Identification of TMEM131L As a Novel Regulator of Thymocyte Proliferation in Humans

Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans

This information is current as Nesrine Maharzi, Véronique Parietti, Elisabeth Nelson, of September 25, 2021. Simona Denti, Macarena Robledo-Sarmiento, Niclas Setterblad, Aude Parcelier, Marika Pla, François Sigaux, Jean Claude Gluckman and Bruno Canque J Immunol 2013; 190:6187-6197; Prepublished online 20

May 2013; Downloaded from doi: 10.4049/jimmunol.1300400 http://www.jimmunol.org/content/190/12/6187

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identiﬁcation of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans

Nesrine Maharzi,*,†,‡ Ve´ronique Parietti,*,†,‡ Elisabeth Nelson,*,†,‡ Simona Denti,*,†,‡ Macarena Robledo-Sarmiento,*,†,‡ Niclas Setterblad,x Aude Parcelier,*,†,‡ Marika Pla,{ Franc¸ois Sigaux,*,†,‡ Jean Claude Gluckman,*,†,‡ and Bruno Canque*,†,‡

In this study, we identify transmembrane protein 131–like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA–mediated silencing of TMEM131L in human CD34+ hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rgnull mice, resulted in both thymocyte hyperproliferation and multiple pre– and post–b-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deﬁcient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and b-catenin intranuclear accumulation. Using T cell factor reporter Downloaded from assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/b-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome- dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-speciﬁc surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent http://www.jimmunol.org/ mechanisms. The Journal of Immunology, 2013, 190: 6187–6197.

ollowing extravasation from the blood, early thymus mature pro–double-negative (DN)1 human thymocytes (CD34hi 2 immigrants establish lymphostromal synapses with cortical CD7++CD1a ) correlates with both the onset of cell proliferation thymic epithelial cells, which triggers proliferation and and upregulation of T lineage–affiliated genes (5). Downstream F int + + 2 drives their specification along the T cell lineage through the si- transition from the DN1 (CD34 CD7 CD5 CD1a ) to DN2 multaneous activation of the IL-7R, c-Kit, sonic hedgehog, Wnt/ (CD34loCD7+CD5+CD1a+) stage coincides with definitive T lin- LEF/T cell factor (TCF), and Notch signaling pathways (1–3). eage commitment and initiation of TCRb D-J rearrangements (6). by guest on September 25, 2021 2 2 Most current evidence suggests that activation of the Frizzled/ Subsequently, thymocytes reach the DN3a (CD34 CD4intCD8a ) LRP6 receptor complex by its cognate ligands (Wnt1–4) reg- stage, when they stop proliferating to complete V-DJb rearrange- ulates survival and proliferation of early thymus immigrants, ment, and pass through the b-selection checkpoint to become 2 whereas Notch1 ligation by Delta-like 4 also drives commitment DN3b thymocytes (CD34 CD4intCD8a+) (3, 7). Later on, post–b- toward the T lineage (4). Acquisition of CD5 by the most im- selection DN3b thymocytes further upregulate CD4, acquire expression of CD8b-chain, and reach the double-positive (DP) stage where rearrangement of the TCRa locus takes place. Although most *Laboratoire De´veloppement du Syste`me Immunitaire de l’Ecole Pratique des evidence indicates that simultaneous termination of Wnt and Notch Hautes Etudes, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, 75571 signaling plays an important role in DN3a proliferation arrest, the Paris, France; †INSERM Unite´ 944, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, 75571 Paris, France; ‡Universite´ de Paris 7/Centre National de la underlying mechanisms remain poorly defined. There is evidence Recherche Scientifique Unite´ Mixte de Recherche 7212, Institut Universitaire that E proteins (E2A, HEB) play an important role in DN3a pro- d’He´matologie, Hoˆpital Saint-Louis, 75571 Paris, France; xPlateforme Technologi- que, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, 75571 Paris, France; liferation arrest, but whether they interfere with Notch or Wnt and {Unite´ Mixte de Recherche en Sante´ 940, Institut Universitaire d’He´matologie, signaling has not yet been investigated (8–11). Ikaros/IKZF1 also Hoˆpital Saint-Louis, 75571 Paris, France participate in the process through both induction of CDKN1B/ Received for publication February 11, 2013. Accepted for publication April 4, 2013. p27KIP1 (12) and interruption of Notch signaling following binding This work was supported by INSERM, the Association pour la Recherche contre le to proximal RBPJ-responsive elements in the HES1 promoter (13– Cancer, the Comite´ de Paris de la Ligue Nationale contre le Cancer, and by the Ecole 16). The mechanisms of Wnt signaling downmodulation in DN3a Pratique des Hautes Etudes. thymocytes are less well characterized. It has been shown that Address correspondence and reprint requests to Prof. Bruno Canque, Institut Uni- versitaire d’He´matologie, Centre Hayem, 1 Avenue Claude Vellefaux, 75475 Paris a p53-dependent ubiquitin E3 ligase complex involving Siah1, SIP Cedex 10, France. E-mail address: [email protected] (CacyBP), Skp1, and Ebi promotes b-catenin degradation in pre–b- The online version of this article contains supplemental material. selection thymocytes (17). However, that SIP-deficient pre–b- Abbreviations used in this article: BM, bone marrow; CDS, coding sequence; CHD, selection thymocytes display increased susceptibility to apo- conserved homology domain; DN, double-negative; DP, double-positive; ECD, ex- ptosis does not support the view that this pathway could play an tracellular domain; ER, endoplasmic reticulum; HPC, hematopoietic progenitor cell; kd, knocked down; L, long; MA-ICD, membrane-anchored intracellular domain; important role in thymocyte proliferation. Consistent with this NSG, NOD-scid/gc2/2; RT-qPCR, quantitative RT-PCR; S, short; shRNA, short hairpin view, conditional ablation of b-catenin does not affect thymocyte RNA; SP, single-positive; TCF, T cell factor; TMEM131L, transmembrane protein proliferation or differentiation (18, 19). 131–like. In this study, we identify transmembrane protein 131–like Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (TMEM131L) as a negative regulator of thymocyte proliferation www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300400 6188 TMEM131L REGULATES HUMAN INTRATHYMIC PROLIFERATION and provide evidence that it corresponds to a proximal inhibitor of (all from BD Biosciences) or CD8-PE-Cy7 (BioLegend) mAbs. The in- the canonical Wnt pathway with which it interferes through intracellular TCRb-chain was detected with the TCRbF1-allophycocyanin duction of lysosome-dependent degradation of the active phos- (Cytognos) mAb and the Cytofix/Cytoperm kit (BD Biosciences). For cell proliferation analysis, mice were injected i.p. with 1 mg BrdU 16 and 10 h phorylated form of the LRP6 coreceptor. before analysis. Cell surface proteins were labeled as above; cells that had incorporated BrdU were detected using a BrdU flow kit (BD Biosciences). Materials and Methods Data were analyzed using FlowJo software. HL60 cells transduced with a lentiviral CMV-Luc reporter (Addgene) Plasmid constructs and cell protein analysis were injected s.c. in nonirradiated NSG mice. Longitudinal follow-up by Full-length TMEM131L coding sequence (CDS) was obtained from the in vivo optical imaging for assessing luciferase activity using the IVIS Deutsches Ressourcenzentrum fu¨r Genomforschung; long (L) and short (S) Spectrum bioluminescence-fluorescence optical imaging system (Caliper isoform CDS were subcloned in frame with the Flag epitope into a pFlag Life Sciences). expression vector. LRP6DN expression vector was provided by X. He Quantitative RT-PCR (Harvard Medical School, Boston, MA). Stabilized b-catenin (S33A, S37A, T41A, and S45A) was provided by W. Held (Ludwig Institute for Quantification of selected cDNA was performed as described (20). Am- Cancer Research, Lausanne, Switzerland). LRP6-YFP, hAxin1, and hDvl1 plification of mRNAs was performed with the following primer pairs and constructs were from C. Niehrs (Deutsches Krebsforschungszentrum, a LightCycler (Roche Diagnostics). TMEM131L, forward, 59-GTAAA- Heidelberg, Germany). LRP6 CDS was subcloned in frame with a Myc CACAAAACCAGCAC-39, reverse, 59-CATACATGTCTGTATTCCCTC- epitope into a pMyc expression vector. Transient transfection of HeLa and 39; CCND1, forward, 59-CGTGGCCTCTAAGATGAAGGA-39, reverse, HEK 293T cells was performed as reported (20). Cell extracts were pre- 59-CGTGGCCTCTAAGATGAAGGA-39; CCND2, forward, 59-GGACA- pared in RIPA buffer, 0.05% SDS (0.05 M Tris [pH 8], 0.2 M NaCl, 1% TCCAACCCTA-39,reverse,59-CGCACTTCTGTTCCT-39; AXIN2,forward, Nonidet P-40, 0.02 M EDTA [pH 8], 0.5% deoxycholate), subjected to 59-CTGGCTCCAGAAGATCACAAAG-39,reverse,59-ATCTCCTCAAAC- SDS-PAGE, transferred by iBlot (Invitrogen), and probed with Abs to ACCGCTCCA-39; c-MYC, forward, 59-TGGAAGAAATTCGAGCTGC-39, Downloaded from TMEM131L (PC1 rabbit Ab, 3F10 mAb), Flag (Sigma-Aldrich), b-cat- reverse, 59-TGGTTCACCATGTCTCCTC-39; HPRT, forward, 59-CAGAG- enin, LRP6, phospho-LRP6 (Ser1490) (all from Cell Signaling Technol- GGCTACAATGTGATGGC-39, reverse, 59- GCTGAGGATTTGGAAAGG- ogy), or b-tubulin (Sigma-Aldrich) Abs. Labeling was developed using GTG-39. PCR products were detected by the SYBR Green dye (Roche) and HRP-conjugated polyclonal Abs (Pierce). Membranes were processed quantified with LightCycler software v.3.5.3 (Roche Diagnostics). using the chemiluminescence SuperSignal West Femto maximum sensi- Luciferase reporter assays tivity reagent (Thermo Scientific).

