By
From the * Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita,
Osaka 565, Japan; Department of Biology, Jichi Medical School, 3311-1 Yakushiji,
Minamikawachi-machi, Kawachi-gun, Tochigi 329-04, Japan
Using the method of gene targeting in mouse embryonic stem cells, regulatory function of
EF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo
by generating the
EF1 mutant mice. The mutated allele of
EF1 produced a truncated form of
the
EF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous
EF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla.
Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of
magnitude accompanying the impaired thymocyte development. The early stage intrathymic
c-kit+ T precursor cells were largely depleted. The following thymocyte development also
seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing
cells. The mutant thymocyte showed elevated
4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant
mice, the CD4
CD8+ single positive cells were significantly reduced relative to CD4+CD8
single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal.
The data indicated that
EF1 is involved in regulation of T cell development at multiple stages.
Recent progress in our understanding of the T cell development clarified a major developmental pathway
in thymus at cellular level: T cell precursors that originate
from hematopoietic stem cells located in fetal liver and in
adult bone marrow migrate and colonize in thymus. Starting from the CD4 Some of these steps have been assigned to specific genes,
and mutant mice of such genes produced by gene targeting
have contributed greatly in defining each regulatory step of
T cell development (2). However, it is obvious that more
knowledge of genetic regulation is required to understand
cellular events in T cell development. The To clarify the regulatory function of Mice.
C57BL/6 and ICR mice were purchased from Japan
SLC Inc. (Shizuoka, Japan) or CLEA Japan Inc. (Tokyo, Japan). All
mice were maintained under specific pathogen-free conditions.
Construction of Targeting Vector.
Cloning and structural analysis of mouse
Gene Targeting.
E14 embryonic stem cells were electroporated and selected in the presence of G418 as described previously
(13). Homologous recombinants were screened using Southern
blot analysis, and obtained at a frequency of one in 107 electroporated cells. The ES cells carrying the mutated Southern and Northern Blot Analysis.
Total DNA from the ES
cells, the yolk sacs and the tails were isolated as described previously (15). The probe used in Southern blot analysis for identification of homologous recombination and genotyping was a 1.5kb EcoRI-XbaI genomic fragment at 0.5 kb upstream of exon 6 (shown in Fig. 2 A). Total RNA of 12.5 d.p.c. embryos were
prepared by a single step isolation procedure (16). 5 µg of the total RNA was separated by electrophoresis in a 1% formaldehyde agarose gel and blotted to a Hybond N (Amersham, Buckinghamshire, England) nylon membrane. The filter was hybridized
with the 32P-labeled 2.5-kb EcoRI fragment of mouse Expression of the An Antiserum against N-proximal Portion of Immunoprecipitation and Western Blot Analysis.
Nuclear extracts
were prepared according to the previous report (19). From each
12.5 d.p.c. embryo, 650 µl (2 µg protein/µl) of the extract was
obtained, and a 100-µl aliquot was reacted with the anti-AREB6
antiserum at room temperature for 2 h. The immunocomplexes
were precipitated with protein A-Sepharose (Pharmacia) and dissolved in 40 µl of the SDS-PAGE buffer. Each 10-µl sample was
separated by SDS-PAGE (7.5 % polyacrylamide) and blotted onto a nitrocellulose filter. The blot was treated with 5% skim-milk in
TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH
8.0), incubated with the anti-AREB6 antiserum, washed by
TBST, reacted with HRP-labeled goat anti-rabbit IgG and processed for ECL chemoluminescence reaction (Amersham).
FACS® Analysis.
Multicolor analysis of lymphocytes was
performed by FACScan® cell sorter as described previously (20).
The following mAb were purchased from PharMingen (San Diego, CA): fluorescein isothiocyanate (FITC)-conjugated antiCD45R, RA3-6B2; FITC-conjugated anti-CD8a, 53-6.7; FITC-
conjugated anti-CD3 Histology and Immunohistology.
Tissues were fixed in Bouin's
fixative and paraffin sections were stained with hematoxylin and
eosin. For immunofluorescent staining of thymus, a thymus was
excised from an 18.5 days post coitus (d.p.c.) embryo, and rinsed
in Hepes-buffered saline (HBS), quickly frozen by dipping into
liquid nitrogen, and then embedded in OCT compound (Miles
Inc., Elkhart, IN). 6-µm-thick cryosections were fixed in 1%
paraformaldehyde in HBS for 30 min. The sections were incubated with affinity-purified rabbit anti- A cDNA coding for mouse Among the lymphoid tissues, the
To generate the In embryonic stages the homozygous mutant embryos developed up to 18.5 d.p.c. with the expected Mendelian frequency. In histological analysis of the embryos
from 10.5 to 18.5 d.p.c., we noted that the thymi of the
homozygous mutant embryos were smaller than normal embryos. Other tissues and organs were normal in morphological and histological inspections. The number of
thymocytes of 18.5 d.p.c. homozygous mutant embryos
was reduced 10-fold (~5 × 105 cells per thymus) compared to the heterozygous and wild-type embryos (~5 × 106 cells per thymus). FACS® analysis of the thymocytes using CD4 and CD8 markers indicated that the development
from the DN to DP cells appeared to be partially inhibited
(data not shown). We observed no difference between wildtype and heterozygous mutant embryos.
In the postnatal period ~80% of the homozygous mutant pups died within 2 d after birth, but the remaining
20% survived and some of them had offspring. After backcrossing to C57BL/6 mice for 6 generations, 11 of the homozygous mutant mice which survived and aged from 3 to
11 wk were analyzed for lymphoid tissue development. As
observed in the fetuses, thymi of all the homozygous mutant mice inspected were greatly reduced in size. In histological sections of the mutant thymi, medulla and cortex were
hardly distinguishable (Fig. 3, A and B); especially, the cortex which usually consists of the densely packed and actively proliferating small thymocytes seemed to be missing
in the mutant thymi (Fig. 3, C and D). Accordingly, the
total cell number of thymus was greatly reduced from 1/100 to 1/500 of the heterozygous littermate (Fig. 4 A). The
spleens of homozygous mutant mice were not significantly
different in size from those of heterozygous littermates, although the number of splenocytes was slightly lower in homozygous mutant mice (Fig. 4 C). As will be shown below, the extent of reduction of the cell number seems to
be accounted for by the reduction of peripheral T cells in
the spleen. Histological inspection of the spleens of the homozygous mutant mice showed the basic architecture to be
normal (data not shown). The lymph node of the homozygous mutant mice was characteristic in the reduced cellularity of the deep cortex where the T cells reside (data not
shown). The number of lymphocytes recovered from a pair
of inguinal lymph nodes of those mice was reduced to ~1/10
of that from the heterozygous littermates (Fig. 4 B).
The heterozygous and wild-type animals were indistinguishable in their histology and cell content of all the lymphoid organs described above. In addition, Using FACS® with various cell surface
markers, we first investigated the cell populations in thymi
from the homozygous mutant and heterozygous control mice.
