* Department of Biological and Technological Research (DIBIT), San Raffaele Scientific Institute (HSR); and Dipartimento di
Biologia e Genetica per le Scienze Mediche, Università di Milano at DIBIT-HSR, I-20132 Milan, Italy
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THYMUS organogenesis depends upon a complex series of interactions among cells of different embryonic origin. The alymphoid epithelial thymic primordium originates from the third pharyngeal pouch and
requires the inductive effect of mesenchyme for appropriate morphogenesis (Auerbach, 1960). Subsequently, the
advent of hemopoietic precursors at day 10.5 of fetal life
initiates the multiple thymocyte-stroma interactions that
critically settle the architectural and functional organization
of the mature organ (van Ewijk, 1991
; Boyd et al., 1993
).
The potential role of various cell adhesion molecules in
governing early stages of thymocyte development, as well
as thymic epithelium organization, has been described.
LFA-1/ICAM-1 interactions have been shown to play a
role in thymocyte maturation and proliferation (Fine and
Kruisbeek, 1991). Thy-1 supports adhesion of thymocyte to thymic epithelial cells through a heterophilic interaction that can be inhibited by sulfated glycans (Hueber et al.,
1992
). The
6
4 (Wadsworth et al., 1992
) and VLA-4
(Sawada et al., 1992
) integrins display a developmentally
regulated pattern of expression with the highest level in
thymocyte before TCR rearrangement, suggesting a role
for these molecules in mediating adhesion of early thymocyte to stroma. Extracellular matrix proteins have been
detected in the thymus; it has been shown that early thymocytes adhere to thymus epithelium through fibronectin
expressed by the stromal cell and that blockade of this interaction has an impact on T cell maturation (Utsumi et al.,
1991
). Additionally, merosin-thymocyte interaction has
been suggested to play a role in T cell development (Chang et al., 1993
). Recently, homophilic E-cadherin interactions have been shown to be critically involved in the
generation of a functional thymic environment and in cellular interactions occurring in the early phases of T cell development (Muller et al., 1997
).
Maturation of T cells is characterized by the progression of double negative (DN)1 precursors expressing neither CD4 nor CD8, to a double positive (DP) CD4+8+
stage after low-level CD8 expression. As defined by interleukin-2 (IL-2) receptor (CD25) and CD44 expression, the
DN stage can be ordered in the following developmental
sequence of phenotypes: DN CD44+25
> DN CD44+25+ > DN CD44
25+ (Godfrey and Zlotnik, 1993
). The attainment of DP stage is defined
selection, because it is
mainly controlled by TCR
gene rearrangement and expression in association with pre-TCR-
(von Boehmer and Fehling, 1997
). Cells that have succeeded in
selection expand and undergo a further recombination event allowing
TCR-
rearrangements to occur (Petrie et al., 1993
), followed by major histocompatibility complex-driven clonotypic
selection (von Boehmer, 1994
). Recombinase- activating-2 gene deficient (RAG-2
/
) mice, in which
TCR-
gene cannot rearrange, display a block at the
CD44
CD25+ stage (Shinkai et al., 1992
). A scarcely populated cortex is apparent and there is absence of the medullary compartment (Holländer et al., 1995
), consistent
with the observed requirement for a functionally "mature"
TCR-CD3 complex for the development of the medulla
(Negishi et al., 1995
). In vivo treatment of RAG-2
/
mice
with anti-CD3
mAb induces transition of DN into DP
thymocytes with cell proliferation, cell size reduction, and
other phenotypic modifications characteristic of this transition (Jacobs et al., 1994
; Shinkai and Alt, 1994
). In this
respect, the RAG-deficient thymus offers a unique opportunity to obtain the earliest lymphoid precursors uncontaminated with later-stage cells, and an organ phenotypically arrested at 14 or 15 d of embryonic life that can be
induced to expand and differentiate.
To identify genes whose expression is selectively regulated during thymus development, we applied a PCR-based
differential screening approach (RNA fingerprinting)
(Malgaretti et al., 1997), comparing thymus RNAs extracted
from untreated RAG-2
/
mice and from the same mutants treated in vivo with anti-CD3
antibodies. Among
other genes displaying differential expression identified in
this way, we have isolated a new member of the immunoglobulin gene superfamily. The gene, named epithelial
V-like antigen (Eva), encodes a putative transmembrane
type 1 glycoprotein, bearing an immunoglobulin V-type
domain. The gene is expressed at high levels in the thymic
epithelium of RAG-2
/
mice and is almost completely
downregulated by in vivo treatment with anti-CD3
mAbs. The gene is expressed in thymus-derived epithelial cell lines and in various epithelia in development and
adulthood. Here, we present the cloning, sequence, and
initial genetic, biochemical, and functional characterization of this new protein of potential relevance as homotypic adhesion molecule in development, specifically in the
early stages of thymus organogenesis.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General Procedures
Standard molecular techniques, including nucleic acid purification, restriction analysis, gel electrophoresis, cloning, subcloning, dideoxy-sequencing, probe radiolabeling, Northern and Southern analysis, and library
screening were performed according to established protocols (Sambrook
et al., 1989).
Animals
4-wk-old C57BL/6 and CD-1 mice were purchased from Charles River
Laboratories (Calco, Italy) and RAG-2/
mice were purchased from
Bomholtgard (Ry, Denmark). All mice were bred and maintained in a
specific pathogen-free facility at DIBIT-HSR Scientific Institute. RAG-2
/
mice were i.v. injected with 50 µg of anti-CD3
mAb.
Cell Lines, Antibodies, and Primers
The following cell lines were used in this study: thymus epithelium-
derived A89A, A2T (Hiai et al., 1985), TEC (Glimcher et al., 1983
), MTE
(Lepesant et al., 1990
), 24B6 (Yanagihara et al., 1981
), and TNC.R3.1
(Nishimura et al., 1990
); thymocyte-derived BW5147 (Hyman and Stallings, 1974
), M15T, and M14T (Primi et al., 1988
); thymic fibroblast 1D4
(Izon et al., 1994
); NIH3T3 (CRL-1658; American Type Culture Collection, Rockville, MD); bone marrow stroma-derived BMS2 (Pietrangeli et
al., 1988
), M210 (provided by A. Aiuti, San Raffaele Scientific Institute,
Milan, Italy), and MS-5 (Itoh et al., 1989
); multipotent hemopoietic stem
cell FDCP mix (Spooncer et al., 1986
); fetal skin-derived dendritic cell
FSDC (Girolomoni et al., 1995
); macrophage cell line MT2 (Lutz et al.,
1994
); mature T cell line M15S (Primi et al., 1988
); monkey COS7 fibroblasts (Gluzman, 1981
); and CHO cells (provided by R. Sitia, San Raffaele
Scientific Institute). All cell lines were maintained in DME (GIBCO
BRL, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 1 mM
sodium pyruvate, 5 × 10
5 M
-mercaptoethanol, 50 U/ml streptomycin,
and 10% FCS.