HEK 293T cells were transfected in six-well plates with ExGen 500 http://www.jimmunol.org/ Immunofluorescence microscopy (Euromedex) or FuGENE 6 (Promega). Luciferase assays were performed Transiently transfected HeLa cells grown on coverslips were washed in PBS using Dual-Luciferase (Promega) and the Galacto-Light b-galactosidase and fixed and permeabilized for 10 min in PBS, 3% Triton X-100, and 3.7% (Applied Biosystems) reporter systems. For analysis of the Wnt/b-catenin pathway, HEK 293T cells were cotransfected with TMEM131L, TOPFlash formaldehyde. Nonadherent cells were plated on poly-L-lysine-coated 3 3 coverslips, fixed in 2.5% paraformaldehyde, permeabilized in 0.1% Tri- luciferase (M72 Super 16 or M51 Super 8 ; both from Addgene), or ton X-100, and labeled with Abs to TMEM131L, calnexin, RAB6, RAB11 FOPFlash luciferase (Upstate/Cell Signaling) and the pCS2-(n)-b-gal construct (gift of S. Jauliac). Wnt signaling was activated by cotransfection (all from Santa Cruz Biotechnology), p58k, b-COP (both from Sigma- D Aldrich), Axin (Millipore), Lck, p-LRP6 (Ser1490), and cleaved Notch1 of pWnt3a, LRP6 N, hDvl, or stabilized b-catenin (S33A, S37A, T41A, and intracellular domain (Val1744) (both from Cell Signaling Technology). S45A (gift of W. Held, Ecole Polytechnique de Lausanne) or by treatment with Wnt3a-conditioned medium (L-Wnt-3A cell line; American Type Culture

