By
From the Basel Institute for Immunology, Postfach CH-4005, Basel, Switzerland
The early growth response (Egr)-1 is a zinc finger-containing transcription factor belonging to
the immediate-early genes. Its expression in CD4/CD8 double negative (DN) immature thymocytes suggests that Egr-1 expression may be involved in early thymocyte development. In
transgenic mice overexpressing Egr-1 in a recombinase-activating gene-deficient background,
thymocytes bypassed the block at the CD25+CD44 DN stage and matured to the immature
CD8 single-positive (ISP) cell stage, but not further to the CD4/CD8 double-positive (DP) cell stage. When these mice were irradiated, thymocytes did develop to the DP stage, suggesting transcriptional induction of additional genes by irradiation that are required to promote
thymocyte development from the ISP to the DP stage. These results provide genetic evidence for two distinct steps during early thymocyte development from the CD25+CD44
DN to the
DP stage. The first step, from the CD25+CD44
DN to the ISP stage, can be entirely promoted by overexpression of Egr-1.
Intrathymic The second phase of thymocyte development begins
with expression of the pre-TCR complex. Several mutant
mice have revealed that expression of the pre-TCR complex plays a key role during subsequent development. Recombinase-activating gene deficient (RAG The molecular events during the second phase of thymocyte development between the two checkpoints for Immediate-early genes, expression of which is rapidly
induced after cell-surface receptor ligation without de novo
protein synthesis (29), are strong candidates for such rapid
response mediators. Many of these genes encode transcription factors such as c-jun and c-fos, as well as the zinc finger-containing early growth response (Egr) family members (30, 31). Of these Egr family members, early growth
response (Egr)-1, also known as krox-24, NGFI-A, zif-268,
and Tis-8, has been studied most intensively for its involvement in T cell function and activation, because of its rapid
expression after TCR ligation (32). However, Egr-1 is
expressed not only in mature T cells, but also in thymocytes. Shao et al. recently reported the expression of Egr-1 in
DN immature thymocytes, suggesting a potential involvement
in In this report, a potential role for Egr-1 expression in
thymocyte development is described. First, endogenous Egr-1
expression in DN thymocytes from wild-type (C57Bl/6)
mice was dissected in detail. Second, by generating RAG-2 Mice.
All mice used here were maintained in the specific
pathogen-free facility of the Basel Institute for Immunology.
Matched sets of littermates with transgene-positive and -negative
genotypes were used in all experiments.
Generation of Transgenic Mice.
A Northern Blot Analysis.
Total RNA was isolated from thymus
using RNAzolB solution (Tel-Test. Inc., Friendswood, TX) either from transgenic mice or negative littermates with a RAG-2+/ Reverse Transcriptase PCR.
Thymocytes were stained for
CD4, CD8, CD25, and CD44, and then appropriate cell populations were sorted by FACstar® (Becton Dickinson, Mountain
View, CA). From 50,000 cells of each population, total RNA
was isolated by RNAzolB solution (Tel-Test, Inc.), and cDNA
was synthesized by reverse transcriptase with random-hexamer
primers. PCR was performed with titrated amounts of this
cDNA as template, using a set of primers: 5 Serological Reagents and Flow Cytometry.
Reagents used for
staining T cells and subsets thereof were as described (39-41, and
references therein). Thymus, lymph node, and spleen cells were
stained with saturating levels of mAbs and analyzed using a FACScan® cytometer.
Cell Cycling Analysis.
100,000 ISP thymocytes were sorted and
fixed in 70% ethanol at 4°C overnight. Cells were collected by
centrifugation and then resuspended in 500 µl of RNase A (0.5 mg/ml in 0.1 M Tris, pH 7.5, 0.1 M NaCl; Sigma Chemical Co.,
St. Louis, MO) and incubated for 30 min at 37°C. 500 µl of pepsin (1 mg/ml in 0.4% HCl; Sigma Chemical Co.) was added,
mixed well, and incubated for 15 min at 37°C. 1 ml of ethidium
bromide (0.02 mg/ml in 0.2 M Tris, pH 8.5, 0.5% BSA) was
then added, mixed well, and incubated for another 15 min at
room temperature with protection from light. After the incubation, the sample was analyzed directly using a FACScan® with the
double discriminating module.