The following mAbs were used in this study: 145-2C11 (anti-CD3)
(Leo et al., 1987
); H129.19 (anti-CD4), 53-6.7 (anti-CD8), AMT-13 (anti-CD25), and AC-40 (anti-actin) (Sigma Chemical Co., St. Louis, MO);
9.3.4.HL.2 (anti-murine CD45) (TIB-122; American Type Culture Collection); G7 (anti-CD90/Thy-1), RA3-6B2 (anti-CD45R/B220) (PharMingen, San Diego, CA); F4/80 (anti-macrophage) (Austyn and Gordon,
1981
); 9E10 (anti-myc, CRL-1279; American Type Culture Collection);
327 (anti-v-src) (Oncogene Science, Inc., Manhasset, NY). Rabbit antiserum specific for EVA was obtained by immunizing rabbit 833 with a peptide corresponding to the 21 NH2-terminal residues of the predicted mature protein. The immune serum was affinity purified on EAH Sepharose
48 (Pharmacia Biotech, Inc., Piscataway, NJ) coupled to the relevant peptide. Rabbit complement was purchased from Cedarlane Labs, Ltd.
(Hornby, Ontario, Canada).
The following primers were used in this study: DR122 5'-GGGACGTCTACG; Eva forward G1.F 5'-CACGACTGGTTGGCTCCGCT, G1.RT
5'-GGGTGGTCTGGGACGGAAAC; Eva reverse G1.A5 5'-GTGTGCACAACGCTGAGCCGG, G1.T3ON 5'-GGGGTGCTGATGGTGTCCTC; actin forward 5'-TGACGGGGTCACCCACACTGTGCCCATCTA,
actin reverse 5'-CTAGAAGCATTTGCGGTGGACGATGGAGG.
Gene Isolation and Sequence Analysis
PCR-based differential screening (RNA fingerprinting) (Welsh et al.,
1995) was conducted as previously described (Corradi et al., 1996
; Malgaretti et al., 1997
) to compare mRNAs of thymi from untreated C57BL/6,
RAG-2
/
mice and RAG-2
/
mice i.v. injected with 50 µg of 145-2C11
mAb. In the case of treated animals, thymi were dissected 2, 6, and 48 h after antibody injection. DR122 was the arbitrary 12 mer primer used. Thymus RNA extracted from adult RAG-2
/
mice was used to construct a
cDNA library cloned into Lambda Zap II (Stratagene, La Jolla, CA). The
cDNA isolated by RNA display (G1) was used as a probe to screen library
filters containing 106 plaques. Four positive clones were sequenced automatically through a sequencer (model 373; Applied Biosystems, Inc., Foster City, CA) and manually by dideoxy sequencing (Sequenase 2.0, USB,
Cleveland, Ohio). Sequences were analyzed through the MacVector software (Oxford Molecular Group) and a contig was assembled through the Sequencher software (DNA Codes). The putative transcription initiation was mapped through the RACE protocol (Frohman et al., 1989
). The resulting nucleotide and peptide sequences were aligned to databases of existing sequences through the BLAST network server (Altschul et al.,
1990
) and through a local Genetics Computer Group (GCG, Madison,
WI) package (Devereux et al., 1984
). Sequence searches were run against
the GenBank, Swissprot, and dbEST databases.
Genetic Mapping
Genetic mapping was achieved using a ([C57BL/6j × SPRET/Ei]F1 ×
SPRET/Ei) backcross (BSS) generated and distributed by The Jackson
Laboratory (Bar Harbor, ME) (Rowe et al., 1994). A 596-bp G1.F/G1.A5
PCR fragment corresponding to the 5' of the Eva cDNA was used as a probe
to identify a Taq1 restriction fragment length polymorphism (RFLP) by
Southern hybridization. Its segregation was followed on 96 DNAs, corresponding to the parentals and 94 N2 progeny, and then linkage analysis was
performed with the MapManager 2.6 program (Manly and Elliott, 1991
).
The human Eva orthologue was mapped in the human genome by
Southern blot analysis on nine different YAC clones from the CEPH library (Albertsen et al., 1990) (735-C-9, 742-F-9, 785-C-6, 822-G-8, 828-G-11,
886-D-9, 901-A-11, 936-D-9, and 969-D-7). DNAs were digested with
EcoRI and then hybridized with a radiolabeled Eva probe spanning the
complete coding region. YAC clones were chosen based on sinteny with
the mouse mapping position.
RNase Protection Assay
An antisense riboprobe was generated by in vitro transcription of the G1
fragment subcloned into pBluescript with T3 RNA polymerase and incorporation of [32P]UTP (800 Ci/mmol) (Promega Corp., Madison, WI). 50 µg
RNA from each of different adult mouse tissues were hybridized, treated, and then PAGE separated as described (Ausubel et al., 1995
). A densitometer and the ImageQuant software (both from Molecular Dynamics,
Inc., Sunnyvale, CA) were used for quantitative analysis of autoradiographs.
Value normalization was achieved through a mouse
-actin riboprobe.
Reverse Transcriptase-PCR
B cells were isolated from a C57BL/6 spleen. After organ disruption, T
cells were eliminated by complement-mediated cell lysis after incubation
with anti-CD4, anti-CD8, and anti-Thy-1 mAbs (Coligan, et al., 1994).
Then, B cells were positively selected with anti-B220 magnetic microbeads
according to miniMACS cell sorting protocol (Miltenyi Biotec, Sunnyvale,
CA). Mature T lymphocytes were positively selected with anti-Thy 1.2 magnetic microbeads from C57BL/6 mesenteric lymphonodes after B-cell
depletion with anti-B220 magnetic microbeads. DN thymocytes were isolated from C57BL/6 thymi by positive selection with anti-CD25 mAb and
goat anti-rat IgG magnetic microbeads. DP, single positive (SP) thymocytes, and macrophages were previously eliminated by complement-mediated lysis after incubation with anti-CD4, anti-CD8, and anti-macrophage mAbs. Macrophages were isolated from the peritoneal cavity
(Coligan et al., 1994). Freshly drawn dendritic cells were obtained as described (provided by F. Galbiati, Hoffmann-La Roche, Milan, Italy)
(Guery et al., 1996
). Embryos at different days of gestation were separated from extraembryonic tissues under a dissection microscope. RNAs
were isolated as described (Chomczynski and Sacchi, 1987
). cDNA was
generated from 1 µg of RNA by using murine Moloney leukemia virus reverse transcriptase (RT) and oligo-dT (16 mer). G1-RT and G1-T3ON
primer pair was used. The size of the amplified fragment on cDNA is 412 bp, whereas no amplified fragment is obtained from genomic DNA.