Labeling was revealed by Alexa Fluor 488– or 594–labeled secondary Abs by guest on September 25, 2021 (Molecular Probes). Slides were counterstained with DAPI and mounted in Collection). After 48 h, luciferase activity was measured, corrected for trans- Vectashield neutral medium. Fluorescence microscopy was performed ei- fection efficiency as determined by quantification of b-galactosidase activity, ther with an Axiovert 2000M or with a confocal LSM510 Meta laser and normalized relative to background FOPFlash luciferase activity. microscope (Carl Zeiss MicroImaging). Images from confocal microscopy were deconvoluted with the AutoDeblur software (ImageQuant). Results Characterization of the TMEM131L protein Cell separation, lentiviral transduction, and in vitro differentiation assays Genome-wide transcriptome analysis of postnatal thymocytes identified TMEM131L as a candidate gene possibly involved in Umbilical cord blood and postnatal thymuses, collected according to in- stitutional guidelines, were processed as described (1, 20). Thymocyte pre–b-selection T cell development (20). Located on human populations were purified based on positive or negative selection with an chromosome 4q31.3, the KIAA0922/TMEM131L locus has 35 autoMACS system (Miltenyi Biotec) or a FACSAria II cell sorter (BD translated exons and two transcription start sites (TSS1-2; Sup- Biosciences). Phenotypic characterization of postnatal thymocytes was plemental Fig. 1A). During intrathymic development, TMEM131L performed by four-step labeling with the anti-TMEM31L 3F10 mAb and appropriate fluorescent Abs. Cells were analyzed with a FACSCanto cy- transcript levels strongly increase from pro–DN1 thymocytes tometer (BD Biosciences) and FlowJo software. to DN3a cells, in which they peak, and drop immediately after Screening of TMEM131L-specific short hairpin (sh)RNAs, sh1 (sense, b-selection in their DN3b successors (Supplemental Fig. 1B, 1C). 59- GCAAGAACTTTCTCGATACAT-39; loop, TTCAAGAGA; antisense, The KIAA0922/TMEM131L locus codes for two proteins referred 59- ATGTATCGAGAAAGTTCTTGC-39) and sh2 (sense, 59-TGAGCA- to thereafter as the L (1609 aa) and S (1461 aa) isoforms (Sup- GTGACATCAATG-39; loop, TTCAAGAGA; antisense, 59-CATTGATG- TCACTGCTCA-39), production of vesicular stomatitis virus–pseudotyped plemental Fig. 1D). The L isoform is a type 1 protein with an lentiviral vectors, lentiviral transduction of CD34+ hematopoietic pro- N-terminal signal peptide (aa 1–40) and a single putative trans- genitor cells (HPCs), and in vitro lymphoid differentiation assays were membrane segment (aa 870–890) separating an extracellular (aa performed as described (20). 41–869) from an intracytoplasmic domain (aa 891–1609). Al- Mice though lacking a signal peptide, the S isoform carries a putative N-

2/2 terminal myristoylation site suggestive of possible association Eight-week-old NOD-scid/gc (NSG) mice (The Jackson Laboratory, with lipid bilayers. Sequence analysis and orthologous alignments Bar Harbor, MI), housed in the pathogen-free animal facility of the Institut Universitaire d’He´matologie, were irradiated with 2.25 Gy 18–24 h before identified three conserved homology domains (CHDs; CHD1, aa i.v. injection of 1.5 3 105 human CD34+ HPCs exposed to the appropriate 1–176; CHD2, aa 696–916; CHD3, aa 1401–1609) and a C- lentiviral vectors and sacrificed 4–12 wk later. Bone marrow (BM) and terminal serine-rich region (aa 1302–1331) (Fig. 1A). To charac- thymus cells were harvested and FACS-analyzed as above. BM cells were terize the TMEM131L protein, we used a polyclonal Ab, PC1, that labeled with CD45-Alexa Fluor 700 (BioLegend), CD34-Pacific Blue (BioLegend), CD7-PE (BD Biosciences), and CD19-allophycocyanin recognizes its cytoplasmic domain, and the 3F10 mAb directed (BioLegend) mAbs. Thymocytes were labeled with CD34-Pacific Blue, at the extracellular N terminus. Ab specificity was verified by CD7-PE, CD4-allophycocyanin, CD3-allophycocyanin, and TCRab-PE immunoblotting HEK 293T cells transfected with Flag-tagged The Journal of Immunology 6189 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Dynamics of TMEM131L expression in thymocytes. (A) Schematic representation of TMEM131L L and S isoforms showing signal peptide (SP; aa 1–41; green), transmembrane domain (TM; aa 870–890; red), serine-rich region (SRR; blue), as well as CHD1–3 (blue lines). Regions recognized by the 3F10 mAb or PC1 polyclonal Ab are indicated by arrows. (B) RT-qPCR analysis of umbilical cord blood CD34+ prothymocytes (PT) and CD34+ CD1a2 (DN1), CD34lo/2CD1a+ (DN2), CD4intCD82 (ISP), CD4+CD8+CD32 (DP32), CD4+CD8+CD3+ (DP3+), CD4+CD82CD3+ (SP4+), or CD42CD8+ CD3+ (SP8+) thymocytes. Expression is normalized relative to that in PTs and is expressed as fold changes. Means of three independent samples are shown. (C and D) Immunoblot analysis. (C) Total thymocytes and magnetically purified CD3+, CD32, and CD32CD82 cell populations were probed with PC1 or b-tubulin (b-tub) Abs. The 180- and 160-kDa bands are duplicated. Note that only limited quantities of material were available for the minor CD32CD82 cell fraction (see b-tub lane). (D) Comparison of TMEM131L detection by the PC1 Ab and 3F10 mAb in CD3+ and CD32 thymocytes. The two Abs were successively hybridized on the same nitrocellulose membrane. (E–H) TMEM131L subcellular relocalization in developing thymocytes. (E) DN1, DN2, ISP, DP32, DP3+, and SP4+/8+ thymocytes were FACS-sorted, fixed, and labeled with PC1 before fluorescence microscopy analysis (original magnification 340). Arrows indicate stage-specific membrane-associated TMEM131L structures. (F and G) Thymocyte populations were sorted, fixed, and labeled with PC1 and Axin Abs and analyzed by confocal microscopy followed by two-dimensional (E) or three-dimensional (F) reconstruction. TMEM131L and Axin colocalize in polarized crescent-like structures detected at the ISP stage. (H) Confocal microscopy analysis of 3F10 mAb-labeled live ISP and DP CD3+ thymocytes. Note that TMEM131L crescent-like structures preferentially localize in cell-to-cell contact zones. (I and J) TMEM131L stage-specific surface detection in thymocytes. Cells were labeled with 3F10 mAb and the appropriate stage-specific markers (CD34, CD1a, CD3, CD4, CD8) before FACS analysis. Gates were set on (I) total or (J) DN1, DN2, ISP, DP32/+, or SP4+/8+ thymocyte populations. Analysis was performed with FlowJo software. 6190 TMEM131L REGULATES HUMAN INTRATHYMIC PROLIFERATION

