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HEMATOPOIESIS
From the Institut Cochin, Département
d'Hématologie, INSERM, CNRS, Paris, France; the Department of
Cell Biology and Genetics, Erasmus University, Rotterdam, The
Netherlands; and the Département d'Hématologie,
Hôpital Henri Mondor, Créteil, France.
The tal-1 gene encodes a basic helix-loop-helix (bHLH)
transcription factor required for primitive and definitive
hematopoiesis. Additionally, ectopic activation of the
tal-1 gene during T lymphopoiesis occurs in numerous cases
of human T-cell acute lymphoblastic leukemia. With the use of
transgenic mice, we show that, in adult hematopoiesis, constitutive
expression of TAL-1 protein causes disorders in the hematopoietic
lineages that normally switch off tal-1 gene expression during their differentiation process. Myelopoiesis was characterized by
a moderate increase of myeloid precursors and by Sca-1 antigen persistence. Although no lymphoid leukemia was observed, T
lymphopoiesis and B lymphopoiesis were severely impaired. Transgenic
mice showed reduced thymic cellularity together with a decrease in
double-positive cells and a concurrent increase in the single-positive
population. B cells exhibited a differentiation defect characterized by
a reduction of the B-cell compartment most likely because of a
differentiation block upstream of the intermediate pro-B progenitor. B
cells escaping this defect developed normally, but transgenic
splenocytes presented a defect in immunoglobulin class switch
recombination. Altogether, these results enlighten the fine-tuning of
TAL-1 expression during adult hematopoiesis and indicate why TAL-1
expression has to be switched off in the lymphoid lineages.
(Blood. 2002;100:491-500) The tal-1 gene (also called
scl) has been implicated in the development of 40% of cases
of childhood T-cell acute lymphoblastic leukemia via chromosomal
translocations or interstitial deletions and via unknown mechanisms in
the absence of detectable chromosomal alterations. Animal models have
demonstrated that TAL-1 can induce T-cell leukemia,1-6 but
this ability is likely to depend on the regulatory sequences used to
generate the transgenic mice, and, in most cases, the need for
additional oncogenic events has been demonstrated.
In addition to its implication for T-cell leukemogenesis, TAL-1 is
present during normal adult hematopoiesis, and its expression has been
shown in progenitors, mature erythroid cells, megakaryocytes, mast
cells, and early myeloid cells.7-9 Its essential role in normal hematopoiesis has been demonstrated by gene inactivation studies, showing that the disruption of the tal-1 gene
induces early embryonic mortality at 9 to 9.5 days of gestation.
Embryos with a null mutation of the tal-1 gene were pale,
experienced growth retardation, and failed to develop any hematopoietic
cells both in the yolk sac and the embryo.10,11 In vitro
differentiation cultures and in vivo chimeric mice using
tal-1 TAL-1 protein belongs to the basic helix-loop-helix (bHLH) family of
transcription factors13 that contain a basic domain allowing DNA biding, and a helix-loop-helix domain responsible for the
protein-protein interactions. Members of the bHLH family are grouped
into 3 classes, depending on their tissue specificity and function.
TAL-1 protein belongs to the tissue-restricted bHLH class II
transcription factors shown to play important roles in cell type
determination and differentiation. TAL-1 DNA binding depends on its
heterodimerization with widely expressed bHLH class I factors E12/E47,
E2-2, and HEB. The third class of the bHLH factors contains the Id
proteins that lack the basic DNA-binding domain and, therefore, may act
as negative regulators by sequestering bHLH class I and II factors in
protein complexes.14,15 However, although class I and II
bHLH factors were thought to require their DNA binding domain for their
function, a study has shown that a mutated TAL-1 protein lacking the
basic domain can rescue hematopoiesis of
tal-1 The Ly-6E.1 and Ly-6A.2 genes are highly
homologous strain-specific alleles of the mouse belonging to the
Ly-6 multigene family. During hematopoiesis, the
Ly-6E/A gene is expressed in the hematopoietic stem cells
(HSCs) of the adult mouse bone marrow (BM) and fetal liver as well as
in multipotent progenitors and mature T and B lymphocytes.17 Transgenic mice studies, using a
Ly-6E.1-LacZ construct, have shown that a 14 kilobase (kb)
Ly-6E.1 genomic fragment is sufficient to recapitulate
endogenous Sca-1 gene expression.18 Thus, we
used these Sca-1 gene regulatory sequences to study the effects of forced hTAL-1 expression in hematopoietic cells and to
understand why tal-1 gene expression is switched off in
specific hematopoietic lineages.