In Situ Hybridization
Mouse embryos of outbred CD-1 strain and thymi from RAG-2/
mice
were embedded in paraffin, sectioned at 6 µm, and then treated for in situ
hybridization as described previously (Teesalu et al., 1996
). The putative
complete Eva coding sequence, subcloned into pBluescript II SK (Stratagene), termed Eva.cds was used to generate sense and antisense 35S-labeled
probes. After hybridization and washes, slides were dipped into Kodak
NTB2 emulsion (Eastman-Kodak, Rochester, NY), exposed at 4°C for 1-3 wk
and, after standard development, stained with toluidine blue and mounted
in DPX mountant (BDH Chemicals Ltd., Dagenham, Essex, UK). Slides
were observed and photographs were taken using an Axiophot microscope
(Carl Zeiss, Inc., Thornwood, NY).
In Vitro Translation and Transfectants
The Eva.cds construct was used for in vitro transcription and translation
experiments in the presence of [35S]methionine, with or without microsomal membranes, as specified by the manufacturer (Promega Corp.). In
some experiments, 10 µl of translation mix were treated with the enzyme
endo--N-acetyl-glucosaminidase H (endo-H) (Boehringer Mannheim
Corp., Indianapolis, IN) as described (Ceriotti et al., 1995
). Samples were
analyzed by SDS-PAGE under reducing conditions and the resolved
products were detected by fluorography.
A construct encoding the putative complete EVA protein tagged at
the COOH terminus with a c-myc-derived epitope (Wong and Cleveland,
1990) was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) and introduced in monkey COS7 fibroblasts by DEAE-dextran-mediated transfection.
48 h after transfection, cells were trypsinized, plated on poly-L-lysine-coated
glass coverslips (105 cells), fixed with 3% paraformaldehyde, permeabilized with 0.15% Triton X-100 in PBS for 5 min, and immunolabeled
with 9E10 anti-myc mAb followed by FITC-conjugated F(ab')2 fragments
of goat anti-mouse IgG (Protos Immunoresearch, San Francisco, CA).
Stable transfectants were obtained by introducing the same Eva.myc construct in CHO cells with the calcium-phosphate precipitation protocol and
maintaining the cells in medium supplemented with G418 (600 µg/ml).
Biochemical Analysis
A89A and TNC.R3.1 cells were washed three times with cold PBS, resuspended in ice-cold nuclease buffer (50 µg/ml Staphylococcal nuclease,
2mM CaCl2, 20 mM Tris-HCl, pH 8.8) for detachment with a cell scraper.
The same nuclease buffer solution was used to harvest multiple plates to
reach the final concentration of 50 × 106 cells/ml. The following antiproteases were added: 100 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin; then 0.3% SDS and 10% -mercaptoethanol were added and
mixed on ice. Finally, 100 µg/ml DNase I and 50 µg/ml RNase A were
added. Lysates were homogenized by passage through a 23-gauge syringe
and were centrifuged in a microfuge for 10 min. COS7 and CHO transfectants were lysed in Triton X-100 lysis buffer (1% Triton X-100, 5 mM
EDTA, 1 mM iodoacetamide, 1 mg/ml BSA, 10 mM Tris, pH 8.8, 0.15 M
NaCl). Samples were subjected to SDS-PAGE under reducing conditions
followed by transfer to nitrocellulose membranes and immunoblotted
with 9E10 mAb and anti-EVA rabbit serum. Bound antibodies were revealed by peroxidase-conjugated goat anti-mouse (Dako Corp., Carpinteria, CA) or -rabbit (Sigma Chemical Co.) Ig sera, using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).
Cell Fractionation
A89A and TNC.R3.1 cells were detached by incubation at 37°C for 10 min in 1 mM PBS-EDTA (10 ml for 50 ml of culture). After two washes in ice-cold PBS, cells were resuspended at 5 × 106/300 µl in hypotonic buffer (20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, and protease inhibitors) and incubated for 10 min on ice. Cells were then disrupted by homogenization on ice with a Dounce homogenizer (30 strokes at low speed and 10 strokes at maximum speed). Salt concentration was adjusted to 150 mM NaCl and intact cells, nuclei, and cytoskeleton were pelleted by centrifugation at 5,000 rpm for 5 min in a microfuge (Eppendorf Scientific Inc., Hamburg, Germany) at 4°C. After two washes in hypotonic buffer, the pellet (P1) was resuspended in Laemmli sample buffer. The low-speed supernatant was centrifuged at 100,000 g for 30 min and the resulting pellet (P100) was considered the membrane fraction, whereas the supernatant (S100) was considered the soluble proteins fraction. Equivalent amounts of all samples were resolved by SDS-PAGE in a 5-15% gradient gel and then immunoblotted with the indicated antibodies.
Cell Aggregation Assay
CHO cells, both pcDNA3- and Eva.myc-transfected, were detached by incubation at 37°C for 10 min in 1mM PBS-EDTA. After two washes in low-Ca
Krebs-Ringer-Hepes (KRH) buffer (125 mM NaCl, 5 mM KCl, 1 mM
MgSO4, 1 mM KH2PO4, 0.1 mM CaCl2, 33 mM glucose, 25 mM Hepes),
cells were resuspended in low-Ca KRH-2% FCS (predialyzed against
low-Ca KRH) to a final concentration of 2 × 106 cells/ml by three passages through an 18-gauge syringe. Single-cell suspensions were incubated
in 2.5-ml polypropylene tubes with gentle rotation for 60 min at 37°C.