TMEM131L L or S isoforms. The PC1 and anti-Flag Abs detected TMEM131L knockdown leads to increased intrathymic both isoforms at the predicted 180- and 160-kDa molecular proliferation masses, whereas the 3F10 mAb recognized the L isoform only NSG mice were then grafted with CD34+ HPCs transduced with (Supplemental Fig. 1E, 1F). Both isoforms were present in most a lentiviral vector producing shRNAs (sh-TMEM) that reduced lymphoid leukemia cell lines analyzed but remained barely de- TMEM131L expression by 60–80% both at the RNA and protein tectable in the granulomacrophagic cell lines, confirming levels (20, 21) (Supplemental Fig. 3A–C). Twelve weeks after graft, TMEM131L lymphoid affliation (Supplemental Fig. 2A). Notably, BM CD45+GFP+ cell percentages and absolute numbers were 2- the S isoform was expressed at higher levels than the L isoform, fold lower in these mice than in those grafted with control suggesting preferential usage of TSS2. Immunofluorescence shRNA-transduced HPCs (Supplemental Fig. 3D), indicating that analysis of Jurkat T cells showed TMEM131L location in discrete TMEM131L–knocked-down (TMEM131Lkd) cells had slightly punctate structures that did not colocalize with markers of the reduced engraftment capacity. However, FACS analysis showed Golgi (58k, b-COP), the endoplasmic reticulum (ER) (calnexin), that TMEM131L silencing did not modify percentages of BM early or late endosomes (RAB6, RAB11), or with Axin1, a known CD34+lineage2 HPCs, CD34+CD19+ pro–B cells, CD34+CD7+ intermediate of the Wnt pathway (Supplemental Fig. 2B, 2C). prothymocytes, or CD14+ monocytes, indicating that the protein Notably, whereas TMEM131L as detected by 3F10 mAb was does not affect multilineage BM hematopoieisis (Supplemental confined to the cytoplasm, the PC1 Ab also stained the nucleus. Fig. 3E, 3F). Rather, TMEM131L silencing resulted in a 10- to 40- Inasmuch as the L isoform comprises a signal peptide, we next fold increase in the absolute numbers of 12-wk-postgraft CD45+ examined its possible expression at the plasma membrane. Indeed, GFP+ thymocytes, corresponding to a 75- to 200-fold enhance- FACS analysis showed surface labeling by 3F10 mAb in most T ment (median, 110-fold) of thymus reconstitution efficiency (Fig. Downloaded from (SupT1, Jurkat) and B (Daudi, Raji, Ramos) cell lines, but not in 2A). Given that TMEM131L silencing did not affect the emer- granulomonocytic HL60 cells (Supplemental Fig. 2D). Subcellu- gence of BM prothymocytes, we reasoned that this reflected in- lar fractionation of Jurkat cells confirmed these data by showing creased proliferation of TMEM131Lkd thymocytes. Analysis of the selective association of the L isoform with the membrane BrdU incorporation showed that, indeed, whereas TMEM131L fraction (Supplemental Fig. 2E). Live cell imaging finally showed silencing did not influence BM cell proliferation, it increased that surface TMEM131L distributed in large membrane clusters CD45+GFP+BrdU+ thymocyte percentages 4-fold irrespective of http://www.jimmunol.org/ that did not colocalize with TCR (CD3) or BCR (CD19) com- their pre– or post–b-selection developmental stage (Fig. 2B and ponents (Supplemental Fig. 2F–H). data not shown). Notably, long-term follow-up of NSG mice kd + Dynamics of TMEM131L expression during intrathymic grafted with TMEM131L CD34 HPCs showed that only few development thymocytes remained at month 5 postgraft, suggesting that increased proliferation ultimately leads to abortive intrathymic Quantitative RT-PCR (RT-qPCR) analysis of pre– and post–b- development (data not shown). Phenotypic characterization of selection thymic populations confirmed the transcriptome data that TMEM131Lkd thymocytes showed a 3- to 5-fold increase in the TMEM131L locus was repressed immediately after passage pre– or early post–b-selection CD4+CD82/lo cell percentages by guest on September 25, 2021 through b-selection (Fig. 1B). Immunoblotting of bulk and three as well as a corresponding 2- and 10-fold drop in DP and magnetically purified thymocyte populations showed, as expected, single-positive (SP) thymocyte percentages, indicative of multi- that TMEM131L expression was largely restricted to the most 2 2 step developmental defects (Fig. 2C, 2D). Consistent with immature CD3 CD8 cells, in which levels of the two isoforms the hyperproliferative phenotype, about a third of CD4+CD8lo/2 were comparable (Fig. 1C, 1D). Interestingly, in pre–b-selection and DP TMEM131Lkd thymocytes expressed low to undetectable thymocytes, the corresponding 180- and 160-kDa bands were cytoplasmic TCRb and lacked surface TCRab/CD3 (Fig. 2E, duplicated, suggesting TMEM131L posttranslational modifi- 2F), which suggests that hyperproliferation also hampers TCR cations. The PC1 Ab detected a third, lower molecular mass S2 rearrangement. Notably, TMEM131L silencing did not mod- isoform (150 kDa) in the latter cells. Immunofluorescence analysis ify the percentages of intrathymic TCR gd+ cells (data not showed that whereas TMEM131L resided in punctate cytoplasmic shown). structures in DN1 and DN2 cells, it was located into large kd crescent-shaped membrane structures in DN3 cells (Fig. 1E), in- TMEM131L thymocytes display deregulated Wnt signaling dicating that it is submitted to stage-specific positional regulation That TMEM131L colocalizes with Axin1 in DN3 thymocytes led during early intrathymic development. The TMEM131L+ struc- us to hypothesize that intrathymic hyperproliferation could also tures also comprised Axin1, a major component of the b-catenin result from abnormal Wnt signaling. TMEM131Lkd thymocytes degradation complex (Fig. 1F, 1G), but did not colocalize with were therefore FACS-sorted from the thymi of mice at 12 wk after CD4-associated Lck tyrosine kinase (data not shown), which graft and analyzed by RT-qPCR (Fig. 3A), which was performed stresses the specificity of their association. Live cell imaging of on bulk unfractionated thymocytes due to their limited cell DN3 thymocytes confirmed that surface TMEM131L+ structures numbers. Consistent with their hyperproliferative phenotype, these predominantly localized in cell-to-cell contact zones (Fig. 1H). thymocytes expressed higher levels of cell-cycle regulators and FACS analysis found, as expected, that only 0.5–1% of thymo- Wnt target genes c-Myc, CCDN1, CCDN2,and Axin2 (22). Nota- cytes, all of which were CD342CD1a+, expressed surface bly, the expression of Wnt receptors (LRP6, Fz7, Fz8) or signaling TMEM131L (Fig. 1H). In line with TMEM131L expression dy- components (CTNNB, JUP, DVL) did not change (data not namics during intrathymic development, TMEM131L+ cell per- shown). To confirm that TMEM131L knockdown actually led to centages increased from #5% to $60% during the transition from deregulated Wnt signaling, the thymocytes were analyzed by the DN1/DN2 to DN3 stages and rapidly decreased after b-se- immunofluorescence for active p-LRP6 expression and the pres- lection (Fig. 1I, 1J). Consequently, only ,0.5% of circulating ence of intranuclear b-catenin. As expected, a majority stained CD3+ T cells displayed membrane TMEM131L (data not shown). positive for p-LRP6 and displayed prototypic intranuclear b-cat- These data indicate that during intrathymic development enin foci (23) (Fig. 3B). These data indicate that deregulated Wnt TMEM131L expression is regulated at transcriptional, posttrans- signaling may contribute to the hyperproliferative phenotype of lational, as well as positional levels. TMEM131L-deficient thymocytes. The Journal of Immunology 6191 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. TMEM131L silencing affects intrathymic development. NSG mice were injected with 1.5 3 105 human CD34+ HPCs transduced by TMEM131L (sh-TMEM) or scrambled (sh-Ctl) shRNA vectors. After 12 wk, thymocytes were FACS analyzed to determine the extent of CD45+GFP+ cell chimerism and for phenotyping. (A) Enumeration of GFP+CD45+ human cells in the thymus of recipient mice (thymocytes; left panel) and thymus reconstitution efficiency (TRE; right panel), defined as the ratio of GFP+CD45+ cell numbers detected in the thymus versus the BM (transduction efficiency, 25 6 4%; six mice per condition; data are from one of four experiments). Similar results were obtained with vectors driving expression of sh-TMEM-1 (experiments 1–4) or sh-TMEM2 RNAs. (B) Thymocyte proliferation. Mice were i.p. injected with 1 mg BrdU 16 and 10 h before FACS analysis. (C and D) Intrathymic T cell development. FACS analysis of CD45+GFP+ thymocytes of individual mice at week 12 after graft. (C) CD4 and CD8 labeling. Gating on bulk CD45+GFP+ thymocytes shows that TMEM131 deficiency elicits intrathymic accumulation of developmentally arrested ISP cells. (D) Relative quantification of thymocyte developmental subsets. Data are presented as percentages of CD45+GFP+ thymocyte sub- (Figure legend continues) 6192 TMEM131L REGULATES HUMAN INTRATHYMIC PROLIFERATION