TCR Rearrangement Assay.
100,000 thymocytes were sorted
from various populations. Cells were digested in cell lysis buffer;
1× SSC, 10 mM Tris, pH 7.4, 1 mM EDTA, 0.5% SDS containing protenase K (Sigma Chemical Co.) at 0.1 mg/ml in a 55°C
water bath with vigorous shaking for 2 h. Samples were treated
with phenol/chloroform solution once and then ethanol precipitated. DNA corresponding to 5,000 cells was used as template for
PCR reactions. PCR reactions were performed for 25, 30, 35, and 40 cycles under the following conditions: 94°C for 1 min,
60°C for 1.5 min, 72°C for 1.5 min in a total 20 µl of volume.
Primers used for V Shao et al. recently described Egr-1 expression
in DN thymocytes of mice using intracellular fluorocytometry and suggested the possible involvement of Egr-1 expression during
Pre-TCR-mediated
signals are necessary to promote development from the
CD25+CD44 The Lck proximal promoter was used to limit transgene
expression primarily to immature thymocytes (37, 43). A
mouse genomic DNA fragment of the Egr-1 gene, including coding exons and intron 1 but no 3 The thymi of Egr-1 transgenic
mice with a RAG-2
DN and ISP cells from transgenic thymi were sorted,
and transgene expression in each population was confirmed
by RT-PCR. To avoid amplification of endogenous Egr-1
transcripts, a set of primers specific for the hGH gene sequence present in transgene transcripts was used. Transgene
expression was detected in either CD25+CD44 As discussed above, once
the To investigate this question, both Egr-1 Tg-RAG In the thymus of Egr-1 Tg- RAG In both types of mice, no mature CD4+ or CD8+ cells
were detected, which is consistent with previous reports
regarding irradiated RAG Seeing a significant population of ISP (and ISP/
DP intermediate) cells in Egr-1 Tg-RAG As shown in Fig. 4, essentially the same percentage of
ISP cells from transgenic and negative littermate mice were
in S or G2/M phase, indicating that cell cycling was not altered by Egr-1 overexpression.
An alternative explanation for the
large population of ISP (and ISP/DP intermediate) cells in
irradiated Egr-1 Tg-RAG
As previously discussed, pre-TCR independent development of thymocytes in RAG The initial observation that endogenous Egr-1 expression is far upregulated in CD25 Despite these results which implicate involvement of
Egr-1 in early thymocyte development, Egr-1 Thus, although expression of Egr-1 is not necessary for To our surprise, though thymocytes
overexpressing Egr-1 bypassed the One might argue that the signals mediated by TCR- The presence of a large population of ISP (and ISP/
DP intermediate) cells in Egr-1 Tg-RAG Newly generated Egr-1 transgenic mice
with a RAG-2/
T cell development can be grossly separated into three phases based on the configuration of
TCR genes and the expression of cell-surface markers including the CD4 and CD8 coreceptors. In the earliest
phase, thymocytes are characterized by TCR loci in germline configuration and thus, no surface expression of any TCR protein. The most immature cells identified in this
first phase are present in small numbers and express c-kit
and low levels of CD4 (1). Thymocyte maturation is accompanied by loss of CD4 and onset of CD44 expression,
immediately followed by CD25 expression (the CD25+CD44+
CD4/CD8 double-negative [DN]1 stage) and finally, down-regulation of CD44 and c-kit expression (2). This final maturation stage of the first phase is defined as the CD25+CD44
DN stage, during which rearrangement of TCR-
gene
begins and functional TCR-
chains are expressed on the
cell surface associated with the pre-TCR-
chain (3) as a
pre-TCR complex. Several cytokine and cytokine receptor-deficient mice have implied that this first phase of thymocyte development appears entirely dependent on growth
factors. IL-7/IL-7 receptor (4, 5), IL-2 receptor-
(common
chain) (6), and c-kit (9) -deficient mice revealed a
mild, but not complete, block of early thymocyte development. More recently, Rodewald et al. reported a complete
block before TCR-
gene rearrangement in c-kit/common
chain double mutant mice (10).