In the homozygous mutant mice, the total thymocyte number was so small (see Fig. 4 A) that whole thymocytes of a
mutant mouse was subjected to the FACS® analysis. A representative set of the results is shown in Fig. 5 A. The proportion of the CD4/CD8 SP or DP cell populations present was different from that found in a control heterozygous littermate: the relative proportion of cells in CD4+CD8+ quadrant was reduced from 88% in normal to 57% in the mutant thymocytes while that of the SP cells was increased from 11 to 38% (Fig. 5 A). The relative frequency of
From forward light scattering data of the FACS® analysis, which indicated the distribution pattern of cell size of a
cell population examined, we found that the cell population of larger size predominantly existed in the homozygous mutant mice whereas most of the thymocytes in control mice consisted of the small cell population typical for
normal mice (Fig. 5 C). The bias toward the larger size of the cell population in the mutant thymocytes seemed to be
due simply to the reduced cell number of the DP thymocytes relative to that of SP cells in the mutant mice since
we noticed that the most abundant cell size of the DP and
SP cells in the control and mutant mice were not quite different (Fig. 5 C). However, cell size distribution pattern of
DN cell population was significantly different between the
control and mutant thymocytes: mutant thymocytes were
not abundant in the cell population of larger size, but instead had much smaller cells compared to the control mice
(Fig. 5 C).
T cells in the spleen and the lymph node were also examined by FACS®. In the mutant spleen, although decrease
of total cell number in the mutant was less conspicuous
than in the thymus (Fig. 4 B), the fraction of the T cells in
the total splenocytes was significantly reduced from ~40%
(heterozygous) to 10% (mutant) as judged by Thy1 (T cell
marker) and B220 (B cell marker) expression (Fig. 6 A).
In the lymph node also, T cells were reduced in number
(Fig. 7 A) and were
Since MHC class I and II antigens on the thymic stromal
cells are essential for the generation of the CD4 and CD8
SP T cells, we examined the expression of the class I (H-2K)
and class II (I-A) antigens on the thymic epithelial cells by
immunohistochemistry using anti H-2K and I-A antibodies.
The levels of expression of these antigens were not different
between Next we examined the functional maturity of the peripheral T lymphocytes by the proliferative response to the
Con A stimulation using the spleen cells as shown in Table 1.
Although the degree of Table 1.
Con A-induced Proliferation of T Cells in Spleen Cells
from a CD8
double negative (DN)1 stage, thymocytes begin to rearrange their TCR genes and express CD3, a TCR coreceptor molecule, then proceed to the
CD4+CD8+ double positive (DP) stage. The DP thymocytes go through positive and negative selections depending on the specificity of the TCR. Finally, the CD4+CD8
or CD4
CD8+ single positive (SP) mature thymocytes are
produced, and these immunocompetent cells migrate out
and populate the peripheral lymphoid organs (1).
EF1 mutant
mice to be reported in this paper has a novel phenotype:
only T cells are affected among hematopoietic lineages and
major defects are found in early T cell precursors, thus defining a new step in T cell development.
EF1 was originally identified as an enhancer binding
factor of the chicken
1-crystallin gene (3).
EF1 is a unique
protein in that it has multipartite DNA-binding motifs, containing two Krüppel-type C2H2 zinc finger clusters located
close to NH2 and COOH termini and a homeodomain in
between (4). Subsequent analysis showed that
EF1 possesses repressive activity on transcription (5), and that
EF1
is expressed besides lens cells in various anlages of developing tissues, such as notochord, myotome, limb bud, and
neural crest derivatives in chickens (4) and mice (Takagi T.,
H. Kondoh, and Y. Higashi, unpublished results), suggesting that
EF1 is involved in regulation of a number of genes
other than the crystallin genes (6).
EF1 and to understand the functional significance of the multipartite DNAbinding motifs in vivo, we have initiated a study using
EF1 mutant mice of several different alleles generated by
the gene targeting technique. So far, we have produced
two
EF1 mutant alleles of mice: one, a null mutation, in
which most of the coding sequence was replaced by bacterial
-galactosidase (Null-LacZ), the other coding for a truncated protein lacking only the COOH-proximal zinc finger clusters (
C-fin). Unexpectedly, as presented in this report, one of the major phenotype of both homozygous mutant
mice was impairment of thymus development: severe hypocellularity in thymus without clear distinction of cortex
and medulla. Since Null-LacZ homozygous mutant mice
are perinatally lethal with skeletal defects (to be published
elsewhere), while ~20% of the
C-fin homozygous mutant mice were born alive and grown up to adulthood, we
analyzed the lymphoid tissues in detail using the surviving
young adult
C-fin homozygous mutant mice. Here we
describe the generation and analysis of
C-fin mutant mice
and demonstrate that the defect of the thymus was ascribed
to depletion of T precursor cells and to aberration of intrathymic development of T cells.
EF1 has been described (9). The targeting vector
(see Fig. 2 A) was constructed as follows. A 0.8-kb SalI-SalI fragment containing a Sau3AI-SalI genomic fragment of the exon 6 sequence (see Fig. 2 A) and a 12-bp SalI-BamHI adapter sequence which is derived from an EMBL3 cloning vector, was subcloned into the SalI site of pBlueScript II to give pSS. The SalI
and XbaI sites at the 5
end of the inserted genomic fragment of
pSS were inactivated by digesting with XbaI, partially with SalI,
blunt-ending by fill-in, and self-ligation. A XbaI linker carrying stop
codons in all three frames (CTAGTCTAGACTAG) was inserted
in the remaining SalI site at the 3
end of the insert to have pSSstop. A XhoI-KpnI fragment of pSTNeoB (10) containing neor
sequence was inserted in the XhoI-KpnI site of the pSSstop, to have pSSstopNEO. In parallel, the 5.4-kb SalI-ApaI genomic fragment, immediately 3
of the Sau3AI-SalI fragment was once cloned
into the pBlueScript II, and regenerated by digesting with Asp718. The resulting fragment was blunt-ended, digested with SalI and cloned into the SalI-EcoRV site of DT-A vector (11), generating pSADT-A plasmid. The pSSstopNEO was digested with the
Asp718, blunt-ended and digested with NotI. The resulting
Asp718 (blunt-ended by Klenow)-NotI fragment was cloned into
the SalI (blunt-ended by Klenow)-NotI sites of the pSADT-A,
generating a final targeting vector. The vector plasmid was linearized with NotI and used for electroporation. The expected targeted gene product lacks the COOH-terminal zinc finger clusters
which have been shown to be essential for the DNA binding of
EF1 protein (12). The neor element has a promoter but lacks the
termination and poly(A) addition signals, so that the neor is expressed only when poly(A) addition signal is supplied by recombination with a host gene. A DT-A cassette (11) was placed in the
3
end of the linearized vector for the negative selection against
integration into nonhomologous genes.