FIGURE 3. TMEM131Lkd thymocytes display deregulated Wnt signaling. (A) RT-qPCR analysis of CD45+GFP+ thymocytes from mice grafted with human CD34+ HPCs transduced by TMEM131L shRNA vectors (sh-TMEM)–transduced HPCs. Ow- ing to limited cell numbers, only bulk GFP+ thymocytes were analyzed. Expression is normalized relative to that in thymocytes of mice injected with scrambled shRNA-transduced HPCs and is expressed as fold changes. Means of three independent bio- logical samples are shown. (B) Immunofluorescence analysis of CD45+GFP+ thymocytes of individual mice 12 wk after grafting with CD341 HPCs transduced by TMEM131L (shTMEM) or scrambled shRNA vectors (sh-Ctl). Fixed and permeabilized cells were labeled with p-LRP6 (upper panel)or b-catenin (lower panel) Abs (original magnification Downloaded from 340). Consistent with abnormal activation of the canonical Wnt signaling, TMEM131L-deficient thymocytes express LRP6 active phosphorylated form (p-LRP6) and b-catenin is predominantly localized in the nucleus. http://www.jimmunol.org/

Membrane-associated TMEM131L inhibits canonical Wnt tivity stimulated by LRP6DN (Fig. 4C), neither isoform inhibited signaling Wnt signaling activated by Dvl or b-catenin (Fig. 4D, 4E), indi- Inasmuch as these results suggested that TMEM131L might cor- cating that TMEM131L neither interfered with Dvl-mediated re- respond to a novel Wnt signaling antagonist, we sought to deter- cruitment of the b-catenin destruction complex nor did it affect mine whether it interfered with the Wnt canonical pathway. b-catenin nuclear translocation or transcription regulatory activity. Cotransfection of U2OS cells with a Notch reporter gene (RBPJ

Analysis of HeLa cells transfected with either of the TMEM131L by guest on September 25, 2021 isoforms (Fig. 4A) showed, as expected, that the L isoform luciferase) and the L or S isoforms showed that neither affected colocalized with calnexin in the ER, which indicated the func- luciferase activity stimulated by a constitutively cleaved truncated D tionality of its signal peptide, whereas the S isoform remained form of Notch1 (Notch E) or by NICD, which underscores the scattered throughout the cytoplasm in small-sized punctate specificity of TMEM131L interactions with the Wnt pathway (data structures. In HEK 293T cells cotransfected with a luciferase TCF not shown). Accordingly, neither isoform interfered with TNF-a or reporter, L isoform expression strongly inhibited luciferase ac- TGF-b signaling in appropriate reporter assays (data not shown). tivity stimulated by either cotransfection of a Wnt3a expression vector or treatment with Wnt3a-conditioned medium (Fig. 4B). In Membrane-associated TMEM131L triggers degradation of contrast, only marginal inhibition occurred in S isoform–trans- phosphorylated LRP6 fected cells, indicating that TMEM131L transmembrane locali- We next investigated whether TMEM131L interfered with LRP6 zation is essential for Wnt signaling inhibition. Of note, FACS expression or subcellular distribution. Cotransfection of Flag- analysis of transiently transfected HEK 293T cells failed to detect tagged TMEM131L isoforms with LRP6-YFP did not affect the presence of the TMEM131L L isoform at the cell surface, LRP6-YFP subcellular localization, predominantly beneath the which suggests that cell type–specific interactants or posttransla- plasma membrane (Fig. 4F). In line with the luciferase data, tional modifications are needed for its export at the plasma mem- whereas the S isoform did not affect LRP6DN perinuclear local- brane (data not shown). ization (26, 27), the L isoform expression almost abrogated To decipher the level at which TMEM131L interferes with LRP6DN labeling (Fig. 4G). Consistent with these findings, canonical Wnt signaling, we examined its effect on luciferase TCF stimulation with Wnt3a-conditioned medium of HeLa cells tran- reporter activity following stimulation by a constitutively active siently transfected with the L isoform also led to both decreased truncated form of LRP6 (LRP6DN) (24) or by downstream cyto- expression and enhanced perinuclear accumulation of LRP6-YFP plasmic components of the Wnt axis, Dvl or b-catenin (25). The (data not shown). Immunoblotting confirmed these results by LRP6DN construct full description may be found in Tamai et al. showing that whereas membrane-associated TMEM131L did not (24). Whereas the L isoform strongly inhibited the reporter ac- affect basal LRP6 expression in cotransfected HEK 293T cells, it