Cells were finally plated on glass coverslips, treated, and then immunolabeled as described above. In similar experiments, cells were labeled with
different fluorescent lipophilic dyes (Sigma Chemical Co.) before aggregation assay (Litvinov et al., 1994): either PKH2 (green fluorochrome) for
Eva.myc-transfectants, or PKH26 (red fluorochrome) for vector-transfected CHO cells, were used. Aggregation index was calculated as D = (N0
Nt)/N0, where N0 is the initial number of particles corresponding to
the total number of cells, and Nt is the number of remaining particles at
the incubation time point t (Shimoyama et al., 1992
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification and Cloning of Eva
To identify regulated genes during thymus development,
we applied a modified RNA fingerprinting protocol (Malgaretti et al., 1997) to thymi from RAG-2
/
mice at different intervals after the injection of anti-CD3
mAb (2, 6, and 48 h). We identified a 436-nucleotide band (G1) that
was selectively amplified in the thymus cDNA from untreated RAG-2
/
mice. The same band was also detected
in cDNA from RAG-2
/
mice 2 and 6 h after treatment,
but was almost absent at 48 h (Fig. 1). The band was gel-excised, reamplified, and then cloned into pBluescript II
SK. Clones were screened as described (Consalez et al.,
1996
) and the plasmid G1.A2 was sequenced manually. A database search was run with BLASTN and BLASTX, using the GCG interface (Devereux et al., 1984
). One mouse
expressed sequence tag (EST) sequence (GenBank/EMBL/
DDBJ accession number AA288427), and several human
ESTs (AA336903, H77296, W61215, H48606, AA088807,
and N70439) displayed clear similarities to the G1 cDNA
(from 99 to 84% identity). cDNA G1.A2 showed an open
reading frame of 286 nucleotides, preceded by an in-frame
stop codon. G1.A2 was used as a probe to screen a custom-made RAG-2
/
thymus cDNA library. Sequence
analysis and assembly of four positive clones revealed a
645-bp open reading frame encoding a deduced 215-amino
acid (aa)-long sequence. Analysis of the predicted aa sequence with PROSITE, BLOCKS, and TMAP indicates
that G1 encodes a type 1 transmembrane glycoprotein
(Fig. 2 A), with a presumptive cleavable 20-aa-long signal
peptide and a putative 25-aa-long transmembrane region
(residues 151-175). Sequence comparison and alignments
done with the PILEUP program showed a putative extracellular region containing a V-like domain: two cysteines
(positions 47 and 123), corresponding to the invariant cysteines that form the intrachain disulfide bond in the Ig domain, are located 76 residues apart. The tryptophan at position 63, as well as other residues, such as glycine 40, arginine 90, leucine 109, and tyrosine 121 are typical features of V domains (Carayannopoulos and Capra, 1993
).
In addition, two potential glycosylation sites and a putative serine phosphorylation site were observed (Fig. 2, A
and B). Based on database search results, we also sequenced the human cDNA clone containing H48606
EST, derived from a fetal liver/spleen expression library
(I.M.A.G.E. clone N°202065). The human-derived sequence revealed the same features of G1 (Fig. 2 A), with
an identity of 79 and 85% at the nucleotide and aa level,
respectively. Moreover, database search results and sequence alignment revealed 33% identity and 45% homology with the myelin protein zero (Po), the predominant myelin protein in the peripheral nervous system (Fig. 2 C)
(Lemke and Axel, 1985
). The G1 nucleotide sequence did
not match any known gene and protein (GenBank, EMBL,
Swissprot, PIR), and thus represents a new gene, encoding
a likely new member of the immunoglobulin superfamily.
Based on the sequence and expression data (see below) we named this new gene Eva, for epithelial V-like antigen.
|
|
Chromosomal Localization of the Mouse and Human Eva Genes
A TaqI polymorphism was used to type the segregation of Eva alleles in the 94 individual N2 progeny of the BSS backcross by RFLP analysis. 90 out of 94 progeny were successfully typed. Linkage analysis unequivocally localized Eva to mouse chromosome (chr.) 9, 2.25 cM distal to Xmv16 (LOD score 22.6), and 1.12 cM proximal to Cyp1a2 (logarithm of the odds [LOD] score 24.4). The data are summarized in Fig. 3.
|
The human Eva orthologue was localized to chr. 11q24 by Southern blot analysis of YAC clones from the CEPH library. The YACs had been identified based on data bank information; in fact, some of the human ESTs homologous to Eva (namely H77296 and H48606, 84% identical to Eva) had been localized on chromosome 11q, between D11S1341 and D11S924, to the human genomic region syntenic to the mouse map position. DNA from nine YAC clones spanning that region were analyzed by Southern hybridization with a probe containing the complete Eva coding region. Of all YACs tested, Eva hybridizes YACs 822-G-8 and 828-G-11. YAC 828-G-11 has been mapped cytogenetically to 11q24 (Rocchi, M., see data available at http://bioserver.uniba.it/fish/rocchi/webbari/2_YAC/800-899/828G11.html).
Regulated Expression of the Eva Gene in
RAG-2/
Thymus
To confirm the expression data obtained by RNA fingerprinting, we performed Northern analysis on total RNAs
extracted from thymi of C57BL/6 and RAG-2/
mice at
different intervals after the injection of anti-CD3
mAb (2, 6, and 48 h). Hybridization was carried out with radiolabeled G1.A2 cDNA. As shown in Fig. 4 A, a 3.4-kb transcript in
untreated RAG-2
/
thymi as well as in RAG-2
/
thymi at
2 and 6 h after injection was revealed. As predicted from
the RNA fingerprinting pattern, no expression was detectable in wild-type and in RAG-2
/
thymi 48 h after treatment. RNase protection assay (RPA) performed on the same
samples (Fig. 4 B) revealed that Eva was also detectable in
wild-type and RAG-2
/
thymi 48 h after treatment upon
a long gel exposure. Densitometric analysis after normalization with actin showed that Eva expression levels decrease
at least 20-fold in RAG-2
/
thymi 48 h after the treatment
(data not shown). Furthermore, to confirm downregulation
of the Eva transcript in this experimental model, we performed radioactive mRNA in situ hybridization experiments. The RAG-2
/
thymus displayed a strong and uniform signal for the Eva transcript (Figs. 5 and 6), reminiscent of
the embryonic thymus pattern (see below), whereas RAG-2
/
thymus 48 h after injection failed to show any signal
above background (Fig. 5, C and D); an analogous result
was also obtained with wild-type thymus (data not shown).
|
|
|
Eva Distribution in Mouse Tissues and Cell Lines
Total RNA purified from adult mouse organs was used in
RPA to characterize the tissue distribution of the Eva
transcript. As shown in Fig. 4 B, high expression levels of
Eva were observed in the liver and gut, lower levels in the
kidney, and very low ones in the lung and brain. No expression was observed by RPA in the spleen and heart.
High levels of expression were also found in gut and liver
from RAG-2/
mice (data not shown). Eva expression
was also detected in the skin and testis by RT-PCR (Table
I). Furthermore, we could detect the Eva transcript in
wild-type fetal thymus and liver at day 13.5 post coitum
(p.c.) and in whole embryos at the early primitive streak
stage (day 7.5 p.c.) (Table I).
|
To assess the cellular specificity of the Eva transcript,
we performed RT-PCR on purified cell populations. Table
I summarizes the results that define the non-T cell nature
of the transcript. In fact, Eva was neither expressed in purified DN/CD25+ thymocytes that constitute the most
abundant cell population in RAG-2/
thymus, nor in mature T cells. Similarly, no expression was observed in mature B lymphocytes, macrophages, dendritic cells, and
bone marrow. To define the histological identity of Eva-expressing cells, we tested various cell lines by RT-PCR.
No expression was observed either in MS-5, M210, and
BMS2 cells (bone marrow stroma-derived), or in FDCP
mix (representing multipotent hemopoietic precursor). Negative results on M15S, NIH3T3, MT2, and FSDC confirmed that Eva is not expressed in T lymphocytes, fibroblasts, macrophages, and dendritic cells. Interestingly, Eva
transcript is clearly expressed in four (TEC, MTE, 24B6,
and TNC.R3.1) out of six thymic epithelial cell lines (Table I and Fig. 4 C).