/
) mice in which
TCR-
genes cannot be rearranged and hence no pre-TCR complex is expressed on the cell surface, have complete block of thymocyte development at the CD25+CD44
stage
(11, 12). In addition, TCR-
/
(13) and pre-TCR-
/
mice (3) show greatly reduced numbers of CD4+CD8+
double-positive (DP) cells. This first checkpoint regulated
by the pre-TCR complex is termed
selection (14).
When the pre-TCR complex is expressed and its signaling
is initiated, thymocytes lose CD25 expression and, in mice,
most start to express CD8 molecules. After this immature
CD8 single-positive (ISP) stage (15), thymocytes begin
to express CD4 molecules and become CD4/CD8 DP
cells. During this process, TCR-
genes undergo rearrangement, and the TCR
/TCR
(TCR-
/
) complex
is expressed on the cell surface. The second checkpoint for
the final steps of thymocyte maturation is positive/negative selection, which depends on the interaction between TCR-
/
and MHC complexes, but is also influenced by the
CD4 and CD8 coreceptors. Cells surviving selection can
achieve development to mature to CD4 or CD8 single-positive cells, exit the thymus, and enter the periphery. Although these developmental processes are well described phenotypically, the molecular mechanisms involved in developmental control is still unclear.
selection and TCR-
/
selection are elusive because a
possible ligand for the pre-TCR complex, which plays a key
role in this period, has not been identified. Although a recent report suggested CD81, a transmembrane 4 superfamily protein, as a potential candidate for the pre-TCR ligand
(19), this is unlikely because CD81-deficient mice (20, 21)
have normal thymocyte development. Alternatively, this issue has been studied intensively using RAG
/
mice. Several experimental manipulations of the mutant mice either by
irradiation (22, 23), CD3 ligation (24, 25) as well as introduction of activated lck (26) or activated ras (27) transgenes
effectively bypass
selection and promote thymocyte development to the DP stage in the absence of pre-TCR expression. All of these stimuli somehow compensate for pre-TCR-
mediated signaling. Because all of these manipulations can
activate the Ras protein (28), a ras-related pathway could
play an important role in
selection as well as in further
developmental progression to the DP stage. Downstream of
Ras, however, multiple pathways may be involved which have
not yet been dissected. Intracellular signals during this rapid
developmental process must be differentially controlled, and
rapidly responding transcriptional regulators able to elicit a
cascade of changes in gene expression should be necessary.
selection (36). Nevertheless, very little functional information is available concerning the possible involvement of
Egr-1 in thymocyte development.
/
transgenic mice that overexpress Egr-1 in immature
thymocytes, the functional effects of Egr-1 expression on
selection as well as further development of thymocytes
were evaluated.
clone containing a 16-kb
genomic DNA fragment including the whole Egr-1 sequence
(clone GE-1) was isolated by screening a genomic DNA library
from 129/SVJ mice (Stratagene Corp., La Jolla, CA) with an Egr-1
cDNA fragment cloned into the EcoRI site of pBluescript (32P-labeled Egr-1 cDNA fragment: positions 121-1,970, numbering according to ref. 33). A 2.3-kb ApaI-BglII fragment was subcloned and then the 5
end ApaI site was modified to a BamHI
site by T4 DNA polymerase and a BamHI linker. The BamHI-
BglII fragment was cloned into the BamHI site of the plck-human
growth hormone (hGH) expression vector (37). The complete plasmid (plck-Egr) was digested with NotI and the transgene fragment
no longer containing vector sequence was purified by Geneclean II
(BIO101, Vista, CA). DNA was microinjected into fertilized eggs
of C57Bl/6 × DBA2 F2 mice (BDF2). Resulting founders were
screened for transgene by Southern blot and PCR. Three independent founders carried the transgene. All three mice were
backcrossed within RAG-2
/
background.
background. 15 µg total RNA was denatured, electrophoresed on 1% agarose gel, blotted on nylon membrane, and
then hybridized with 32P-labeled Egr-1 cDNA fragment.