Fig. 2.
C-fin
EF1 mutant allele generated by homologous recombination. (A) The last
three exons (6) of the mouse
EF1 gene encoding the homeodomain and the C-proximal zinc
finger cluster are shown (top) together with the targeting vector (middle) and the resulting genomic structure of the homologous recombinant (bottom). Stop codons and the neor cassette are
inserted in the middle of the 6th exon, downstream of the homeodomain in the targeting vector. A DT-A cassette (11) was added at the 3
end of the vector for negative selection against random insertion of the vector. neor, neomycin resistance gene cassette; DT-A, the Diphtheria
toxin A chain expression cassette. The diagnostic BglII fragments detected in Southern blots using the probe (indicated by the thick bar) are shown. Restriction sites of SalI, ApaI, and Sau3AI
in the genomic DNA used for the targeting vector construction are also shown (see Materials
and Methods). (B) Proteins coded by wild-type allele (wt) and the mutated (
C-fin) allele are
schematically shown. (C) DNAs isolated from wild-type mice (+/+), a recombinant ES clone
(A84), and mice heterozygous (+/
) or homozygous (
/
) for the mutant
EF1 gene were
digested with BglII and subjected to Southern blot analysis using the indicated probe. (D) Total RNAs (5 µg each) from wild-type (+/+), heterozygous (+/
), and homozygous (
/
) embryos (12.5 d.p.c.) were analyzed by Northern blotting using a mouse
EF1 cDNA (clone
M12) as probe. Only the larger size mRNA (
EF1+neor) resulting from the insertion of neor
was detected in a homozygous embryo, while only the normal size of
EF1 mRNA was present
in a wild type, and both were in a heterozygous embryo. (E) Nuclear extracts from wild-type
(+/+), heterozygous (+/
) and homozygous (
/
) 12.5 d.p.c. embryos were immunoprecipitated and analyzed by Western blotting for
EF1 and \xc6 C-fin protein using anti-
EF1 antiserum which can react to N-proximal portion of
EF1 (see Materials and Methods). As size references, the nuclear extracts from the COS cells transfected with expression vectors of wild-type
(wt/COS) and
C-fin
EF1 protein (
C-fin/COS) were also electrophoresed in parallel.
[View Larger Versions of these Images (0 + 0 + 0 + 0 + 0K GIF file)]
EF1 allele were
injected into blastocysts from (C57BL/6 × C3H) F1 female
mated with C57BL/6 male, and transferred to ICR pseudopregnant recipient mice. Resulting male chimeras were bred to ICR
female mice to have heterozygous mice. The heterozygous mice
were crossed with ICR or separately with C57BL/6 to keep the
heterozygous pedigrees and intercrossed to generate homozygous
mutant mice. Back-crossing to C57BL/6 has been done for six
generations to date to obtain the mutant animals in C57BL/6 genetic background. Genotypes were determined by PCR analysis of DNA from ear punching (14) or by Southern blot analysis
of tail DNA.
EF1
cDNA containing the sequence from exon 3 to exon 8 (9).
EF1 cDNAs in Cultured Cells.
A cDNA coding for \xc6 C-fin
EF1 protein was constructed by inserting the
XbaI linker with stop codons in the SalI site of exon 6 of fulllength cDNA in the same way as targeting vector construction.
The full-length and \xc6 C-fin
EF1 cDNAs were inserted into the
NotI site of pCDM8 (17) and transfected to COS-7 cells by lipofection as described (18).
EF1.
An antiserum
against human homologue of
EF1, AREB6, was used to detect
the N-proximal region of mouse
EF1. HpaI-NheI (1041-2498) fragment of human AREB6 cDNA (6) was blunt-ended and subcloned into the SmaI site of pGEX-3X to produce a GST-AREB6
fusion protein. An antiserum against this fusion protein, which
cross-reacts to mouse
EF1, was used for Western blot analysis.
,145-2C11; FITC-conjugated anti-Gr-1, RB6-8C5; FITC-conjugated anti-CD25, 7D4; PE-conjugated
anti-Thy 1.2, 53-2.1; PE-conjugated anti-CD4, RM4-5; PEconjugated anti-
/
TCR, H57-597; PE-conjugated anti-Mac-1,
M1/70; PE-conjugated anti-CD44, IM7; Biotin-conjugated antiIgM, R6-60.2, biotin-conjugated anti-c-kit receptor, 2B8. Biotin-conjugated anti-
4 integrin (CD49d), MFR4.B. FITC-conjugated anti-IgD was purchased from Nordic Immunological Laboratories (Capistrano Beach, CA). Three-color analysis for c-kit,
4 integrin, and CD44/CD25 expression was done using
Cy-Chrome-labeled streptavidin (PharMingen) as the third fluorescence dye and analyzed by FACScan® cell sorter.
EF1 antibodies and biotinylated rat monoclonal anti-CD4 and anti-CD8 antibodies, then
with FITC-labeled anti-rabbit Ig and Texas red-conjugated streptavidin anti-rat Ig in TBST containing 10% skim milk with washings by TBS between the steps. Finally, the specimens were mounted in Gelvatol (PBS containing 20% polyvinylalcohol, 20% glycerol, and 2.5% 1,4 diazabycyclo-[2,2,2]-octane) and examined under a microscope.
Expression of EF1 in Embryo and in Lymphoid Organs.
EF1 was isolated by crosshybridization with chicken
EF1 cDNA that was isolated
and characterized in our laboratory (9). Comparison of the
mouse
EF1 sequence with the chicken and other species
revealed that the two zinc finger clusters and the homeodomain are highly conserved (9). Expression pattern of the
mouse
EF1 in the embryos was almost identical to that of
chicken (4): mesodermal tissues (e.g., notochord, somite,
limb bud mesenchyme); neural crest derivatives (e.g., dorsal root ganglia, cephalic ganglia); a part of the central nervous system (hindbrain, motor neurons in the spinal cord). In Northern blot analysis of adult tissues,
EF1 mRNA was
detected in all solid tissues examined (data not shown).
EF1 transcripts were
detected in thymocytes, but not in splenocytes consisting of
only mature T and B cells (Fig. 1 A, lanes 2 and 3). Bone
marrow cells contained a low but detectable level of the
transcripts (Fig. 1 A, lane 1). Immunohistological analysis
showed that
EF1 protein was expressed in most of the
thymocytes including cells stained with the mixtures of
anti-CD4 and anti-CD8 antibodies (Fig. 1 B). Taken together with the results of Northern blot analysis, it was indicated that
EF1 is expressed in some of the bone marrow
cells and in most of the thymocytes including CD4- and
CD8-expressing cells, but once the cells migrate out
EF1
expression is lost.
Fig. 1.