DNA constructs and transgenic mice
Northern analysis
Western blot analysis Cellular extracts of spleen, thymus, kidney, and Western blotting were performed as previously described20 by using the mouse anti-TAL-1 monoclonal antibody BTL-136 that detects murine and human TAL-1,9 the mouse anti-hTAL-1 monoclonal antibody BTL-73 specific for the human protein,9 and a rabbit antimouse horseradish peroxidase-linked antibody (Promega, Madison, WI). Immunoblots were then developed by using electrochemiluminescence (Amersham, Buckinghamshire, United Kingdom).Purification and activation of splenic B lymphocytes Splenic cells were depleted of T cells by using anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and activated for 3 days in minimal essential medium containing 10%
fetal calf serum, 50 mM  -mercaptoethanol, 1% penicillin and streptomycin, and lipopolysaccharide (25 µg/mL).
Flow cytometric analysis Peripheral blood was taken from the ophthalmic vein and washed 3 times in phosphate-buffered saline (PBS). After incubation with the relevant antibodies ready-made flow-activated cell sorter (FACS) lyse solution (Becton Dickinson, Franklin Lakes, NJ) was added. Thymus, spleen, and BM were removed from mice and homogenized in RPMI 1640 containing 1% fetal calf serum, 50 µM-mercaptoethanol, and 1%
penicillin and streptomycin. Anucleated cells were lysed by osmotic
shock in lysis buffer (NH4Cl 155 mM, KHCO3 10 mM, EDTA 0.1 mM) and washed twice with complete medium. Nondissociated cells and tissue debris were filtered on a 70-µm nylon cell strainer (Falcon, 35-2350). Cells were immunophenotyped by using conjugated monoclonal antibodies and standard techniques. The following antibodies were used: anti-CD4-fluorescein isothiocyanate (FITC),
CD8-phycoerythrin (PE), CD45R (B220)-FITC, CD45R (B220)-PE-Cy5,
CD19-PE, interleukin 7 receptor (IL-7R)-biotin,
c-kit-allophycocyanin (APC), CD11b (MAC-1)-APC, CD11b-FITC,
Sca-1-FITC, and streptavidin-Cy-Chrome complex. All antibodies used
were purchased from PharMingen. A total of 10 000 events were analyzed
on a FACScalibur (Becton Dickinson) permitting 4-color analysis.
Real-time-polymerase chain reaction For quantification of transgene expression in different hematopoietic lineages and animal lines, BM cells and thymocytes were sorted by flow activation with the use of anti-B220-PE-Cy5 (CD45R), anti-TER-PE, anti-CD11b-FITC (MAC), anti-CD4-FITC, and anti-CD8-PE antibodies (all from PharMingen). After TRIZOL extraction of total messenger RNAs (mRNAs) and standard reverse transcription (RT) quantitative polymerase chain reaction (PCR) was performed with the LC FastStart DNA Master SYBR Green I reaction kit (Roche Diagnostics, Basel, Switzerland; catalog no. 2 239 264). The following primers were used: hprt 5', GCT GGT GAA AAG GAC CTC T; 3', CAC AGG ACT AGA ACA CCT GC; htal-1 5', GCC GGA TGC CTT CCC TAT GTT; 3', AAT GAC CAG GAG GAG GAG GG; mtcra 5', ACA GAC AGC ATT CTG GGA AAG C; 3', ATC TTG CAG GTG AAG CTT GT; pT(5', TCA GGT GTC AGG CTC TAC GA; and 3', CCC ATA GGT GAA GGC GTC TA.MgCl2 was used at a final concentration of 4 M. The annealing temperature was 60°C. All samples were run in duplicates. Melting curve analysis and quantification calculation were performed by using fluorimetric online detection with the LightCycler (Roche Diagnostics). Immunohistochemistry Thymus paraffin-embedded sections were prepared in the histopathology department of Henri Mondor Hospital. Sections were dewaxed and microwave treated. HTAL-1 protein was revealed by using the mouse monoclonal