populations. Means 6 SD of eight representative mice from two of four experiments. *p , 0.05 (Student t test). (E and F) TCR expression. (E) Expression of intracytoplasmic TCRb by CD4+CD8+ DP (upper panel) or CD4+CD8lo/2 ISP (lower panel) thymocytes. Cells were labeled as above, ﬁxed, and permeabilized before labeling with an allophycocyanin-conjugated anti-TCRb mAb. Gates were set on DP CD45+GFP+ cells. Percentages of each pop- ulation are indicated. (F) Expression of surface TCR/CD3 complexes by post–b-selection DP and mature SP thymocytes. Gates are set on CD4+CD8+ DP or CD4+/2CD8+/2 SP thymocytes. CD3+ cell percentages are indicated. The Journal of Immunology 6193 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Membrane-associated TMEM131L antagonizes Wnt signaling. (A) Subcellular localization of TMEM131L isoforms. HeLa cells were transfected with Flag-tagged S or L isoforms and fixed and stained with Abs to Flag or calnexin. Confocal imaging (original magnification 340) shows S isoform distribution among cytoplasmic aggregates, whereas the L isoform colocalizes with calnexin in the ER. (B–E) Effect of TMEM131L L or S isoforms on luciferase TCF reporter activity in HEK 293T cells following activation of Wnt signaling by cotransfection (B) with pWnt3a or treatment with Wnt3a-conditioned medium, or with (C) LRP6DN, (D) Dvl, or (E) b-catenin (0.2 mg each). Normalized luciferase activities of the TCF/b-catenin reporter TOPFlash are reported as arbitrary units, with the control (transfection with empty vector) being defined as 1. Means 6 SD of triplicate wells from one of three experiments. (F and G) Confocal microscopy analysis of HeLa cells transfected with Flag-tagged TMEM131L isoforms and (F) LRP6-YFP or (G) constitutively active LRP6DN. Cells were fixed and stained with Abs to Flag or p-LRP6 48 h after transfection (images were obtained as above; original magnification 340). Cotransfection with the L isoform abrogates LRP6DN detection. elicited almost complete degradation of LRP6DN, the expression pression, showing the specificity of this effect (Fig. 5C). We then of which was only marginally altered by the S isoform (Fig. 5A, examined the effect of membrane-associated TMEM131L on en- 5B). As expected, neither isoform interfered with b-catenin ex- dogenous LRP6 in Wnt3a-treated HEK 293T cells (Fig. 5D). 6194 TMEM131L REGULATES HUMAN INTRATHYMIC PROLIFERATION

FIGURE 5. Membrane-associated TMEM- 131L triggers p-LRP6 degradation. (A–C)HEK 293T cells were cotransfected with vectors encoding Flag-tagged TMEM131L isoforms and (A)hLRP6(1mgeach),(B) constitutively active truncated LRP6DN, or (C) stabilized b-catenin (0.5 mg each). Whole-cell extracts were immunoblotted 48 h later using Abs to Flag, LRP6, p-LRP6, b-catenin, or b- tubulin. (D) Effect of membrane-associated TMEM131L on endogenous LRP6. HEK 293T cells transfected as above with Flag-tagged L isoform were cultured for 48 h. Wnt signaling was activated by adding Wnt3a-conditioned Downloaded from medium at the indicated time points. Whole- cell extracts were immunoblotted with appropriate Abs; the arrow indicates p-LRP6. (E and F) TMEM131L induces p-LRP6 lysosome- dependent degradation. HEK 293T cells cotransfected with the TMEM131L L isoform, http://www.jimmunol.org/ LRP6DN, and the TCF/b-catenin reporter TOPFlash were treated with graded doses of

NH4Cl. Whole-cell extracts were assayed for (E) luciferase activity or (F) immunoblotted with Abs to Flag, p-LRP6, or b-tubulin. by guest on September 25, 2021

Kinetic assays showed that, indeed, the L isoform not only pre- well as to almost complete LRP6DN degradation (Fig. 6B, 6C). vented p-LRP6 appearance but also enabled degradation of low– That the MA-ICD construct displayed only marginal activity in molecular mass LRP6 species. Because LRP6 can be internalized this context demonstrates that TMEM131L-mediated inhibition in a clathrin-dependent manner (28, 29), we speculated that of the Wnt pathway does not depend on its intrinsic signaling TMEM131L-induced degradation of p-LRP6 could depend on the activity. That LRP6DN construct comprises only a limited portion lysosomal system. Treatment with NH4Cl, which inhibits lyso- of its juxta-membrane domain (aa 1370–1613) (24) led us to hy- somal protein degradation, but not with proteasome inhibitors pothesize that TMEM131L-mediated inhibition of Wnt signaling lactacystin or MG132 (data not shown), both restored luciferase could depend on the membrane-proximal extracellular CHD2 activity and antagonized TMEM131L-mediated LRP6DN degra- domain. Transfection of HEK 293T cells with a membrane- dation in the cotransfected cells (Fig. 5E, 5F). anchored derivative of CHD2 showed, as expected, robust inhi- Thus, membrane-associated TMEM131L antagonizes canonical bition of luciferase activity stimulated by LRP6DN (Fig. 6D). Wnt signaling by triggering lysosome-dependent degradation of p- Consistent with these data, the membrane-anchored CHD2 con- LRP6. struct also elicited dose-dependent LRP6DN degradation in cotransfected HEK 293T cells, although less efﬁciently than did p-LRP6 degradation depends on the TMEM131L CHD2 the ECD construct (Fig. 6E). domain To determine whether Wnt inhibition depends on TMEM131L TMEM131L ECD limits HL60 acute myeloid leukemia cell signaling, we used a truncated form of TMEM131L that lacks most proliferation in vivo of the intracellular domain (aa 1:920, referred to as the extracellular To further document the interaction between TMEM131L and Wnt domain [ECD]) and a reciprocal membrane-anchored version of its signaling, we used HL60 cells, which express barely detectable cytoplasmic part (membrane-anchored intracellular domain [MA- endogenous TMEM131L (see Supplemental Fig. 2A) and depend ICD], aa 1–40/870–1609) (Fig. 6A, left panel). Transient expres- on Wnt/b-catenin signaling for in vivo expansion in immunode- sion assays showed that the two forms had the expected molecular ﬁcient mice (30). The cells were doubly transduced with lentiviral mass (ECD, 101 kDa; MA-ICD, 87 kDa) and located in the ER vectors driving ECD or MA-ICD expression and a constitutively (Supplemental Fig. 4). When tested in luciferase TCF reporter active luciferase reporter, before being s.c. injected into NSG assays, only the ECD construct antagonized Wnt signaling, leading mice. Longitudinal monitoring of tumor development based on to dose-dependent inhibition of the luciferase reporter activity as luciferase signal intensity showed that only the ECD (but not the The Journal of Immunology 6195