EF1 expression in adult lymphoid tissues. (A) Total RNAs were prepared from splenocytes (lane 1), thymocytes (lane 2), and bone marrow
cells (lane 3) of 8-wk-old wild-type C57BL/6 mice. Each 5-µg RNA sample was analyzed by Northern blotting using a mouse
EF1 cDNA as probe.
The position of the origin of electrophoresis,
EF1 mRNA and ribosomal RNAs are indicated. The same filter was rehybridized for glyceraldehyde-
3-phosphate dehydrogenase (G3PDH) mRNA to control the amount of loaded mRNAs. (B) A section of thymus of 18.5 d.p.c. embryo was doubly
stained with anti-
EF1 antibody (green) and a mixture of anti-CD4 and anti-CD8 antibodies (orange). Note that the majority of the thymocytes had
EF1
in the nuclei, together with CD4/CD8 on cell surface. Bar, 10 µm.
[View Larger Versions of these Images (0 + 0K GIF file)]
EF1 Mutant Mice Which Lack the Zinc Finger Cluster Proximal to COOH Terminus of
EF1 Protein.
EF1 \xc6 C-fin mutant allele, we constructed the targeting vector shown in Fig. 2 A. It was expected that the product of the recombinant gene lacks the
C-proximal zinc finger cluster required for high affinity
DNA binding (Fig. 2 B and see reference 12). Germ-line
male chimeras were produced from homologous recombinant E14 ES cells (e.g., A84 in Fig. 2 C), and heterozygous
mutant animals were generated by crossing these chimeras
with C57BL/6 or ICR female mice. The heterozygous mice appeared normal in growth, fertility, behavior, and
morphology of internal organs. Southern blot analysis of the
yolk sac DNA from the 12.5 d.p.c. embryos generated by
crossing the heterozygous mice showed the set of the hybridizing bands expected for the homozygous embryos
(Fig. 2 C). Then we examined the
EF1 mRNA from the
homozygous mutant embryos (12.5 d.p.c.) and compared them with the littermates of other genotypes. As shown in
Fig. 2 D, transcripts from the mutated allele were longer
than those from wild-type allele as expected from insertion
of the neor sequence. The heterozygous embryos had both
transcripts while the homozygous mutant embryos possessed only the longer transcript. To further confirm the mutation of
EF1 gene by homologous recombination, we
examined the
EF1 protein in the mutant embryos by
Western blot analysis (Fig. 2 E). The mutant protein is expected to be smaller by 40 kD than the wild-type
EF1
protein. It was demonstrated that wild-type and homozygous mutant embryos had only full-length and truncated
forms of
EF1, respectively, while the heterozygous embryos had both. All these observations indicated that
EF1
gene was mutated as designed, and that the truncated form
of the mutant protein was synthesized no less efficiently
than the wild-type form.
EF1 Mutant
Mice.
Fig. 3.
Histology of thymus of EF1 mutant mouse. Thymi of 6-wk-old heterozygous control (A, C) and homozygous mutant (B, D) mice were fixed
in Bouin's fixative and stained with hematoxylin and eosin. The control thymus had developed distinct medulla and cortex (A), while mutant thymi had
uniform parenchyma with light staining as seen in medulla of the control thymus (B). Note also the differences of size and cellularity between them. The
control thymus had a typical cortex which consists of the densely packed and actively proliferating small thymocytes as shown in higher magnification (C), while the mutant thymus seemed to lack its architecture (D). Bars: (A and B) 200 µm; (C and D) 40 µm.
[View Larger Version of this Image (0K GIF file)]
Fig. 4.
Total cell count of lymphocytes in lymphoid organs of EF1
mutant mice in comparison with control heterozygous littermates. Total
lymphocyte numbers in thymi (A), spleens (B), and lymph nodes (C) of
6-11 wk were plotted. Note severe reduction of the total lymphocyte
numbers in the mutant thymi (~100-fold) and lymph nodes (~10-fold)
while the difference in cell number was less pronounced in the spleen.
[View Larger Version of this Image (0K GIF file)]
EF1 mutant of
another allele, Null-LacZ, which lacked almost all of the
coding sequence, also exhibited the reduced size of the
thymi and impaired T cell development at fetal stages as observed in the \xc6 C-fin mutant mice (Takagi T., H. Kondoh,
and Y. Higashi, unpublished results). These observations exclude the possibility that the T cell defect in the homozygous \xc6 C-fin mutant mice is due to the specific effect
of the truncated protein.
EF1
Mutant Mice.
/
TCR+CD3+
cells in the mutant was higher than in the control heterozygous littermate (Fig. 5 B), which was consistent with the
fact that the proportion of SP cells was higher in the mutant mice compared with the control heterozygous littermate as described above.
Fig. 5.
FACS® analysis of thymocytes from a EF1 mutant and a
control heterozygous littermate. Thymocytes from 6-wk-old homozygous mutant (left) and heterozygous littermate (right) were analyzed by staining
with the combination of mAbs: (A) PE-anti-CD4 vs. FITC-anti-CD8;
(B) PE-anti-
/
TCR vs. FITC-anti-CD3 to assess developmental stages
of thymocytes. Numbers in parentheses indicate the percentage of cells
within the quadrant defined by fluorescence of cell surface markers. The
thymocytes were also analyzed by the forward light scattering for the estimation of cell size (C). The histograms for whole or a portion of the thymocytes that were logically gated for the DN, DP, and SP cells in A are
shown with combination of heterozygous (+/
) and mutant (
/
) thymocyte data: abscissa, forward light scattering and ordinate, relative cell number.
[View Larger Versions of these Images (0 + 0K GIF file)]
Fig. 6.
FACS® analysis of splenocytes from a EF1 mutant and a control heterozygous littermate. Splenocytes from 6-wk-old homozygous mutant (left) and heterozygous littermate (right) were doubly stained with the
following combinations of mAbs; (A) PE-anti-Thy1 vs. FITC-anti-B220
to assess fractions of T and B cells; (B) PE-anti-CD4 vs. FITC-anti-CD8
to assess CD4, CD8 expression in T cell population; (C) biotinylated antiIgM plus streptoavidin-PE vs. FITC-anti-B220 and; (D) biotinylated
anti-IgM plus streptoavidin-PE vs FITC-anti-IgD to assess development
of B cells. Numbers in parentheses have the same indication as in Fig. 5.
[View Larger Version of this Image (0K GIF file)]
/
TCR+CD3+ (Fig. 7 C), indicating
full maturity, but the ratio of CD4+CD8
SP to CD4
CD8+
SP cells at the age of 3-8 wk was 5 in the mutant as compared to the ratio of 2 in normal (Fig. 7 B). This bias toward CD4+CD8
cell population tended to be greater in
older mice (e.g., 20 wk, data not shown). In such old mice,
the bias also became evident in the spleen though there
seemed to be no such bias in the spleen of young adult
mice at the age of 3-8 wk (Fig. 6 B). The decrease of the
T cells commonly observed in the peripheral lymphoid organs may be ascribed to the limited supply of T cells from
the thymus.