antibody BTL-73 and the Mouse-on-Mouse Iso-IHC Kit (Inno Genex TH, San Ramon, CA).Nuclear extracts and electrophoretic mobility shift assay Nuclear extracts were obtained as previously described.21 Protein concentrations were assayed with the Pierce Coomassie Protein Assay reagent (Perbio, Helsingborg, Sweden). Binding reactions for the electrophoretic mobility shift assay were performed as previously described.20 For each reaction 15 µg of nuclear extract was used. For antibody supershift assays, 1 µL monoclonal anti-TAL-1 antibody (BTL-73) was added to the binding reaction. The sequence of the tal-1 probe used is (upper strand): 5'-ACCTAACAGA TGGTCGGCT-3'.Nonobese diabetic, severe combined immunodeficient mice transplant experiments Grafts were enriched for HSCs by treating transgenic and littermate control donors with 150 mg/kg 5-fluorouracil. On day 5 after treatment BM cells were harvested and depleted of CD4+ or CD8+ cells by using magnetic beads (Miltenyi) to prevent graft-versus-host disease. Cells (5 × 104) were injected into the ophthalmic vein of nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice previously sublethally irradiated with 2.5 Gy. Weekly full blood counts were obtained starting at 6 weeks. BM cells were harvested from recipients 8 weeks after transplantation, and hematologic reconstitution was assessed by flow cytometry.
Ly-6E.1-htal-1 and
Ly-6E.1-  bhtal-1)
followed by the bovine growth hormone gene polyA sequence into the Cla
I site of the 14-Kb Ly-6E.1 cassette (Figure 1A).17,19 The
bhtal-1 mutant was kindly given to us by Dr E. MacIntyre
(Laboratoire d'hématologie, Hôpital Necker, Paris, France)
and was derived by using oligonucleotide-mediated mutagenesis of the
wild type to delete the basic domain containing the amino acids 187 to
199 as previously described.22,23 The
Ly-6E.1-htal-1 or the Ly-6E.1-bhtal-1
fragments were microinjected into the pronuclei of fertilized
(C57BL/6 × DBA/2) F1 oocytes. Two transgenic lines were generated
with the Ly-6E.1-htal-1 construct, line 6 (L6) and line 8 (L8). Southern blot analysis of tail DNA showed that L6 contained 10 copies of the transgene, whereas L8 contained 2 copies (data not
shown). Although the females of L6 were infertile, making it impossible
to generate a homozygous line 6, we could obtain, at low frequency,
homozygous descendants for line 8. Generation of bhtal-1
transgenic mice was difficult because of low transgene transmission.
One founder did not transmit, whereas another passed on the transgene
to only 1 of 40 offspring. The third founder transmitted the transgene
to 10 of 68 descendants, establishing the transgenic line b L3. The
transgene copy number of b L3 was found to be 6 copies (data not
shown). Because we could only generate one bhtal-1
transgenic animal line, the conclusions regarding the DNA binding
domain must be guarded, as possible integration effects cannot be ruled
out. Although the general physical appearance of the transgenic lines
was normal, an obvious kinking and short length of the tail was
observed in 100% of high-copy L6 mice, 70% of the 2 copy heterozygous
L8, and 100% of the homozygous 4 copy L8 mice (Figure 1B). b L3
transgenic mice did not have kinked tails. Taken together with the
known expression pattern of the Ly-6E.1 cassette in the
müllerian ducts and tail during embryogenesis, these results
suggest that ectopic expression of hTAL-1 induces female infertility,
tail growth retardation, and bone abnormalities and that the severity
of this phenotype is, in part, TAL-1 dose dependent.