FIGURE 6. Degradation of p-LRP6 depends on the TMEM131L extracellular domain. (A) Schematic representation of membrane-associated (MA)-ECD and MA-ICD (left panel), and MA-JMD (right panel) Downloaded from constructs. (B and C) Effect of ECD or MA-ICD proteins on LRP6DN-activated Wnt/b-catenin signaling. Cell transfections, (B) luciferase reporter assays, and (C) immunoblots were performed as indicated in Fig. 5. (D and E) Effect of MA-JMD on LRP6DN-activated Wnt/b-catenin signaling. HEK

293T cells were transfected with the corresponding http://www.jimmunol.org/ constructs at the indicated doses. Luciferase reporter assays and immunoblots were performed as indicated above. by guest on September 25, 2021

MA-ICD) construct limited tumor growth during a 3-wk obser- (37) or ICAT (38) or conditional ablation of HMG box tran- vation period (Fig. 7A). Between day 18 and 22 after injection a 4- scription factors LEF1/TCF7 (39–41) both arrest intrathymic de- to 7-fold decrease in total luciferase activity was noted in mice velopment prior to, or immediately after, b-selection. The role of grafted with ECD-transduced HL60 cells relative to the control or the b-catenin coactivator in thymocyte proliferation and differ- MA-ICD conditions (Fig. 7B). Surprisingly, the MA-ICD con- entiation is more controversial. Although it has been shown that struct did not limit tumor growth and even exerted a weak stim- immature thymocytes display intranuclear b-catenin (23) and that ulatory effect in recipient mice. Collectively, these results identify b-catenin conditional stabilization allows thymocytes to bypass TMEM131L as a novel antagonist of canonical Wnt signaling and the requirement for pre-TCR signaling to reach the DP stage, suggest that its stage-specific membrane translocation in DN3 neither b-catenin ablation nor b- and g-catenin combined absence thymocytes may contribute to limit pre–b-selection thymocyte affects intrathymic development (18, 19). One possible explana- proliferation. tion is that a still undefined alternate b-catenin–independent noncanonical pathway regulates intrathymic LEF1/TCF7 activity. Discussion As reported for Notch1 (42), Wnt signaling undergoes important Wnt signaling plays a key role in thymocyte proliferation and stage-specific fluctuations during intrathymic development. survival (31). In the mouse, Wnt family members are abundantly Reports based on in vivo assessment of TCF reporter activity or expressed in the thymus, and ablation of Wnt1, Wnt3a, or Wnt4 b-catenin transgenic expression in developing thymocytes have (32, 33), or transgenic expression of Wnt antagonists Dkk1, provided evidence that, during intrathymic development, Wnt Kremen (23, 34, 35), or soluble Frizzled receptors (36), decrease signaling follows a biphasic dynamics with peak levels corre- thymocyte proliferation and lead to pre–b-selection intrathymic sponding to pre– and post–b-selection cell proliferation. Consis- differentiation arrest. Similarly, transgenic expression of Axin1 tent with this view, Wnt signaling reaches maximum levels in 6196 TMEM131L REGULATES HUMAN INTRATHYMIC PROLIFERATION

FIGURE 7. TMEM131L extracellular domain inhibits Wnt signaling in vivo. Longitudinal quantitative analysis of bioluminescence imaging (BLI). NSG mice were injected s.c. with 1 3 106 HL-60 cells doubly transduced with a luciferase reporter and TMEM131L ECD or MA-ICD constructs. (A) Representative dorsal views of NSG mice at day 22. The optical signal is expressed as photons per second per square centimeter of body surface analyzed. (B) Relative BLI signal fold changes from day 4 to day 22 after injection. *p , 0.05 for ECD versus control or MA-ICD versus control (Student t test). Downloaded from

DN1 and DN2 thymocytes (23), ceases in DN3 thymocytes (17), creased levels of Wnt target genes c-Myc, CCND1, CCDN2,and re-increases in pre-TCR expressing thymocytes, and ends in Axin2. That TMEM131Lkd thymocytes also displayed intranuclear + + post–b-selection CD4 CD8 early DP thymocytes (43, 44). Thus, foci of stabilized b-catenin and expressed the active phosphory- http://www.jimmunol.org/ during Tab lymphocyte development, cessation of Wnt signaling lated form of the LRP6 coreceptor confirmed the deregulation of coincides with the two successive phases of TCR rearrangement. Wnt signaling, raising thus the hypothesis that TMEM131L could Whereas most current evidence indicates that Wnt inhibition in correspond to an antagonist of the Wnt pathway. post–b-selection thymocytes relates to downmodulation of pre- Consistent with this view, the membrane-associated TMEM131L TCR signals, the mechanisms that dampen Wnt signaling in L isoform selectively interfered with proximal Wnt signaling by DN3a thymocytes remain poorly defined. inducing lysosome-dependent degradation of active phosphory- In this study, we identify TMEM131L as a previously unreported lated LRP6. Surprisingly, TMEM1311L-mediated inhibition of negative regulator of thymocyte proliferation, and we provide Wnt was dependent on membrane-proximal CHD2 domain sharing evidence that the protein acts through mixed Wnt-dependent and close homology with the TMEM131 protein, which suggests that by guest on September 25, 2021 -independent mechanisms. The KIAA0922/TMEM131L locus both proteins might behave as Wnt antagonists. Given that codes for two major protein isoforms, that is, the L and S iso- TMEM131L-mediated degradation of p-LRP6 depends on lyso- forms, which differ as to their subcellular localization. Whereas somes, and because it has been shown that CKII-mediated phos- both isoforms are detected among cytoplasmic granular structures, phorylation at Ser1579 shunts LRP6 endocytosis toward a clathrin- only the L isoform bearing an N-terminal signal peptide corre- dependent inhibitory pathway (28), one may also speculate that sponds to a prototypic type 1 transmembrane protein and is exported TMEM131L perturbs LRP6 intracellular trafficking. Of note, we at the cell surface. During human thymopoiesis, TMEM131L failed by coimmunoprecipitationtodetectTMEM131Ldirect undergoes stage-specific surface translocation in DN3 thymo- physical interaction with p-LRP6 (N. Maharzi, unpublished obser- cytes where it assembles in crescent-shaped membrane struc- vations). Thus, the precise mechanism by which TMEM131L tures that also comprise Axin, a major component of the promotes p-LRP6 degradation remains to be determined. That b-catenin degradation complex. This led us to hypothesize that TMEM131L cytoplasmic domain is not required for p-LRP6 TMEM131L could participate in proliferation arrest, possibly by degradation also suggests that its function largely exceeds Wnt interacting with the Wnt pathway. Phenotypic characterization of inhibition. In line with this idea, we have recently shown that NSG mice grafted with TMEM131Lkd CD34+ HPCs showed that the MA-ICD construct modulates expression of a wide array of such was the case. Whereas TMEM131L silencing did not inter- lymphoid lineage-affiliated genes upon transfer in CD34+ HPCs fere with xenogeneic hematopoiesis or B cell development in the (N. Maharzi, unpublished observations). BM, it resulted in a complex thymic phenotype characterized by In conclusion, the data presentedinthisstudyidentify TMEM131L enhanced thymocyte proliferation, leading to significant increase both as a previously unknown regulator of intrathymic proliferation in thymic cellularity, as well as to multistage developmental ar- and differentiation, as well as a novel antagonist of the Wnt rest. Consistent with intracellular TCRb-decreased expression, pathway. Our working model is that transcriptional activation of a significant fraction of CD4intCD8lo/2 TMEM131Lkd thymocytes the TMEM131L/KIAA0922 locus in early thymic immigrants leads remained stuck between the pre– and early post–b-selection de- to cytoplasmic accumulation of the protein until the DN3 stage. velopmental stages, and about half of the post–b-selection DP At that time, TMEM131L translocates to the cell surface where it TMEM131Lkd thymocytes lacked surface TCR/CD3 and failed assembles in large clusters and becomes functionally active in to differentiate into mature SP T lymphocytes. Interestingly, regulating thymocyte proliferation and/or differentiation through TMEM131L silencing generated an even more severe phenotype mixed Wnt-dependent and -independent mechanisms. in fetal thymic organ culture assays, in which most ex vivo– generated thymocytes were arrested before the DP stage Acknowledgments (V. Parietti, unpublished results). Consistent with their hyper- We are most grateful to D. Trono (Ecole Polytechnique Fe´de´rale, Lausanne, proliferative phenotype, TMEM131Lkd thymocytes expressed in- Switzerland) for providing the lentiviral vectors. We thank J. Larghero The Journal of Immunology 6197

