, R. A. Diamond, and E. V. Rothenberg. M. S. Wolfe, L. A. Turka, and W. S. Pear. 2003. Notch signaling augments T cell 1999. Precise developmental regulation of Ets family transcription factors during responsiveness by enhancing CD25 expression. J. Immunol. 171:2896. speciﬁcation and commitment to the T cell lineage. Development 126:3131. 50. Raya, A., Y. Kawakami, C. Rodriguez-Esteban, M. Ibanes, D. Rasskin-Gutman, 32. He, Y. W., C. Beers, M. L. Deftos, E. W. Ojala, K. A. Forbush, and M. J. Bevan. J. Rodriguez-Leon, D. Buscher, J. A. Feijo, and J. C. Izpisua Belmonte. 2004. 2000. Down-regulation of the orphan nuclear receptor ROR␥t is essential for T Notch activity acts as a sensor for extracellular calcium during vertebrate left- lymphocyte maturation. J. Immunol. 164:5668. right determination. Nature 427:121. 33. Li, C., C. Iosef, C. Y. Jia, V. K. Han, and S. S. Li. 2003. Dual functional roles 51. Harman, B. C., E. J. Jenkinson, and G. Anderson. 2003. Entry into the thymic for the X-linked lymphoproliferative syndrome gene product SAP/SH2D1A in microenvironment triggers Notch activation in the earliest migrant T cell pro- signaling through the signaling lymphocyte activation molecule (SLAM) family genitors. J. Immunol. 170:1299. of immune receptors. J. Biol. Chem. 278:3852. 52. Lorsbach, R. B., J. Moore, S. O. Ang, W. Sun, N. Lenny, and J. R. Downing. 34. Pivniouk, V., E. Tsitsikov, P. Swinton, G. Rathbun, F. W. Alt, and R. S. Geha. 2003. Role of Runx1 in adult hematopoiesis: analysis of Runx1-IRES-GFP 1998. Impaired viability and profound block in thymocyte development in mice knock-in mice reveals differential lineage expression. Blood 103:2522. lacking the adaptor protein SLP-76. Cell 94:229. 53. Sato, T., R. Ito, S. Nunomura, S. Ohno, K. Hayashi, M. Satake, and S. Habu. 35. Sosinowski, T., N. Killeen, and A. Weiss. 2001. The Src-like adaptor protein 2003. Requirement of transcription factor AML1 in proliferation of developing downregulates the T cell receptor on CD4ϩCD8ϩ thymocytes and regulates pos- thymocytes. Immunol. Lett. 89:39. itive selection. Immunity 15:457. 54. Kim, W. Y., M. Sieweke, E. Ogawa, H. J. Wee, U. Englmeier, T. Graf, and Y. Ito. 36. Mitnacht, R., M. Tacke, and T. Hunig. 1995. Expression of cell interaction mol- 1999. Mutual activation of Ets-1 and AML1 DNA binding by direct interaction ϩ ϩ of their autoinhibitory domains. EMBO J. 18:1609.