Fig. 7.
FACS® analysis of lymph node lymphocytes from a EF1 mutant and a control heterozygous littermate. Inguinal lymph node cells from
6-wk-old homozygous mutant (left) and heterozygous littermate (right)
were doubly stained with the following combinations of mAbs; (A) PE-
anti-Thy1 vs. FITC-anti-B220; (B) PE-anti-CD4 vs. FITC-anti-CD8;
(C) PE-anti-
/
TCR vs. FITC-anti-CD3. Numbers in parentheses
have the same indication as Fig. 5.
[View Larger Version of this Image (0K GIF file)]
EF1 \xc6 C-fin mutant and control mice, although
the expressions of both MHC antigens were detected in
medullary region in the control mice while in the whole
region in the mutant mice (data not shown). This difference may suggest and is consistent with the histological observation (Fig. 3) that the small thymi in the mutant mice
lacked the typical cortex comprising the densely packed small
thymocytes, most of which are DP and do not express the
MHC antigens highly.
cpm of the mutant mice was
about one-third of that of the control mice, it seemed that
the decrease in
cpm in the mutant mice was simply due
to reduction of the number of T cells in the spleen cells of
homozygous mutant mice (Fig. 6). The sensitivity to Con
A stimulation and the size of colonies of the proliferating T cells were not different between the control and mutant
mice (data not shown). Thus, functional maturity of the peripheral T lymphocytes accumulated in the
EF1 homozygous mutant mice did not seem to be affected at least in the
responsiveness to the Con A stimulation.
EF1
C-fin Mutant and a Control Heterozygous Littermate
Genotype
Animal*
Con A (
)
Con A (+)
cpm
(+/
)
1
1,280
30,600
29,350
2
900
31,120
30,220
(
/
)
1
560
11,140
10,580
2
200
9,110
8,910
*
Numbers 1 and 2 represent different animals from different litter of 1 and 2, respectively. Animals of the same number belong to the same littermate.
Con A (5 µg/ml) was added to the spleen cell culture in a 96-well microtiter plate (1 × 105 cells/well), and incubated for 24 h. Pulse labeling
with [3H]thymidine (0.5 µCi/ml) was carried out for 12 h. Incorporation of [3H] thymidine in each well was measured using Top-Counter
(Packard). Values represented were average cm of the triplicate samples.
In contrast to T cells, the development of B cells and
myeloid cells was not significantly affected by the EF1
mutation. B cells were comparable in number among the
spleens of homozygous, heterozygous and wild-type littermates. The level of IgM and IgD expression in splenocytes
was normal in the homozygous mutant mice (Fig. 6, C and
D). The population of the B cells and myeloid cells in the
bone marrow was normal as analyzed using B220 (low expression), Mac1 and Gr-1 markers (data not shown). Hematocrit value of the homozygous mutant mice was also
similar to the wild-type animals (data not shown). Thus, the
defects in the
EF1 mutant mice appeared specific to T lymphocytes.
The FACS® analysis of the mutant thymocytes
using CD4 and CD8 markers indicated that, despite the severe decrease of the total cell number, intrathymic development of the T lymphocytes was not arrested at a specific
stage. This is in contrast to the cases of knockout mice
lacking molecules that are essential for the development
and function of T lymphocytes (e.g., TCR- [21], CD3 [22]). We suspected that the T precursor cells at a very
early stage, for instance, before the rearrangement of the
TCR genes, might be affected and decreased in the
EF1
mutant mice, and that a small fraction of the T precursor
cells which escaped from the block by the
EF1 mutation
proceeded to subsequent development.
To address this point, we analyzed the c-kit expression
in the CD4CD8
DN cell population by three-color
FACS® analysis. The early intrathymic T precursor cells
which migrate from the bone marrow express c-kit receptor as other hematopoietic progenitors (23, 24). As shown
in Fig. 8, A and B, only 15% of the CD4
CD8
cell population in the mutant thymus was assigned as c-kit+, while
>50% of CD4
CD8
cells were c-kit+ in the control heterozygous littermate. Taking into account the difference in
the total thymocyte number (Fig. 4) and in the proportion of DN cells (Fig. 5 A), it was concluded that early T cell
precursor was depleted in the thymus of
EF1 mutant mice.
We analyzed further the DN cell population by staining
CD44 and CD25 antigens. It is known that a combination
of expression states of these markers defines a stage of thymocyte development within the DN cell population which
proceeds in the following order: CD44+CD25 CD44+
CD25+
CD44
CD25+
CD44
CD25
(25, 26). The
CD44+CD25
population contains the earliest intrathymic
T precursor cells, but also include the cells of non-T cell
lineages. It is also known that the CD44+ CD25+ cells are
largest in size and express c-kit most highly, while advancement of the cells to CD44
CD25+ and CD44
CD25
stages results in smaller cell size and in lower c-kit expression (26, 27). As shown in Fig. 8 C, CD44+CD25+ cells, which
correspond to the high c-kit+ population was greatly reduced from 12 to 3% in the mutant thymocytes as expected. We also noticed that the relative proportion of
CD25+ cells in DN cell population was reduced at least to
50% of that of control mice.
Then, what gene might be affected in EF1 mutant
mice to cause this severe reduction of early T cell precursors? Given that
EF1 can be a repressor of E2-box sequence (7, 12), genes known to have E2-box sequences in
their promoter region are good candidates as a regulatory
target of
EF1. Among them,
4 integrin gene is especially
interesting since its promoter contains multiple E2-box sequences (28), and it is known to have an essential role in
lymphocyte migration which may include a pathway from bone marrow to thymus (29). Furthermore,
4 integrins have recently been suggested to have some functions
in intrathymic development of T lymphocytes (32, 33).
We thus examined the expression of several cell surface
markers including integrins in the mutant thymocytes to
look for the expression of those proteins affected by EF1
mutation. We found that the expression of
4 integrin
(CD49d) was significantly increased (Fig. 9): two- to threefold increase in
/
TCR
/low/CD3
/low immature cells of
the mutant, and to a lesser extent in
/
TCRhigh/CD3high
more mature cells (Fig. 9).
The data presented in this report demonstrates that EF1
is essential for normal T cell development. In
EF1 mutant
mice, total lymphocyte number in thymus was reduced to
~1% of that of normal mice. The cell populations found in
the thymus of the mutant mice, though reduced in number, represented those of T cells with advanced development, such as CD4+CD8+ DP and CD4+/CD8+ SP cells.