Localization of Eva Transcript during Mouse Development
We used radioactive in situ hybridization to localize Eva transcripts in mouse 8.5- and 9.5-d p.c. embryo implantation sites and in 12.5-16.5 d p.c. embryos. At 8.5-d p.c., abundant Eva expression was found in the extraembryonic ectoplacental cone and giant trophoblasts (Teeslau, T., manuscript in preparation), which are trophoectoderm- derived cells important for embryo implantation and placenta formation. In mouse embryos, Eva expression was found in a number of epithelial structures: surface ectoderm, and epithelia lining gastrointestinal and respiratory tracts as well as salivary glands (Fig. 6). At 13.5-d p.c., a prominent signal is evident in the developing thymus (Fig. 6, A and B), and persists through the later embryonic stages analyzed (data not shown).
Characterization of the EVA Protein
To validate sequence analysis data and assess the size of
the peptide translated from Eva cDNA corresponding to
the open reading frame, we performed in vitro transcription and translation, the latter in the presence or absence
of microsomal membranes (Fig. 7 A). The translation
product was subjected to SDS-PAGE analysis and two
peptide bands, the more abundant migrating with a molecular mass of 32 kD and a weaker one of 30 kD, were obtained only in the presence of microsomal membranes
(Fig. 7 A, lane 6). Lack of protein product in Fig. 7 A, lane
5, at this exposure time, represented the transitional arrest
by the excess of signal recognition particles described for
secretory proteins in reticulocyte lysate system (Wolin and
Walter, 1989). This observation suggested that EVA is a
membrane-bound protein. The observed protein size was greater than the predicted one (24.5 kD), suggesting posttranslational modifications of the peptide product. Consistently, treatment of the translated polypeptide with endo-H
enzyme resulted in the appearance of two additional smaller
bands of 30 and 24 kD, respectively, likely to be deglycosylation products (Fig. 7 A, lane 8).
|
To further substantiate EVA expression at the cell surface, a construct encoding EVA in frame with a COOH-terminal myc epitope tag (Eva.myc) was generated and transfected into cultured COS cells. A cellular immunofluorescence experiment run on permeabilized transfectants revealed that EVA is clearly present at the cell plasma membrane (Fig. 7 B).
An anti-myc immunoblot on Triton X-100 lysates of CHO cells transfected with the Eva.myc construct revealed a band with a molecular weight of 31-33 kD (Fig. 7 B). The predicted size of the recombinant protein is 25.7 kD, thus the difference between the predicted and the observed size (i.e., 6-8 kD) is in agreement with the one observed among the differently glycosylated products obtained in the in vitro translation experiments. Furthermore, both the recombinant proteins, Eva.cds and Eva.myc, were recognized in immunoblotting by a rabbit antiserum raised against an NH2-terminal peptide of EVA (data not shown and Fig. 7 C). The same antiserum was tested in Western blot analysis on SDS lysates of TNC.R3.1 and A89A cells. Three bands with molecular weights of 32, 30, and 24 kD, respectively, likely corresponding to different glycoforms of EVA, were revealed in the TNC.R3.1 sample and were absent in A89A lysate. The same results were obtained under nonreducing conditions (data not shown), implying that EVA is expressed at the cell surface, and is devoid of interchain disulfide bridges.
Cell Separation and Homotypic Adhesiveness of EVA
The poor recovery of EVA in the soluble fraction of Triton X-100 cell lysates of thymic epithelial cell lines (data
not shown) prompted us to separate cells into soluble and
particulate fractions to determine the solubility of EVA
and assess its association with the cytoskeleton, as it has
been proposed for the Po protein (Wong and Filbin, 1994).
TNC.R3.1 and A89A cells were lysed in hypotonic buffer
and then separated in P1 (containing nuclei and cytoskeleton), P100 (membranes), and S100 (soluble proteins) fractions, as detailed in Materials and Methods. Immunoblots
of these fractions with anti-EVA rabbit serum 833 revealed the almost exclusive presence of EVA in the P1
fraction of TNC.R3.1 cells at this exposure time (Fig. 7 D).
Only long exposures of the membrane revealed the presence of EVA in the P100 fraction, implying that EVA is
predominantly insoluble in hypotonic buffer. The same
membranes were stripped and hybridized with anti-actin
and anti-Src mouse mAbs. Fig. 7 D shows the presence of
actin in the P1 and S100 fractions, as expected, and of Src
exclusively in the P100 fraction, demonstrating the effectiveness of the lysis and the efficient isolation of cell membranes. The same result was obtained using TEC cells
(data not shown). Whether the insolubility of EVA is due
to formation of large homotypic aggregates (see below) or
to association with cytoskeleton is under investigation.
To evaluate the adhesive properties of EVA-expressing cells, we performed an aggregation assay in which EVA. myc.transfected CHO and mock-transfected CHO cells were labeled with different lipophilic fluorochromes. Single-cell suspensions were allowed to aggregate and the presence of cell clusters was monitored by microscopic examination. As shown in Fig. 8 A, nonrecombinant pcDNA3-transfected CHO cells were scattered as a single-cell suspension, whereas EVA-expressing CHO cells formed large cell clusters after 60 min of incubation (Fig. 8 B). Large aggregates (> 10 cells) were observed only in the case of Eva-transfected cells and the aggregation index appeared to be fourfold higher than the one observed with parental cells (Fig. 8 C). Same results were obtained also when the aggregation assay was performed with unlabeled cells (data not shown). When monocellular suspensions of the two cell lines were mixed in a 1:1 ratio, EVA-expressing cells aggregated on their own (Fig. 8 D) in a 90-min assay, but not at 30 min, and random incorporation of parental CHO into aggregates was observed within a range between 10 and 20% (data not shown). Furthermore, when the two unlabeled cell lines were mixed at a 1:1 ratio and stained with FITC-labeled 9E10 anti-myc mAb, the obtained cell clusters were selectively fluorescent, whereas scattered cells were detected only by phase-contrast analysis (Fig. 8, E and F). These results demonstrate that EVA expression can mediate cell aggregation, most likely through an homotypic interaction.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinase-deficient mice display a T cell maturation
arrest at an early lymphoid precursor stage (DN CD25+
CD44) (Shinkai et al., 1992
). Since organogenesis depends on thymocyte-stroma cross-talk, this thymocyte defect also has an impact on the stromal component of the
thymus, which fails to develop a medullary compartment (Holländer et al., 1995
). Moreover, it is clear from various experimental systems that thymocytes at different maturation stages exert a distinct influence on stromal epithelial
cells (Boyd et al., 1993
; Ritter and Boyd, 1993
). In RAG-2
/
mice, massive transition to the thymocyte DP stage
can be induced by in vivo cross-linking of CD3, which is
expressed at low level on the membrane of RAG-2
/
thymocytes (Jacobs et al., 1994
; Shinkai and Alt, 1994
). This treatment induces the characteristic phenotypic modifications occurring in thymocytes during the physiological
transition (Nikolic-Zugic, 1991
) and determines the expansion of the cortex that becomes histologically indistinguishable from the wild-type counterpart (data not shown).