-TGCTCCTGGCTTTTGGCCTGCTCTG (positions 26-50 of the hGH
cDNA sequence, from ATG, the start codon) and 5
-GTTGTGTGAGTTTGTGTCGAACTT (positions 534-511) for transgene expression, 5
-GCAGATCTCTGACCCGTTCGG (positions 300-320 of the mouse Egr-1 cDNA, according to ref. 33)
and 5
-CCGAGTCGTTTGGCTGGGATA (positions 630-610)
for Egr-1 expression, 5
-ATGGATGACGATATCGCTGCG (positions 80-101, regarding ref. 38), and 5
-CATGTTCAATGGGGTACTTCA (positions 300-280) for
actin expression. PCR
reactions were performed as described; 10% of each reaction was
analyzed on a Southern blot with either an hGH, Egr-1, or
actin
cDNA probe.
rearrangement detection were 5
-CAGAAGGTGCAGCAGAGCCCAGAA (V
5H), 5
-ACTGTCTCTGAAGGAGCCTCTCTG (V
2C), 5
-ACCCAGACAGAAGGCCTGGTCACT (V
F3), and 5
-GACCCTATTACTCACATACTTGGCTTG (V
TT11); and for V
rearrangement
detection were, 5
-CCTGATTGGTCAGGAAGGGC (V
6),
5
-TCCCTGATGGGTAGAAGGCC (V
8), and 5
-TAACACGAGGAGCCGAGTGC (J
2.5), as described (42). 10% of each
reaction was analyzed on a Southern blot with a 32P-labeled internal oligo as a probe, for V
, 5
-GAAAGCAGAGTCCCAATTCCAAAG, for V
, 5
-CTGGCCCAAAGTACTGGGTG. As
a control, the
actin gene was amplified from each template using primers 5
-TTGAGACCTTCAACACCCCAG (positions
450-471, according to ref. 37) and 5
-CGAAGTCTAGAGCAACATAGC (positions 750-730), and then hybridized with a
actin cDNA fragment.
Endogenous Egr-1 Expression in DN Thymocytes of Wild-type Mice.
selection in immature thymocytes (36). Because the pre-TCR-expressing population in DN thymocytes
can be distinguished by CD25/CD44 expression, endogenous Egr-1 expression was dissected in DN thymocytes from
wild-type C57Bl/6 (B6) mice by semiquantitative reverse
transcriptase PCR (RT-PCR), using sorted CD25+CD44
cells that precede pre-TCR expression, and CD25
CD44
cells that express the pre-TCR complex. As shown in Fig.
1, much higher endogenous Egr-1 expression was detected
in CD25
CD44
cells than in CD25+CD44
cells. Egr-1
was expressed at even higher levels in DP cells than in CD25
CD44
DN cells, consistent with the recent report (36).
Fig. 1.
Increased endogenous Egr-1 expression in
CD25CD44
DN thymocytes
of wild-type (C57Bl/6) mice.
CD25+CD44
DN, CD25
CD44
DN, and DP thymocytes
were sorted from C57Bl/6 (B6)
mice, total RNA was isolated,
and the expression of Egr-1 gene was assessed by semiquantitative RT-PCR. The amount of template cDNA for PCR corresponds to 5 × 104,
5 × 103, or 103 cells from the left lane to the right in each type of cell.
[View Larger Version of this Image (46K GIF file)]
/
Background.
to the CD25
CD44
stage as revealed in
several mutant mice. For instance, in RAG-2
/
mice
lacking pre-TCR expression, thymocyte development is
blocked at the CD25+CD44
stage (12). The increased expression of Egr-1 seen in CD25
CD44
DN thymocytes
(Fig. 1) might reflect induction of Egr-1 expression by pre-TCR engagement, which may help to promote development of DN thymocytes from the CD25+CD44
to the
CD25
CD44
stage. To test this hypothesis, transgenic
mice designed to overexpress Egr-1 in immature thymocytes were generated and bred to RAG-2
/
mice (12).
noncoding sequence was used to insure efficient and stable expression of
transcripts (44, 45). Three independent lines of transgenic
founder mice were obtained. Phenotypes of these original
transgenic mice will be detailed elsewhere (Miyazaki, T.,
and U. Müller, manuscript in preparation). All three lines were backcrossed to RAG-2
/
mice. All progeny carrying
the transgene were of normal size and appeared healthy. As
observed for many other transgenes driven by this promoter (43), transgene expression in the thymus was detected at levels far exceeding the endogenous Egr-1 when
analyzed by Northern blotting using thymic RNA from
transgenic and negative littermate mice, both of which had
a RAG-2+/
background (data not shown). All three lines
showed essentially the same expression levels.