This phenotype is in contrast to the previously reported mutant mice of T cell development, e.g., RAG (34, 35),
TCR-
/
(21, 36), CD3 (22, 37). In these previous cases
where the mutated genes were essential for critical steps of
T cell development, advancement of thymocytes to subsequent stages was blocked resulting in accumulation of the
cells arrested at the critical stages. The total thymocyte
number tended to be smaller as the blocked stage became
earlier. In the RAG-2-deficient mice, for instance, thymocytes were decreased 100-fold as was observed in
EF1
mutant mice, and the existing thymocytes were mostly DN cells (34). The fact that the majority of thymocytes in
EF1 mutant mice expressed CD4, CD8 markers despite the severe reduction in total thymocyte number led us to postulate that the major defect of this mutant lay at a very early
stage, much earlier than that at which the TCRs were required, and a small fraction of the cells that somehow escaped from the
EF1 mutation could go through the maturation stages.
The idea that the EF1 mutation impairs early T cell development was supported by the observation on c-kit+
cells in the DN cell population that are considered to be
the earliest intrathymic T precursor cells migrating from bone
marrow (24). This particular cell population was largely depleted from the mutant thymocytes (Fig. 8 B). Consistent
with this observation, the CD44+CD25+ cells, a subset of
cells in the early stage of the DN cell population expressing
c-kit strongly (26), were also significantly reduced (Fig. 8 C).
The cell size distribution of the mutant DN thymocytes measured by the forward light scattering was shifted to
smaller than control (Fig. 5 C), which is again in agreement
with the observation mentioned above because the c-kit+
DN thymocytes have been shown to have the largest cell
size in the DN cell population (26). Furthermore,
EF1 was
expressed in the thymocytes (Fig. 1 A, lane 2) and in the
bone marrow cells (Fig. 1 A, lane 1), though at a low level
in the latter. All these observations support the idea that
EF1 plays an essential role in the very early stages of T cell
development so that abrogation of
EF1 results in the severe decrease of T cell populations, accompanied by the
significant loss of immature (CD4
CD8
c-kit+) intrathymic
T precursor cells.
It is interesting to note that the recently reported mutant
mice of IL-7 receptor (IL-7R) (38), IL-2 receptor -chain
(IL-2R
) (39), and Jak3 (40), all showed severe reduction in the thymocyte number, and yet produced all DN,
DP, and SP cells, similar to
EF1 mutant mice. Since IL-7R,
IL-2R
, and Jak3 molecules are thought to be essential signaling molecules in expansion of the thymocytes at a very
early stage before rearrangement of the TCR genes, it would
be interesting to see if
EF1 is involved in such an signaling
pathway.
Besides the early stage of T cell development, EF1 may
have additional regulatory roles in the later stage of intrathymic T cell development. The proportion of DN cells
in total thymocytes was higher, and the ratio of DP to SP
cells was lower in the
EF1 mutant mice. It was also noted
that the mutant thymus did not develop medullo-cortical
distinction, probably, due to lack of the typical cortex
which usually consists of the densely packed and actively
proliferating small thymocytes. The defects of
EF1 mutants are different in these two points from those of IL-7, IL-2R
, and Jak3 mutant mice: the proportion of the four
cell populations marked by CD4, CD8 expression are identical to the normal mice and the thymi of those mice develop distinct structures of cortex and medulla, though the
thymi themselves were very small and the thymocyte numbers are ~1% of the normal mice as the
EF1 mutant mice
(38, 39). These differences support the involvement of
EF1 in not only early but also late stages of thymocyte development. Reconstitution of the hematopoietic system in
wild-type host using mutant hemopoietic tissues will clarify
if the defects in early and late T cell development can be ascribed to lymphocytes or stroma.
In peripheral lymphoid tissues the proportion of CD4
CD8+ T lymphocytes relative to CD4+CD8
cells was
significantly reduced from ~0.5 (wild type) to 0.2-0.1 (mutant). In older mice, the majority of the peripheral T cell population was occupied by CD4+CD8
cells (data not
shown). Since the MHC class I and II antigens, which are
required for generation of the functional CD4
CD8+ and
CD4+CD8
SP T cells, were expressed at the normal level
on thymic epithelial cells of the mutant mice (data not
shown), and actually the CD4/CD8 SP T cells were produced in the mutant thymus (Fig. 5 A), survival of mature
CD4
CD8+ cells may be affected in peripheral tissues of
EF1 mutant mice.
An interesting observation was the increase of 4 integrin (CD49d) expression in the mutant thymocyte (Fig. 9).
4
1 integrins are expressed in CD34+ hematopoietic progenitors (43) and regulate lymphocyte attachment and the
homing process in the endothelial microenvironment (30, 31). Thymocytes also express
4
1 integrins (44) and stagedependent regulation of
4 integrin expression in developing thymocytes has been demonstrated (32, 33), suggesting
its possible roles in T cell development. The regulatory region of the
4 integrin promoter has been analyzed and a
negative element effective in lymphocytes has been identified between positions
400 and
300 in the 5
-flanking
sequence (28, 45). The
EF1 binding sequence, CACCT,
is present at
357 in a region conserved between human and mouse (45), which may be the target site of
EF1. It is possible, therefore, that derepression of
4 integrin expression caused by
EF1 mutation may affect homing of T precursor cells or intrathymic development of T cells as reported here.
nil-2-a, which seems to be a truncated form of EF1,
was reported to function as a repressor of the IL-2 gene transcriptional regulation in human T cell jarkat cell line (46).
The impairment of T cell development observed in the
EF1deficient mice could result from the incomplete regulation
of the IL-2 gene expression. Deregulated expression of the
IL-2 gene in transgenic mouse system, however, did not
show any T cell deficiency (47, 48). Moreover, the IL-2
gene knockout mice showed no effects on thymocyte and peripheral T cell subsets (49). Thus,
EF1 mutant phenotype is not explained by altered regulation of the IL-2 gene.
It has been reported that EF1 (and its human homologue, ZEB) is expressed in myeloma and B-lymphoma cell
line (7). This is not totally consistent with our observation
that
EF1 mRNA were not detected in the Northern blot
analysis of the splenocytes. It is possible that expression of
EF1 might be correlated to neoplastic transformation of
B cells, or, alternatively, that
EF1 mRNA in matured B
or T cells are too little to be detected by our Northern blot
analysis using total RNA.
Although we still do not know the reason for neonatal
death observed in part of the \xc6 C-fin mutant mice, it was
rather unexpected that homozygous mutant embryos carrying the \xc6 C-fin allele developed normally except for the
T cell defect, given the fact that EF1 acts as a competitive
repressor against bHLH activator proteins, which are widely
involved in embryogenesis (12). It should be mentioned
that Sna, a mouse homologue of the Drosophila snail and escargot, is expressed at high levels in cephalic neural crest,
limb bud mesenchyme, and somites of 9.5 d.p.c. embryo
and later in a variety of mesenchymal tissues (50), showing
close resemblance to the expression pattern of
EF1. Moreover, consensus binding sequence of Drosophila snail and
escargot proteins contain CACCTG E2 box sequence and
actually these proteins counteracts E2-box-mediated activation by heterodimer of Scute and Daughterless bHLH proteins (51, 52). It is possible that loss of normal
EF1 protein
was compromised by the mouse Sna protein.