We have exploited the possibility to time a synchronous
T cell maturation event offered by this experimental system, with the aim of identifying regulated genes in the
course of thymus development. RNA fingerprinting at different intervals after the injection of anti-CD3 mAb allowed us to isolate a new member of the immunoglobulin
gene superfamily named Eva, whose expression is markedly downregulated at 48 h after the treatment, when thymocytes have completely downregulated the IL-2 receptor
(CD25) and are in the CD4-8lo stage, immediately preceding the acquisition of the DP phenotype. EVA is expressed in the epithelial component of the organ, thus is
apparently downregulated by a signal, or extinction of a
signal, coming from maturing thymocytes. Considering cytokines as potential inducers of EVA expression in the
thymus epithelium, it is interesting to note that acquisition
of DP phenotype, which constitutes the bulk population in
wild-type thymus, is accompanied by the loss of cytokines'
production (Zlotnik and Moore, 1995
). This phenomenon was also observed in RAG-2
/
mice for IFN
and TNF
48 h after anti-CD3 injection (our unpublished observations).
EVA bears a characteristic V-type domain and two potential N-glycosylation sites in the extracellular domain; a
putative serine phosphorylation site for casein kinase 2 is
also present in the cytoplasmic tail; those sites could account for posttranslational modification of the molecule. It
shows a high degree of homology (45%) with the myelin
Po protein, which is critically involved in compaction of
peripheral nervous system myelin via interactions through
both its extracellular and cytoplasmic domains (Lemke and Axel, 1985). It has been shown that the immunoglobulin-like domain of Po mediates homophilic interactions of
the molecule (Filbin et al., 1990
); moreover, it has been
proposed that the association of the cytoplasmic tail of Po
with the cytoskeleton strengthens the homophilic adhesive
interaction by inducing conformational changes and clustering of the extracellular domain (Wong and Filbin, 1996
). The reported homology between Po and EVA
prompted us to investigate functional similarities between
the two molecules. Analogous to that observed with Po,
we could show that EVA is largely insoluble in thymus-
derived epithelial cell lines and that, after transfection of
CHO cells, is able to mediate homophilic cell-cell adhesion.
In the thymus, the importance of epithelial interactions
for appropriate organ histogenesis has been recently underlined by a study describing the absence of thymus organogenesis in vitro, in a reaggregated organ culture system,
by blocking a homophilic interaction of E-cadherin (Muller
et al., 1997). Various other cell adhesion molecules have
been hypothesized to play a role in thymus morphogenesis
(Patel and Haynes, 1993
); the structural and functional
properties of EVA are particularly attractive in this respect, offering a likely new candidate for such a role. The
strong downregulation of Eva expression induced by thymocyte maturation and expansion might suggest a role for
EVA in compaction of the thymic epithelial framework
during early thymus organogenesis, a situation that is aberrantly procrastinated in RAG-deficient mice. The EVA-dependent adhesiveness of epithelium would not be needed once the thymocyte compartment has sufficiently
expanded. Suggestively, in the embryonic thymus, we
could detect the highest level of Eva expression at 14-d
p.c., which can be considered a developmental stage analogous to the one at which RAG-2
/
mice are arrested, immediately preceding the expression of a rearranged TCR
chain.
At present, we cannot rule out the possibility that EVA
binds through a lectin-like interaction, as hypothesized for
Po (Filbin and Tennekoon, 1991) and/or with an heterophilic ligand expressed on thymocyte, as it has been shown
for E-cadherin (Karecla et al., 1995
). If this were the case,
EVA downregulation by DP transition would reflect the
need of thymocytes to move to a more appropriate stage-specific niche in the organ. Accordingly, it has been shown that the progress of thymocyte maturation renders the
cells less adhesive to stroma (Nishimura et al., 1990
).
Finally, Eva expression in several epithelia suggests other potential roles for EVA in addition to the putative ones envisaged during thymus organogenesis. The early expression of Eva during embryogenesis and its selective epithelial localizations are particularly significant in this respect.
![]() |
Footnotes |
---|
Address all correspondence to Maria Guttinger, DIBIT-HSR, via Olgettina 58, I-20132 Milan, Italy. Tel.: (39) 226-434-797. Fax: (39) 226-434-786. E-mail: guttinger.maria{at}hsr.it (note change)
Received for publication 23 December 1997 and in revised form 30 March 1998.
T. Teesalu's present address is Department of Virology, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland.We thank A. Kruisbeck (Netherlands Cancer Institute, Amsterdam, The Netherlands) and P. Castagnoli (University of Milan, Milan, Italy) for cell lines; E. Barbier (Pateur Institute, Paris, France) for skillful advice in biochemistry; C. Sala (San Raffaele Scientific Institute) for YAC clones; K. Servis (University of Lausanne, Lausanne, Switzerland), P. Cordioli (Istituto Zooprofilattico, Brescia, Italy), S. Fabbrini, S. Levi (both from San Raffaele Scientific Institute), P. Panina (Hoffmann-LaRoche, Milan, Italy), and A. Ceriotti (Consiglio Nazionale delle Ricerche, Milan) for reagents and experimental advice; and L. Adorini (Hoffman-LaRoche, Milan), F. Blasi (University of Milan), and R. Pardi (San Raffaele Scientific Institute) for critically reading the manuscript. We are especially grateful to A.G. Siccardi (University of Milan) for constant support and encouragement.