(Banque de Sang Placentaire, Hoˆpital Saint Louis, Paris, France) and 19. Jeannet, G., M. Scheller, L. Scarpellino, S. Duboux, N. Gardiol, J. Back, G. Katz (Fondation Geńe´rale de Sante´, Paris, France) for providing um- F. Kuttler, I. Malanchi, W. Birchmeier, A. Leutz, et al. 2008. Long-term, multilineage hematopoiesis occurs in the combined absence of b-catenin and g-cat- bilical cord blood. For providing reagents and advice, we are grateful to enin. Blood 111: 142–149. X. He (Harvard Medical School, Boston, MA), C. Niehrs (Deutsches 20. Parcelier, A., N. Maharzi, M. Delord, M. Robledo-Sarmiento, E. Nelson, Krebsforschungszentrum, Heidelberg, Germany), S. Jauliac (Centre Na- H. Belakhdar-Mekid, M. Pla, K. Kuranda, V. Parietti, M. Goodhardt, et al. 2011. tional de la Recherche Scientifique Unite´ Mixte de Recherche 7212 and AF1q/MLLT11 regulates the emergence of human prothymocytes through cooperative interaction with the Notch signaling pathway. Blood 118: 1784–1796. INSERM Unite´ 940, Institut Universitaire d’He´matologie, Paris, France), 21. Parietti, V., E. Nelson, G. Telliam, S. Le Noir, M. Pla, M. Delord, V. Vanneaux, W. Held (Ecole Polytechnique de Lausanne, Lausanne, Switzerland), M. Mohtashami, E. A. Macintyre, J. C. Gluckman, et al. 2012. Dynamics of C. Brou (Institut Pasteur, Paris, France), B. Kopan (Washington University human prothymocytes and xenogeneic thymopoiesis in hematopoietic stem cell- null School of Medicine, St. Louis, MO), M. Li-Weber (German Cancer engrafted nonobese diabetic-SCID/IL-2rg mice. J. Immunol. 189: 1648–1660. 22. Li, V. S., S. S. Ng, P. J. Boersema, T. Y. Low, W. R. Karthaus, J. P. Gerlach, Research Center, Heidelberg, Germany), S. Cohen (Institute of Molecular S. Mohammed, A. J. Heck, M. M. Maurice, T. Mahmoudi, and H. Clevers. 2012. and Cellular Biology, Singapore), H. Bellen (Baylor College of Medicine, Wnt signaling through inhibition of b-catenin degradation in an intact Axin1 Houston, TX), and M. Goodhardt and X. Carnec (Institut Universitaire complex. Cell 149: 1245–1256. d’He´matologie, Paris, France). 23. Weerkamp, F., M. R. Baert, B. A. Naber, E. E. Koster, E. F. de Haas, K. R. Atkuri, J. J. van Dongen, L. A. Herzenberg, and F. J. Staal. 2006. Wnt signaling in the thymus is regulated by differential expression of intracellular Disclosures signaling molecules. Proc. Natl. Acad. Sci. USA 103: 3322–3326. The authors have no financial conflicts of interest. 24. Tamai, K., X. Zeng, C. Liu, X. Zhang, Y. Harada, Z. Chang, and X. He. 2004. A mechanism for Wnt coreceptor activation. Mol. Cell 13: 149–156. 25. Niehrs, C. 2012. The complex world of WNT receptor signalling. Nat. Rev. Mol. Cell Biol. 13: 767–779. References 26. Cruciat, C. M., B. Ohkawara, S. P. Acebron, E. Karaulanov, C. Reinhard, 1. Haddad, R., F. Guimiot, E. Six, F. Jourquin, N. Setterblad, E. Kahn, M. Yagello, D. Ingelfinger, M. Boutros, and C. Niehrs. 2010. Requirement of prorenin re- Downloaded from C. Schiffer, I. Andre-Schmutz, M. Cavazzana-Calvo, et al. 2006. Dynamics of ceptor and vacuolar H+-ATPase-mediated acidification for Wnt signaling. Sci- thymus-colonizing cells during human development. Immunity 24: 217–230. ence 327: 459–463. 2. Maillard, I., T. Fang, and W. S. Pear. 2005. Regulation of lymphoid development, 27. Yamamoto, H., H. Komekado, and A. Kikuchi. 2006. Caveolin is necessary for differentiation, and function by the Notch pathway. Annu. Rev. Immunol. 23: Wnt-3a-dependent internalization of LRP6 and accumulation of b-catenin. Dev. 945–974. Cell 11: 213–223. 3. Dik, W. A., K. Pike-Overzet, F. Weerkamp, D. de Ridder, E. F. de Haas, 28. Jiang, Y., X. He, and P. H. Howe. 2012. Disabled-2 (Dab2) inhibits Wnt/ M. R. Baert, P. van der Spek, E. E. Koster, M. J. Reinders, J. J. van Dongen, et al. b-catenin signalling by binding LRP6 and promoting its internalization through

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