In conclusion, T cells, but not other hematopoietic lineage cells, are depleted in EF1 mutant mice although they
are thought to be derived from common stem cells. Possibly,
EF1 has an important role in growth and differentiation of early T cells in the bone marrow, in the homing
process and/or in development in the thymus. To date, the
early T precursor cells have been understood only poorly.
EF1 mutant mice should thus provide a unique tool to study early T cell development from the aspect of transcriptional regulation.
Address correspondence to Yujiro Higashi, Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565, Japan.
Received for publication 29 October 1996 and in revised form 7 February 1997.
1 Abbreviations used in this paper: DN, double negative; DP, double positive; d.p.c., days post coitum; FITC, fluorescein isothiocyanate; HBS, Hepes-buffered saline; SP, single positive.We thank Dr. A. Shimono for his help in the targeting experiments. We also thank Dr. T. Yasui and Mr. K. Yoshida for useful advises on FACS® analysis, and Drs. A. A. Postigo and D. C. Dean for drawing our attention to the 4 integrin promoter.
This work was supported by grants from the Ministry of Education, Science and Culture of Japan to Y. Higashi, T. Takagi, R. Sekido, and H. Kondoh, and from the Science and Technology Agency of Japan to Y. Higashi. T. Takagi and R. Sekido are recipients of a fellowship from the Japan Society for the Promotion of Science for Japanese Junior Scientists.
1. | Robey, E., and B.J. Fowlkes. 1994. Selective events in T cell development. Ann. Rev. Immunol. 12: 675-705 [Medline]. |
2. | Pfeffer, K., and T.W. Mak. 1994. Lymphocyte ontogeny and activation in gene targeted mutant mice. Ann. Rev. Immunol. 12: 367-411 [Medline]. |
3. |
Funahashi, J.-I.,
Y. Kamachi,
K. Goto, and
H. Kondoh.
1991.
Identification of nuclear factor ![]() ![]() |
4. |
Funahashi, J.-I.,
R. Sekido,
K. Murai,
Y. Kamachi, and
H. Kondoh.
1993.
![]() ![]() |
5. |
Kamachi, Y., and
H. Kondoh.
1993.
Overlapping positive
and negative regulatory elements determine lens-specific activity of the ![]() |
6. |
Watanabe, Y.,
K. Kawakami,
Y. Hirayama, and
K. Nagano.
1993.
Transcription factors positively and negatively regulating the Na, K-ATPase ![]() |
7. | Genetta, T., D. Ruezinsky, and T. Kadesch. 1994. Displacement of an E-box-binding repressor by basic Helix-LoopHelix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer. Mol. Cell. Biol. 14: 6153-6163 [Abstract]. |
8. | Franklin, A.J., T.L. Jelton, K.D. Shelton, and M.A. Magnuson. 1994. BZP, a novel serum-responsive zinc finger protein that inhibits gene transcription. Mol. Cell. Biol. 14: 6773-6788 [Abstract]. |
9. |
Sekido, R.,
T. Takagi,
M. Okanami,
H. Moribe,
M. Yamamura,
Y. Higashi, and
H. Kondoh.
1996.
Organization of the
gene encoding transcriptional repressor ![]() |
10. | Kato, K., Y. Takahashi, S. Hayashi, and H. Kondoh. 1987. Improved mammalian vectors for high expression of G418 resistance. Cell Struct. Funct. 12: 575-580 [Medline]. |
11. | Yagi, T., S. Nada, N. Watanabe, H. Tamemoto, N. Kohmura, Y. Ikawa, and S. Aizawa. 1993. A novel negative selection for homologous recombination using diphtheria toxin A fragment gene. Anal. Biochem. 214: 77-86 [Medline]. |
12. |
Sekido, R.,
K. Murai,
J.-I. Funahashi,
Y. Kamachi,
A. Fujisawa-Sehara,
Y. Nabeshima, and
H. Kondoh.
1994.
The
![]() ![]() |
13. | Sawai, S., A. Shimono, K. Hanaoka, and H. Kondoh. 1991. Embryonic lethality resulting from disruption of both N-myc alleles in mouse zygotes. New Biologist 3: 861-869 [Medline]. |
14. | Qi, S.-L., K. Akagi, K. Araki, J.-i. Miyazaki, and K.-i. Yamamura. 1990. Rapid identification of transgenic mice with PCR amplification of DNA from ear punching. Methods Mol. Cell. Biol. 2: 119-122 . |
15. |
Higashi, Y..
1985.
Changes of chromatin conformation around
mouse interferon-![]() |
16. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156-159 [Medline]. |
17. | Seed, B.. 1987. An LFA-3 cDNA encodes a phospholipidlinked membrane protein homologous to its receptor CD2. Nature (Lond.) 329: 840-842 [Medline]. |
18. | Kamachi, Y., S. Sockanathan, Q. Liu, M. Breitman, R. Lovell-Badge, and H. Kondoh. 1995. Involvement of SOX proteins in lens-specific activation of crystallin genes. EMBO (Eur. Mol. Biol. Organ.) J. 14: 3510-3519 [Abstract]. |
19. | Schreiber, E., P. Matthias, M.M. Muller, and W. Schaffner. 1989. Rapid detection of octamer binding protein with "miniextracts", prepared from a small number of cells. Nucleic Acids Res. 17: 6419 [Medline]. |
20. | Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, and H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1: 167-178 [Medline]. |
21. |
Mombaerts, P.,
A.R. Clarke,
M.A. Rudnicki,
J. Iacomini,
S. Itohara,
J.J. Lafaille,
L. Wang,
Y. Ichikawa,
R. Jaenisch,
M.L. Hooper, and
S. Tonegawa.
1992.
Mutations in T-cell antigen
receptor genes ![]() ![]() |
22. |
Malissen, M.,
A. Gillet,
J. Trucy,
E. Viver,
C. Boyer,
F. Kontgen,
N. Brun,
G. Maza,
E. Spanopoulou,
D. GuyGrand, and
B. Malissen.
1993.
T cell development in mice
lacking the CD3-![]() ![]() |
23. | Ogawa, M., Y. Matsuzaki, S. Nishikawa, S.I. Hayashi, T. Kunisada, T. Sudo, T. Kina, H. Nakauchi, and S.I. Nishikawa. 1991. Expression and function of c-kit in hemopoietic progenitor cells. J. Exp. Med. 174: 63-71 [Abstract]. |
24. | Matsuzaki, Y., J.-I. Gyotoku, M. Ogawa, S.-I. Nishikawa, Y. Katsura, G. Gachelin, and H. Nakauchi. 1993. Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation. J. Exp. Med. 178: 1283-1292 [Abstract]. |
25. | Pearse, M., L. Wu, M. Egerton, A. Wilson, K. Shortman, and R. Scollay. 1989. A murine early thymocyte developmental sequence is marked by transient expression of the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 86: 1614-1618 [Abstract]. |
26. |
Godfrey, D.I.,
J. Kenedy,
T. Suda, and
A. Zlotnik.
1993.