/
background (Egr-1 Tg-RAG
)
were significantly larger than those of RAG-2
/
negative
littermates (NL-RAG
). The absolute number of thymocytes was 3.5 (± 0.5) × 106 in Egr-1 Tg-RAG
(n = 10), compared to 1.2 (± 0.3) × 106 in NL-RAG
(n = 10). Typical staining profiles of thymocytes are presented in
Fig. 2 a. The most impressive aberration in the transgenic thymi was the presence of a large population (30%) of
CD4
CD8+ cells. These CD8+ cells are CD25-negative (Fig.
2 b) and display lower heat-stable antigen (HSA) levels than
those on DN cells, but higher levels than those on DP cells
(Fig. 2 c), indicating that they are ISP transitional stage thymocytes (15). Consistently, size profiles of total thymocytes
show that smaller cells appeared (Fig. 2 d) and a significant
population (18%) of CD25
CD44
DN cells was detected
in Egr-1 Tg-RAG
thymi (Fig. 2 e). Thus, overexpression
of Egr-1 promoted thymocyte development from the DN
to the ISP stage, overcoming the
block in the RAG-2
/
background. In accordance with this, small numbers of ISP
cells appeared to express CD4 also, falling into the CD4/
CD8-DP quadrant (Fig. 2 a). These cells form, however, a
very small (2%), indistinct population, possibly representing
the intermediate stage cells between the ISP and the DP.
Neither mature single-positive thymocytes nor peripheral
T cells were detected in either Egr-1 Tg-RAG
or NL-RAG
mice. ISP cells isolated from Egr-1 Tg-RAG
could not develop to the DP stage spontaneously in in vitro
culture for 12-24 h (data presented in following section as
Fig. 3 d).
Fig. 2.
Development of ISP cells induced by Egr-1 expression in
RAG-2/
mice. Thymocyte suspensions from 6-8-wk-old transgenic
mice or negative littermates, both with a RAG-2
/
background, were
stained and analyzed by fluorocytometry. (a) CD4/CD8 profiles. Numbers indicate relative percentages of positive cells within a quadrant. The
average of the total thymocyte numbers of 10 of each type of mice are indicated above the profiles. (b) CD25 expression of total thymocytes from
transgenic or negative littermates (top) or CD8+ cells from transgenic
mice. (c) HSA histograms. (d) Side scatter (SSC)/forward scatter (FSC)
profiles indicating cell size. Only living cells were gated. (e) CD25/CD44
profiles of DN cells. Thymocytes were stained with anti-CD25-Tricolor,
anti-CD44-FITC, anti-CD4-PE, and anti-CD8-PE. PE-negative cells
(CD4/CD8 DN cells) were gated and analyzed for CD25/CD44 expression. Numbers indicate relative percentages of positive cells within a
quadrant.
[View Larger Versions of these Images (39 + 21 + 42K GIF file)]
Fig. 3.
Development of DP cells induced by irradiation. Both transgenic mice and negative littermates with a RAG-2/
background were
sublethaly irradiated (600 rads). 4 wk after irradiation, their thymocyte
suspensions were stained and analyzed by a fluorocytometer. (a) CD4/
CD8 profiles. Numbers indicate relative percentages of positive cells
within a quadrant. The average of the total thymocyte numbers of seven
of each type of mice are indicated above the profiles. (b) CD25 expression
on total thymocytes. (c) Peanut-agglutinin binding capacity of each subpopulation. (d) In vitro culture of ISP cells. 50,000 ISP cells were sorted
from irradiated or nonirradiated transgeic mice with a RAG-2
/
background and cultured in 200 µl of complete culture medium for 12-24 h.
After the culture period, cells were stained for CD4 and CD8, and analyzed by fluorocytometer. This experiment was repeated three times and
the results were reproducible. A representative result is shown.