![]() |
Abbreviations used in this paper |
---|
aa, amino acid;
DN, double negative;
DP, double positive;
endo-H, endo--N-acetyl-glucosaminidase H;
EST, expressed sequence tag;
Eva, epithelial V-like antigen;
KRH, Krebs-
Ringer-Hepes;
p.c., post coitum;
Po, myelin protein zero;
RAG-2
/
, recombinase-activating-2 gene deficient;
RPA, RNase protection assay;
RT, reverse transcriptase.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Albertsen, H.M., H. Abderrahim, H.M. Cann, J. Dausset, D. Le Paslier, and D. Cohen. 1990. Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc. Natl. Acad. Sci. USA 87: 4256-4260 [Abstract]. |
2. | Altschul, S., W. Gish, W. Miller, E. Myers, and D. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410 |
3. | Auerbach, R.. 1960. Morphogenetic interactions in the development of the mouse thymus gland. Dev. Biol. 2: 271-285 . |
4. | Austyn, J.M., and S. Gordon. 1981. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11: 805-815 |
5. | Ausubel, F.M., R. Brent, R.E. Kingstone, D.D. Moore, J.A. Smith, and K.R. Struhl. 1995. Current Protocols in Molecular Biology. John Wiley and Sons, New York. |
6. | Boyd, R.L., C.L. Tucek, D.I. Godfrey, D.J. Izon, T.J. Wilson, N.J. Davidson, A.G. Bean, H.M. Ladyman, M.A. Ritter, and P. Hugo. 1993. The thymic microenvironment. Immunol. Today 14: 445-459 |
7. | Carayannopoulos, L., and J.D. Capra. 1993. Immunoglobulins. Structure and function. In Fundamental Immunology. 3rd edition. W.E. Paul, editor. Raven Press, New York. 283-314. |
8. | Ceriotti, A., E. Pedrazzini, M. De Silvestris, and A. Vitale. 1995. Import into the endoplasmic reticulum. Methods Cell Biol. 50:295-308. San Diego Academic Press. |
9. |
Chang, A.C.,
S. Wadsworth, and
J.E. Coligan.
1993.
Expression of merosin in
the thymus and its interaction with thymocytes.
J. Immunol.
151:
1789-1801
|
10. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159 |
11. | Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober. 1994. Current Protocols in Immunology. E.R. Coico, editor. John Wiley and Sons, New York. 3.1.2-3.4.2 |
12. | Consalez, G.G., A. Corradi, S. Ciarmatori, M. Bossolasco, N. Malgaretti, and C.L. Stayton. 1996. A new method to screen clones from differential display experiments prior to RNA studies. Trends Genet 12: 455-456 |
13. | Corradi, A., L. Croci, C.L. Stayton, M. Gulisano, E. Boncinelli, and G.G. Consalez. 1996. cDNA sequence, map, and expression of the murine homolog of GTBP, a DNA mismatch repair gene. Genomics 36: 288-295 |
14. | Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-395 [Abstract]. |
15. | Filbin, M., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Role of myelin Po protein as a homophilic adhesion molecule. Nature 344: 871-872 |
16. | Filbin, M.T., and G.I. Tennekoon. 1991. The role of complex carbohydrates in adhesion of the myelin protein Po. Neuron. 7: 845-855 |
17. |
Fine, J.S., and
A.M. Kruisbeek.
1991.
The role of LFA-1/ICAM-1 interactions
during murine T lymphocyte development.
J.Immunol.
147:
2852-2859
|
18. | Frohman, M.A., T.R. Downs, P. Chomczynski, and L.A. Frohman. 1989. Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: Increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse. Mol. Endocrinol. 3: 1529-1536 [Abstract]. |
19. | Girolomoni, G., M.B. Lutz, S. Pastore, C.U. Assmann, A. Cavani, and P. Ricciardi-Castagnoli. 1995. Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin. Eur. J. Immunol. 25: 2163-2169 |
20. | Glimcher, L.H., A.M. Kruisbeek, W.E. Paul, and I. Green. 1983. Functional activity of a transformed thymic epithelial cell line. J. Immunol. 17: 1-11 . |
21. | Gluzman, Y.. 1981. SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23: 175-182 |
22. | Godfrey, D.I., and A. Zlotnik. 1993. Control points in early T-cell development. Immunol. Today 14: 547-553 |
23. | Guery, J.C., F. Ria, and L. Adorini. 1996. Dendritic cells but not B cells present antigen complexes to class II-restricted T cells after administration of protein in adjuvant. J. Exp. Med. 183: 751-757 [Abstract]. |
24. | Hiai, H., Y. Nishi, H. Kaneshima, Y.O. Buma, and Y. Nishizuka. 1985. Thymic lymphoid-stromal cell complexes in mice: In vitro assay and mechanism of the complex formation. Exp. Hematol. 13: 215-220 |
25. | Holländer, G.A., B. Wang, A. Nichogiannopoulou, P.P. Platenburg, W. van Ewijk, S.J. Burakoff, J.C. Gutierrez-Ramos, and C. Terhorst. 1995. Developmental control point in induction of thymic cortex regulated by a subpopulation of prothymocytes. Nature 373: 350-353 |
26. |
Hueber, A.O.,
M. Pierres, and
H.T. He.
1992.
Sulfated glycans directly interact
with mouse Thy-1 and negatively regulate Thy-1-mediated adhesion of thymocytes to thymic epithelial cells.
J. Immunol.
148:
3692-3699
|
27. | Hyman, R., and V. Stallings. 1974. Complementation patterns of Thy-1 variants and evidence that antigen loss variants "pre-exist" in the parental population. J. Natl. Cancer Inst. 52: 429-436 |
28. | Itoh, K., H. Tezuka, H. Sakoda, M. Konno, K. Nagata, T. Uchiyama, H. Uchino, and K.J. Mori. 1989. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp. Hematol. 17: 145-153 |
29. |
Izon, D.J.,
L.A. Jones,
E.E. Eynon, and
A.M. Kruisbeek.
1994.
A molecule expressed on accessory cells, activated T cells, and thymic epithelium is a
marker and promoter of T cell activation.
J. Immunol.
153:
2939-2950
|
30. | Jacobs, H., D. Vandeputte, L. Tolkamp, E. de Vries, J. Borst, and A. Berns. 1994. CD3 components at the surface of pro-T cells can mediate pre-T cell development in vivo. Eur. J. Immunol. 24: 934-939 |
31. |
Karecla, P.I.,
S.J. Bowden,
S.J. Green, and
P.J. Kilshaw.
1995.
Recognition of
E-cadherin on epithelial cells by the mucosal T cell integrin ![]() ![]() ![]() ![]() |
32. | Lemke, G., and R. Axel. 1985. Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin. Cell 40: 501-508 |
33. | Leo, O., M. Foo, D.H. Sachs, L.E. Samelson, and J.A. Bluestone. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA. 84: 1374-1378 [Abstract]. |
34. | Lepesant, H., H. Reggio, M. Pierres, and P. Naquet. 1990. Mouse thymic epithelial cell lines interact with and select a CD31lowCD4+CD8+ thymocyte subset through an LFA-1-dependent adhesion-de-adhesion mechanism. Int. Immunol. 2: 1021-1032 |
35. | Litvinov, S.V., M.P. Velders, H.A.M. Bakker, G.J. Fleuren, and S.O. Warnaar. 1994. Ep-CAM: A human epithelial antigen is a homophilic cell-cell adhesion molecule. J. Cell Biol. 125: 437-446 [Abstract]. |
36. | Lutz, M.B., F. Granucci, C. Winzler, G. Marconi, P. Paglia, M. Foti, C.U. Assmann, L. Cairns, M. Rescigno, and P. Ricciardi-Castagnoli. 1994. Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity. J. Immunol. Meth. 174: 269-279 |
37. |
Malgaretti, N.,
O. Pozzoli,
A. Bosetti,
A. Corradi,
S. Ciarmatori,
M. Panigada,
M.E. Bianchi,
S. Martinez, and
G.G. Consalez.