ecules by immature rat thymocytes during passage through the CD4 8 com- Downloaded from partment: developmental regulation and induction by T cell receptor engagement 55. Haks, M. C., S. M. Belkowski, M. Ciofani, M. Rhodes, J. M. Lefebvre, S. Trop, of CD2, CD5, CD28, CD11a, CD44 and CD53. Eur. J. Immunol. 25:328. P. Hugo, J. C. Zuniga-Pflucker, and D. L. Wiest. 2003. Low activation threshold as a mechanism for ligand-independent signaling in pre-T cells. J. Immunol. 37. Kirchner, J., and M. J. Bevan. 1999. ITM2A is induced during thymocyte selec- 170:2853. tion and T cell activation and causes downregulation of CD8 when overexpressed ϩ ϩ 56. Aifantis, I., F. Gounari, L. Scorrano, C. Borowski, and H. von Boehmer. 2001. in CD4 CD8 double positive thymocytes. J. Exp. Med. 190:217. Constitutive pre-TCR signaling promotes differentiation through Ca2ϩ mobiliza- 38. Lucas, J. A., L. O. Atherly, and L. J. Berg. 2002. The absence of Itk inhibits tion and activation of NF-␬B and NFAT. Nat. Immunol. 2:403. positive selection without changing lineage commitment. J. Immunol. 168:6142. 57. Kim, M. G., F. A. Flomerfelt, K. N. Lee, C. Chen, and R. H. Schwartz. 2000. A 39. Rajagopal, K., C. L. Sommers, D. C. Decker, E. O. Mitchell, U. Korthauer, putative 12 transmembrane domain cotransporter expressed in thymic cortical http://www.jimmunol.org/ A. I. Sperling, C. A. Kozak, P. E. Love, and J. A. Bluestone. 1999. RIBP, a novel epithelial cells. J. Immunol. 164:3185. Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation. J. Exp. 58. Kakuta, S., S. Shibata, and Y. Iwakura. 2002. Genomic structure of the mouse Med. 190:1657. 2Ј,5Ј-oligoadenylate synthetase gene family. J. Interferon Cytokine Res. 22:981. 40. Carter, J. D., B. G. Neel, and U. Lorenz. 1999. The tyrosine phosphatase SHP-1 59. Singer, K. H., S. M. Denning, L. P. Whichard, and B. F. Haynes. 1990. Thymo- influences thymocyte selection by setting TCR signaling thresholds. Int. Immu- cyte LFA-1 and thymic epithelial cell ICAM-1 molecules mediate binding of nol. 11:1999. activated human thymocytes to thymic epithelial cells. J. Immunol. 144:2931. 41. Wilkinson, B., J. Y. Chen, P. Han, K. M. Rufner, O. D. Goularte, and J. Kaye. 60. Smith, J. B., and H. R. Herschman. 1996. The glucocorticoid attenuated response 2002. TOX: an HMG box protein implicated in the regulation of thymocyte genes GARG-16, GARG-39, and GARG-49/IRG2 encode inducible proteins con- selection. Nat. Immunol. 3:272. taining multiple tetratricopeptide repeat domains. Arch. Biochem. Biophys. 42. Bettini, M., H. Xi, J. Milbrandt, and G. J. Kersh. 2002. Thymocyte development 330:290. in early growth response gene 1-deficient mice. J. Immunol. 169:1713. 61. Vanguri, P., and J. M. Farber. 1990. Identification of CRG-2: an interferon-

43. Kuo, C. T., M. L. Veselits, and J. M. Leiden. 1997. LKLF: a transcriptional inducible mRNA predicted to encode a murine monokine. J. Biol. Chem. by guest on October 2, 2021 regulator of single-positive T cell quiescence and survival. Science 277:1986. 265:15049. 44. Grasis, J. A., C. D. Browne, and C. D. Tsoukas. 2003. Inducible T cell tyrosine 62. Vogel, U., B. Denecke, S. M. Troyanovsky, R. E. Leube, and E. C. Bottger. 1995. kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen Transcriptional activation of psoriasis-associated cytokeratin K17 by interfer- receptor. J. Immunol. 170:3971. on-␥: analysis of ␥-interferon activation sites. Eur. J. Biochem. 227:143. 45. Chang, C. H., and R. A. Flavell. 1995. Class II transactivator regulates the ex- 63. Gattass, C. R., L. B. King, A. D. Luster, and J. D. Ashwell. 1994. Constitutive pression of multiple genes involved in antigen presentation. J. Exp. Med. expression of interferon-␥-inducible protein 10 in lymphoid organs and inducible 181:765. expression in T cells and thymocytes. J. Exp. Med. 179:1373. 46. Klug, D. B., C. Carter, E. Crouch, D. Roop, C. J. Conti, and E. R. Richie. 1998. 64. Lopez, F., J. Rougemont, B. Loriod, A. Bourgeois, L. Loi, F. Bertucci, P. Interdependence of cortical thymic epithelial cell differentiation and T-lineage Hingamp, R. Houlgatte, and S. Granjeaud. 2004. Feature extraction and signal commitment. Proc. Natl. Acad. Sci. USA 95:11822. processing for nylon DNA microarrays. BMC Genomics 5:38.