[View Larger Versions of these Images (35 + 28K GIF file)]
, CD25
CD44
, or ISP cells (data not shown).
/
Background.
block is bypassed in a RAG
/
thymus, either by irradiation, CD3-ligation, or by expression of activated lck
or ras transgenes, thymocytes do mature to the DP stage,
whereas in Egr-1 Tg-RAG
mice, the vast majority of
thymocytes develop to the ISP stage, but not to the DP
stage. Two potential explanations can be considered for
this difference. First, overexpression of Egr-1 is sufficient to
drive development of DN thymocytes to the ISP stage, but
other gene(s) might be required to promote ISP cells to the
DP stage. Second, overexpression of Egr-1 at ISP stage
might interfere with further development of ISP cells towards the DP stage.
and
NL-RAG
mice were sublethally irradiated (600 rads) and
their thymi were examined. As observed previously (22),
the thymic cellularity in both Tg and negative littermates
was strikingly decreased 2-3 d after irradiation. Most of the
thymocytes were dead when examined by trypan blue exclusion (data not shown), and very few living ISP cells
were detected in irradiated Tg thymi by fluorocytometric analysis (data not shown). 4 wk after irradiation, however,
thymi were significantly enlarged as compared to nonirradiated thymi both in Tg and negative littermates, and the
absolute numbers of thymocytes were 1.8 (± 0.3) × 107 in
Tg versus 1.0 (± 0.2) × 107 in negative littermates (n = 7 each). As shown in Fig. 3 a, in both types of mice, an obvious DP population appeared which was 1.2 (± 0.2) × 107
cells in Tg and 0.8 (± 0.1) × 107 cells in negative littermates. Large numbers of total thymocytes both in Tg and
negative littermates did not express CD25 as shown in Fig.
3 b. These results indicate that overexpression of Egr-1 does not block thymocyte development from the ISP to
the DP stage and, therefore, perhaps additional gene(s) induced by irradiation are required to drive ISP cells to the
DP stage.
4-wk after irradiation, a substantial population (20%) of CD4
/lowCD8+ cells
was detected. This population differs from the ISP cells in
nonirradiated Egr-1 Tg- RAG
thymi, since the ISP cells
disappeared soon after irradiation. However, these cells are
apparently repopulated ISP and ISP/DP intermediate stage
cells because their peanut agglutinin-binding capacity
which is reduced as thymocytes mature, is even higher than
that of DP cells (Fig. 3 c), indicating that these CD4
/low
CD8+ cells are more immature than DP cells. This is also
supported by the observation that these CD4
/lowCD8+
cells bear higher levels of HSA than do DP cells, but lower
levels than do DN cells (data not shown). In addition, ISP
cells isolated from irradiated Egr-1 Tg-RAG
mice spontaneously mature to the DP stage in 12-24 h of culture (Fig.
3 d), indicating these immature cells have the potential to
mature into DP cells, in sharp contrast to ISP cells from
nonirradiated Egr-1 Tg-RAG
mice which do not develop to the DP cell stage in identical culture conditions
(Fig. 3 d).
/
mice (22, 23). TCR V
and
V
rearrangements were examined in each sorted population and, as expected (22, 23), no rearrangement could be
detected in any thymocyte population, either from irradiated transgenic or negative littermates with a RAG-2
/
background (data not shown).
mice even after
irradiation, which have the potential to further develop to
the DP stage, I wondered if overexpression of Egr-1 might
lead the ISP cells to be hyperproliferative, resulting in enlargement of this population in the profiles. To asses this question, the cell cycle of ISP cells was determined. To
compare the fraction of cycling cells between Egr-1 overexpressing ISP and normal ISP cells, RAG+ background
Tg (Egr-1 Tg-RAG+) and NL-RAG+ mice were used. In
RAG+ background animals, ISP cells can be distinguished
as CD8+ TCRlow. These cells were sorted from Egr-1 Tg-RAG+ and NL-RAG+ mice, and their cell cycling condition was examined by DNA content .
Fig. 4.