Expression of htal-1 and
bhtal-1 in the b L3 transgenic mice was the same as in
L6 animals, but the expression level was lower (data not shown). We
next performed real-time RT-PCR on total BM of the 10 copy L6, the
heterozygous 2 copy L8, and wild-type littermates. This assay revealed
that the htal-1/hprt mRNA ratio of the L6 animals was 6 times the expression level ratio of the L8 mice (Table
1). Thus, the transgene copy numbers in
the 2 animal lines correlated well with their htal-1 mRNA
expression level. To assess the transgene expression in different
hematopoietic lineages, we quantified htal-1 mRNA of
FACS-sorted BM cells and thymocytes of L6 mice and their wild-type
littermate controls. Real-time RT-PCR showed a htal-1/hprt
mRNA ratio of 1 for B220+ BM cells and a ratio of 2 for the
Mac+ population. The overall ratio in total thymocytes was
0.4 (data not shown). Real-time RT-PCR on double-negative (DN),
double-positive (DP), CD4+ single-positive (SP), and
CD8+ SP cells showed that the transgene was expressed in
all thymocyte subsets in increasing order: DP, DN, and SP cells. There
was no significant difference in the mRNA expression level of CD4 and CD8 SP cells (data not shown). Hence, htal-1 mRNA expression
was present in all the hematopoietic lineages studied and is in
accordance with previously published data on the expression pattern of
Ly-6E.1.18
To examine whether the expression of hTAL-1 protein was correlated with the expression of htal-1 mRNA, protein extracts from different organs were analyzed by Western blot. A correlated expression between the htal-1 mRNA and the hTAL-1 protein levels was found in all the positive organs tested, except for the kidney that expressed a high level of mRNA but did not express the hTAL-1 protein (Figure 1D). A comparative study of TAL-1 protein expression was performed by using spleen nuclear extracts of the 10 copy L6 and the 2 copy heterozygous L8 mice. The protein level detected in L6 animals was 5 times the level of L8 mice, thus showing the correlation between transgene copy numbers and protein expression level in the spleen (Figure 1E). A faint hTAL-1 expression was found in the total BM by Western blotting, and immunohistochemical staining of BM paraffin-embedded sections showed the presence of positive mononuclear cells in this organ (data not shown). Peripheral blood FACS analysis of transgenic mice reveals abnormalities in circulating lymphocytes and myeloid cells To determine whether ectopic expression of htal-1 orbhtal-1 under the control of the Sca-1
regulatory sequence induces a defect in hematopoiesis, blood of
12-week-old transgenic and nontransgenic age- and sex-matched
littermates was collected and subjected to full blood counts. This
analysis showed an increase in the total number of white blood cells in
the transgenic versus nontransgenic mice (3000/µL ± 200 versus
2200/µL ± 200, respectively), whereas the erythroid
compartment, assessed by hemoglobin measurements and red blood cell
numbers, was not significantly altered (data not shown).
FACS analysis, performed on 12-week-old L6 mice and their age- and
sex-matched wild-type controls, revealed alterations of the peripheral
blood cell distribution (Table 2). After
gating on the total white blood count, we found a 1.7- and 2.4-fold
increase in CD4+ and CD8+ cells, respectively,
leading to an alteration of the CD4/CD8 ratio of 1.2 versus 0.8 in
nontransgenic versus transgenic animals. Additionally, there was a 60%
reduction in the absolute number of B cells identified by anti-B220
antibody. Interestingly, FACS analysis with anti-CD11b showed that most
of the L6 CD11b+ cells continued to express the cell
surface marker Sca-1, whereas wild-type CD11b+ cells were
negative for this antigen in the peripheral blood. All full blood
counts and FACS analysis were performed on 6 transgenic and
nontransgenic littermates.