A
developmental pathway involving four phenotypically and
functionally distinct subsets of CD3![]() ![]() ![]() |
27. |
Godfrey, D.I.,
A. Zlotnik, and
T. Suda.
1992.
Phenotypic
and functional characterization of c-kit expression during intrathymic T cell development.
J. Immunol.
149:
2281-2285
|
28. |
Rosen, G.D.,
J.L. Barks,
M.F. Iademarco,
R.J. Fisher, and
D.C. Dean.
1994.
An intricate arrangement of binding sites
for the Ets family of transcription factors regulates activity of
the ![]() |
29. | Savangner, P., B.A. Imhof, K.M. Yamada, and J.-P. Thiery. 1986. Homing of hematopoietic precursor cells to the embryonic thymus: characterization of an invasive mechanism induced by chemotactic peptides. J. Cell. Biol. 103: 2715-2727 [Abstract]. |
30. |
Berlin, C.,
R.F. Bargatze,
J.J. Campbell,
V.H. von Andrian,
M.C. Szabo,
S.R. Hasslen,
R.D. Nelson,
E.L. Berg,
S.L. Erlandsen, and
E.C. Butcher.
1995.
![]() |
31. |
Arroyo, A.G.,
J.T. Yang,
H. Rayburn, and
R.O. Hynes.
1996.
Differential requirements for ![]() |
32. |
Sawada, M.,
J. Nagamine,
K. Takeda,
K. Utsumi,
A. Kosugi,
Y. Tatsumi,
T. Hamaoka,
K. Miyake,
K. Nakajima,
T. Watanabe,
S. Sakakibara, and
H. Fujiwara.
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
|
33. |
Salomon, D.R.,
C.F. Mojcik,
A.C. Chang,
S. Wadsworth,
D.H. Adams,
J.E. Coligan, and
E.M. Shevach.
1994.
Constitutive activation of integrin ![]() ![]() |
34. | Shinkai, Y., G. Rathbun, K.P. Lam, E.M. Oltz, V. Stewart, M. Mendelsohn, 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 68: 855-867 [Medline]. |
35. | Mombaerts, P., J. Iacomini, R.S. Johnson, K. Herrup, S. Tonegawa, and V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68: 869-877 [Medline]. |
36. |
Philpott, K.L.,
J.L. Viney,
G. Kay,
S. Rastan,
E.M. Gardiner,
S. Chae,
A.C. Hayday, and
M.J. Owen.
1992.
Lymphoid development in mice congenitally lacking T cell receptor ![]() ![]() |
37. |
Ohno, H.,
T. Aoe,
S. Taki,
D. Kitamura,
Y. Ishida,
K. Rajewsky, and
T. Saito.
1993.
Developmental and functional
impairment of T cells in mice lacking CD3![]() |
38. | Peschon, J.J., P.J. Morrissey, K.H. Grabstein, F.J. Ramsdell, E. Maraskovsky, B.C. Gliniak, L.S. Park, S.F. Ziegler, D.E. Williams, C.B. Ware, et al . 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960 [Abstract]. |
39. | DiSanto, J.P., W. Muller, G.D. Guy, A. Fischer, and K. Rajewsky. 1995. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc. Natl. Acad. Sci. USA 92: 377-381 [Abstract]. |
40. | Nosaka, T., J.M.A. van Deursen, R.A. Tripp, W.E. Thierfelder, B.A. Whitthuhn, A.P. McMickle, P.C. Doherty, G.C. Grosveld, and J.N. Ihle. 1995. Defective lymphoid development in mice lacking Jak3. Science (Wash. DC) 270: 800-802 [Abstract]. |
41. | Russell, S.M., N. Tayebi, H. Nakajima, M.C. Riedy, J.L. Roberts, M.J. Aman, T.-S. Migone, M. Noguchi, M.J. Markert, R.H. Buckley, et al . 1995. Mutation of Jak3 in a patient with SCID: Essential role of Jak3 in lymphoid development. Science (Wash. DC) 270: 797-800 [Abstract]. |
42. | Thomis, D.C., C.B. Gurmiak, E. Tivol, A.H. Sharpe, and L.J. Berg. 1995. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science (Wash. DC) 270: 794-797 [Abstract]. |
43. | Miyake, K., I.L. Weissman, J.S. Greenberger, and P.W. Kincade. 1991. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J. Exp. Med. 173: 599-607 [Abstract]. |
44. | Utsumi, K., M. Sawada, S. Narumiya, J. Nagamine, T. Sakata, S. Iwagami, Y. Kita, H. Teraoka, H. Hirano, M. Ogata, T. Hamaoka, and H. Fujiwara. 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]. |
45. |
Audet, J.F.,
J.Y. Masson,
G.D. Rosen,
C. Salesse, and
S.L. Guerin.
1994.
Multiple regulatory elements control the basal
promoter activity of the human ![]() |
46. | Williams, T.M., D. Moolten, J. Burlein, J. Romano, R. Bhaerman, A. Godillot, M. Mellon, F. J. Rauscher III, and J.A. Kant. 1991. Identification of a zinc finger protein that inhibits IL-2 gene expression. Science (Wash. DC) 254: 1791-1794 [Medline]. |
47. | Ishida, Y., M. Nishi, O. Taguchi, K. Inaba, N. Minato, M. Kawaichi, and T. Honjo. 1989. Effects of the deregulated expression of human interleukin-2 transgenic mice. Int. Immunol. 1: 113-120 [Medline]. |
48. | Akiyama, M., M. Yokoyama, M. Katsuki, S. Habu, and T. Nishikawa. 1993. Lymphocyte infiltration of the skin in transgenic mice carrying the human interleukin-2 gene. Arch. Dermatol. Res. 285: 379-384 [Medline]. |
49. | Schorle, H., T. Holtschke, T. Hünig, A. Schimple, and I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature (Lond.) 352: 621-624 [Medline]. |
50. |
Smith, D.E.,
F. Franco Del Amo, and
T. Gridley.
1992.
Isolation of Sna, a mouse gene homologous to the Drosophila
genes snail and escargot: its expression pattern suggests multiple
roles during postimplantation development.
Development
116:
1033-1039
|
51. | Mauhin, V., Y. Lutz, C. Dennefeld, and A. Alberga. 1993. Definition of the DNA-binding site repertoire for the Drosophila transcription factor SNAIL. Nucleic Acids Res 21: 3951-3957 [Abstract]. |
52. | Fuse, N., S. Hirose, and S. Hayashi. 1994. Diploidity of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot. Genes Dev. 8: 2270-2281 [Abstract]. |