Normal cell cycling in ISP cells of transgenic mice. ISP
(CD8+TCRlow) thymocytes from transgenic mice or negative littermates
with a RAG-2+/+ background were sorted and their DNA content was
determined. The high peaks at ~75 of relative intensity represent the G1
phase DNA and the other smear part at ~200 represents the S or G2/M phase DNA. The amount of S or G2/M phase DNA was essentially same in transgenic mice and negative littermate controls.
[View Larger Version of this Image (22K GIF file)]
thymi is that the high level of
Egr-1 expression might boost the thymocyte maturation
from the DN to the ISP stage. This question was assessed
by comparing the CD25/CD44 profiles of DN cells in both types of mice 4 wk after irradiation. Fig. 5 shows histograms of CD25 expression in CD44
DN thymocytes.
In irradiated NL-RAG
thymi, two clear populations
(CD25
and CD25+ ) could be identified and seven (± 2.0; n = 8) percent of CD44
DN cells exhibited the
CD25
phenotype. In irradiated Egr-1 Tg-RAG
thymi,
however, CD25
and CD25+ populations were not clearly
separated, and a large population of intermediate (CD25low
CD44
) cells bearing broadly differing levels of CD25
were observed. Using the same gate as for the negative littermate cells, CD25
cells were 22 (± 1.6; n = 8) percent
of CD44
DN cells. In addition, the relative peak intensity
of the CD25+ population was 1.7 times higher in negative
littermates than in Tg mice, indicating that many of the
CD25+CD44
cells had shifted to the CD25
CD44
stage. This shift was detected even in nonirradiated Egr-1
Tg-RAG
DN cells (see Fig. 2 e). These results suggest
that the maturation may be boosted in Egr-1 Tg DN thymocytes. An increase in the number of CD4
CD8low in irradiated Tg thymi (6 [± 0.8] × 105 in Tg versus 1.7 [± 0.5] × 104 in negative littermate thymus [n = 8 each]),
might also reflect the boost also during maturation from the
CD25
CD44
DN to the ISP cell stage.
Fig. 5.
More CD25/lowCD44
DN cells in irradiated transgenic
thymus than in irradiated negative littermate thymus. Thymocyte suspensions from either irradiated transgenic or irradiated negative littermate mice 4 wk after irradiation were stained for CD4, CD8, CD25, and CD44 and analyzed by fluorocytometry. CD44
DN cells were gated and
their CD25 expression is presented. Percentages of the CD25-negative
population (the gate for which was created using the negative littermate's
histogram as a standard) are indicated.
[View Larger Version of this Image (28K GIF file)]
Egr-1 Overexpression Is Sufficient to Promote Development of
CD25+CD44 DN Cells to the CD25
CD44
DN and
Further to the ISP Stage.
/
mice has been achieved by various experimental manipulations, including irradiation, CD3 ligation, and introduction
of several transgenes. These stimuli are thought to compensate for signaling through the pre-TCR complex, though
the precise molecular mechanisms are unknown. Because
all of these manipulations can activate Ras (28), activation
events downstream of the Ras pathway might be strong
candidates for key players in promoting thymocyte development beyond
selection. We are, however, entirely ignorant of the gene regulation/transcription involved in this
maturation control. In this report, an immediate-early
gene, Egr-1, is presented as a primary transcription factor,
overexpression of which does initiate thymocyte development in RAG-2
/
mice.
CD44
DN cells in comparison to the immediately preceding CD25+CD44
DN
population, suggested that Egr-1 might be induced after ligation of the pre-TCR complex and a set of genes which
are transcriptionally regulated by Egr-1 could possibly play
key roles in promoting CD25+CD44
DN cells to develop
to the CD25
CD44
stage. This idea was well supported
by transgenic mice overexpressing Egr-1 with a RAG-2
/
background in which CD25+CD44
DN cells achieved
maturation to the CD25
CD44
and further to the ISP
stage. Because several reports have indicated that Egr-1 expression is upregulated by irradiation (46) and ras activation
(47, 48), the observation in these transgenic mice is consistent with the previously reported ras-related promotion of
thymocyte development in RAG
/
mice.