1997.
Mmotl, a new helix-loop-helix transcription factor gene displaying a sharp expression boundary
in the embryonic mouse brain.
J. Biol. Chem.
272:
17632-17639
|
38. | Manly, K.F., and R.W. Elliott. 1991. RI Manager, a microcomputer program for analysis of data from recombinant inbred strains. Mamm. Genome. 1: 123-126 |
39. | Muller, K.M., C.J. Luedecker, M.C. Udey, and A.G. Farr. 1997. Involvement of E-cadherin in thymus organogenesis and thymocyte maturation. Immunity 6: 257-264 |
40. | Negishi, I., N. Motoyama, K. Nakayama, S. Senju, S. Hatakeyama, Q. Zhang, A.C. Chan, and D.Y. Loh. 1995. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376: 435-438 |
41. |
Nikolic-Zugic, J..
1991.
Phenotypic and functional stages in the intrathymic development of ![]() ![]() |
42. |
Nishimura, T.,
Y. Takeuchi,
Y. Ichimura,
X.H. Gao,
A. Akatsuka,
N. Tamaoki,
H. Yagita,
K. Okumura, and
S. Habu.
1990.
Thymic stromal cell clone with
nursing activity supports the growth and differentiation of murine CD4+8+
thymocytes in vitro.
J. Immunol.
145:
4012-4017
|
43. | Patel, D.D., and B.F. Haynes. 1993. Cell adhesion molecules involved in intrathymic T cell development. Sem. Immunol. 5: 283-292 . |
44. |
Petrie, T.,
F. Livak,
D.G. Schatz,
A. Strasser,
I.N. Crispe, and
K. Shortman.
1993.
Multiple rearrangements in T cell receptor ![]() |
45. | Pietrangeli, C.E., S. Hayashi, and P.W. Kincade. 1988. Stromal cell lines which support lymphocyte growth: Characterization, sensitivity to radiation and responsiveness to growth factors. Eur. J. Immunol. 18: 863-872 |
46. |
Primi, D.,
B. Clynes,
E. Jouvin-Marche,
J.P. Marolleau,
E. Barbier,
P.A. Cazenave, and
K. B. Marcu.
1988.
Rearrangement and expression of T cell receptor and immunoglobulin loci in immortalized CD4![]() ![]() |
47. | Ritter, M., and R.L. Boyd. 1993. Development in the thymus: It takes two to tango. Immunol. Today 14: 462-469 |
48. | Rowe, L.B., J.H. Nadeau, R. Turner, W.N. Frankel, V.A. Letts, J.T. Eppig, M.S. Ko, S.J. Thurston, and E.H. Birkenmeier. 1994. Maps from two interspecific backcross DNA panels available as a community genetic mapping resource. Mamm. Genome. 5: 253-274 |
49. | Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. C. Nolan, editor. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 545 pp. |
50. |
Sawada, M.,
J. Nagamine,
K. Takeda,
K. Utsumi,
A. Kosugi,
Y. Tatsumi,
T. Hamaoka,
K. Miyake,
K. Nakajima,
T. Watanabe, et al
.
1992.
Expression of
VLA-4 on thymocytes. Maturation stage-associated transition and its correlation with their capacity to adhere to thymic stromal cells.
J. Immunol.
149:
3517-3524
|
51. |
Shinkai, Y., and
F.W. Alt.
1994.
CD3![]() ![]() ![]() ![]() |
52. | Shinkai, Y., G. Rathbun, K.P. Lam, E.M. Oltz, V. Stewart, M. Mendelshon, J. Charron, M. Datta, F. Young, A.M. Stall, and F.W. Alt. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 58: 855-867 . |
53. | Shimoyama, Y., A. Nagafuchi, S. Fujita, M. Gotoh, M. Takeichi, S. Tsukita, and S. Hirohashi. 1992. Cadherin disfunction in a human cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness. Cancer Res. 52: 5770-5774 [Abstract]. |
54. | Spooncer, E., C.M. Heyworth, A. Dunn, and T.M. Dexter. 1986. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation 31: 111-118 |
55. | Teesalu, T., F. Blasi, and D. Talarico. 1996. Embryo implantation in mouse: Fetomaternal coordination in the pattern of expression of uPA, uPAE, PAI-1 and alpha 2MR/LRP genes. Mech. Develop. 56: 103-116 |
56. | Utsumi, K., M. Sawada, S. Narumiya, J. Nagamine, T. Sakata, S. Iwagami, Y. Kita, H. Teraoka, H. Hirano, M. Ogata, et al . 1991. Adhesion of immature thymocytes to thymic stromal cells through fibronectin molecules and its significance for the induction of thymocyte differentiation. Proc. Natl. Acad. Sci. USA. 88: 5685-5689 [Abstract]. |
57. | van Ewijk, W.. 1991. T-cell differentiation is influenced by thymic microenvironments. Annu. Rev. Immunol. 9: 591-615 |
58. | von Boehmer, H.. 1994. Positive selection of lymphocytes. Cell 76: 219-228 |
59. | von Boehmer, H., and H.J. Fehling. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15: 433-452 |
60. |
Wadsworth, S.,
M.J. Halvorson, and
J.E. Coligan.
1992.
Developmentally regulated expression of the ![]() |
61. | Welsh, J., N. Rampino, M. McClelland, and M. Perucho. 1995. Nucleic acid fingerprinting by PCR-based methods: applications to problems in aging and mutagenesis. Mutant. Res. 338: 215-229 |
62. | Wolin, S.L., and P. Walter. 1989. Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate. J. Cell Biol. 109: 2617-2622 [Abstract]. |
63. | Wong, M.H., and M. Filbin. 1994. The cytoplasmic domain of the myelin Po protein influences the adhesive interactions of its extracellular domain. J. Cell. Biol. 126: 1089-1097 [Abstract]. |
64. | Wong, M.H., and M. Filbin. 1996. Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain. J. Cell. Biol. 134: 1531-1541 [Abstract]. |
65. | Wong, P.C., and D.W. Cleveland. 1990. Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M. J. Cell Biol. 111: 1987-2003 [Abstract]. |
66. | Yanagihara, K., T. Kajitani, K. Kamiya, and K. Yokoro. 1981. In vitro studies on the mechanism of leukemogenesis. I. Establishment and characterization of cell lines derived from the thymic epithelial reticulum cell of the mouse. Leukemia Res. 5: 321-329 |
67. | Zlotnik, A., and T.A. Moore. 1995. Cytokine production and requirements during T-cell development. Curr. Opin. Immunol. 7: 206-213 |