/
mice
showed no defect in thymocyte development (49; my unpublished results). Potential compensation for the role of
Egr-1 by other Egr family members, such as Egr-2, -3, and
-4, which are reported to be expressed in the thymus (36),
could explain normal thymocyte development in Egr-1
/
mice. Because the sequence of the zinc finger DNA-binding site is highly conserved in all members of the Egr family
(29), the set of expressed genes that are required to promote DN thymocyte development could be induced by
other members of the Egr family, even in the absence of
Egr-1.
selection, overexpression allows thymocytes to overcome
selection and mature to the ISP stage.
block and developed
to the ISP stage in a RAG
/
background, these cells could
not further mature to the DP stage. All other manipulations
reported that induce thymocyte development in RAG
/
mice drive thymocyte maturation past the
block to the
DP stage. However, irradiation did promote maturation of
DP cells in Egr-1 Tg-RAG
, demonstrating that overexpression of Egr-1 itself does not impair thymocyte maturation from the ISP to the DP stage. Thus, transcriptional regulation by Egr-1 is sufficient to promote maturation of
thymocytes from the CD25+CD44
DN to the ISP stage,
but additional regulations that can be induced by irradiation are necessary to drive ISP cells to the DP stage. Several
immediate-early genes other than Egr-1, such as c-jun and
c-fos, are upregulated by irradiation (28, 46). In addition, p53 expression, which can rescue both the rearrangement
of the TCR-
locus and thymocyte development to the
DP stage in scid mutant mice is also induced by irradiation
(50), though induction of rearrangement is clearly not a
factor in the RAG-2
/
background animals. On the other
hand, null-mutant mice of an high-mobility group box
protein family member, T cell factor 1, revealed a maturation block of thymocytes at the ISP stage, and this protein
appeared indispensable for thymocyte development from
the ISP to the DP stage (51). Though it is not known
whether T cell factor 1 is induced by irradiation, it is a potential candidate as a key player in this developmental process. Further studies are required to identify the responsible
gene(s) expression for thymocyte maturation from the ISP
to the DP stage.
/
instead of pre-TCR, might be responsible for the development from ISP to DP cells, because the rearrangement of
chain becomes active and low levels of TCR-
/
expression can be detected on the cell surface at the ISP stage. It
appears unlikely, however, because thymocytes do develop
to the DP stage even in TCR-
/
mice (13).
mice after irradiation revealed an unexpected effect of Egr-1 overexpression on immature thymocytes. These cells have the potential to develop to the DP stage, and it is unlikely that there
is a distinct population that is resistant to induction of maturation by irradiation. In addition, this phenotype was not
due to the hyperproliferation of ISP cells because cell cycling was not affected by overexpression of Egr-1. The
shifted balance of CD25+CD44
versus CD25
CD44
DN cell populations and increased number of CD4
CD8low
cells in irradiated Tg thymi suggest that overexpression of
Egr-1 may boost thymocyte maturation from CD25+
CD44
DN to the ISP stages, resulting in an enlarged ISP
population in the thymic profiles. The precise mechanism
of this boost effect by Egr-1 overexpression is unclear.
However, not only CD25 downregulation and CD8 upregulation are affected, because these changes were accompanied by reduction of cell size and HSA downregulation. In addition, Egr-1-binding domains have not been identified either in CD8 or CD25 gene promoter regions. Recently, Eibel and colleagues have generated transgenic mice
which overexpress Egr-1 in B cell lineage under the control of immunogloblin promoter/enhancer. In these transgenic mice, B cell maturation seems to be advanced (Eibel,
H., personal communication). This result appears analogous with the boosted thymocyte maturation in our transgenic
mice.
/
background provide several novel insights, two of which are worth reemphasizing. First, overexpression of Egr-1 is sufficient to promote the development of CD25+CD44
DN thymocytes to the ISP stage,
beyond
selection. Second, there are two distinct steps
regulating thymocyte maturation between the DN and DP
stages, the turning point of which appears to be at the ISP
stage. Egr-1 transgenic mice may provide a model for further dissection of the gene regulation involved in early thymocyte development.
Address correspondence to Toru Miyazaki, Basel Institute for Immunology, Grenzacherstrasse 487, Postfach CH-4005, Basel, Switzerland. Phone: 41-61-605-1294; FAX: 41-61-605-1364; E-mail: miyazaki{at}bii.ch
Received for publication 7 May 1997 and in revised form 3 July 1997.
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