Thus, abnormalities were only found in hematopoietic cells that normally do not express TAL-1, leading us to investigate hematopoietic and lymphopoietic organs. Sca-1 antigen expression persists on granulocytes and monocytes After gating on the myeloid population, 100% of CD11b+ cells in the peripheral blood of L6 transgenic mice expressed the cell surface antigen Sca-1. In the BM, we found a moderate increase of transgenic CD11b+ BM cells. To define the mechanism underlying this moderate expansion of the myeloid pool more closely, we performed methylcellulose cultures of total BM and showed that the granulocyte-macrophage colony number was similar with the use of transgenic or wild-type BM, whereas the size of the transgenic colonies appeared larger, thus indicating an increase in the proliferation potential of the transgenic granulomacrophagic precursors (data not shown). By looking at the cell surface antigen, we found that, although only 5% to 10% of the CD11b+ cells expressed the Sca-1 antigen in the wild-type mice and the low copy L8 line, more than 50% of the CD11b+ cells were positive for Sca-1 in the high copy L6 andb L3 line (data not shown). As the BM
Sca-1 cells corresponded partly to macrophages
(identified by the F4-80 specific antibody) and as the Sca-1 antigen is
normally expressed on immature hematopoietic progenitors, the
persistence of Sca-1 expression could indicate a block in granulocytic
differentiation. This phenomenon has already been observed in
granulopoietic cells that constitutively and inappropriately expressed
Tal-1.24 We, therefore, performed cytospins of
CD11b+ peripheral blood cells and of
Sca-1/CD11b+ BM cells and showed that their morphologic
stage of differentiation was similar to wild-type CD11b+
cells (Figure 2 and data not shown). By
looking at the transgene expression using the BTL-73 anti-TAL-1
antibody, we found that the transgene was extinguished in mature
monocytes and granulocytes, indicating why we did not see a block in
granulocytic differentiation.
Ectopic hTAL-1 or b L3 mice of different ages showed that the
transgenic animals presented with a smaller thymus (Figure 3B) and a
decrease of absolute thymocytes numbers
(160 × 106 ± 10 × 106 versus
110 × 106 ± 10 × 106 at 4-8 weeks;
80 × 106 ± 10 × 106 versus
40 × 106 ± 10 × 106 at 12 weeks in
wild-type and transgenic mice, respectively). FACS analysis of the
total thymic population did not show, in the younger animals, any
significant alterations in thymocyte subset distribution. In contrast,
12-week-old transgenic mice presented a 50% decrease of the DN cell
population, a 30% to 50% decrease in the absolute number of DP cells,
whereas the same absolute number of SP CD4 or CD8 cells was observed in
transgenic and nontransgenic mice (Figure 3C). Hence, taken together,
these data indicate a relative increase in the number of SP cells with regard to the more immature cell populations. To investigate the causes
for this relative increase further, we performed FACS analysis on the
CD8 SP cells that consist of 2 distinct type of cells: CD8 immature
single-positive (ISP) cells expressing a low level of T-cell receptor
(TCR-) and representing cells undergoing differentiation just
before the DP stage and CD8 mature SP cells characterized by a high
level of TCR- expression and representing differentiated cells. The
expression level of TCR- was analyzed in CD8 SP cells, showing a 2- to 3-fold decrease of ISP cells in transgenic animals (data not shown)
and indicating that the relative increase in the CD8 SP cells was not
because of an increase in ISP cells.
As signaling by the pre-T-cell receptor The absence of T-cell leukemia in transgenic mice may be related to low hTAL-1 expression in transgenic T cells. Thus, we compared the hTAL-1 expression level in Jurkat and L6 transgenic thymocytes by Western blotting and showed that the hTAL-1 protein level in Jurkat was at least 4 times higher compared with the L6 transgenic thymocytes (Figure 3D). Assuming that the level of TAL-1 expression in Jurkat cells represented a threshold to cause tumors, Ly-6E.1 promoter weakness may be one reason why no T-cell neoplasia occurred in our mouse model. Alternatively, other variables such as mouse strain, transgene integration site, or copy number have to be considered. Ectopic hTAL-1 expression leads to a significant reduction of early B-cell precursors As blood analysis showed a significant reduction in B lymphocytes, we performed FACS analysis on the lymphoid gate of the BM of 12-week-old L6 transgenic mice and age- and sex-matched littermate controls and showed a 40% to 50% reduction in the relative and absolute number of B220+ CD19+ cells. These results were independent of the copy number and were also observed in theb L3 transgenic mice (Figure 4A).
To define the stage of a possible block in B-cell differentiation, we
performed multicolor fluorescent staining on the BM of L6 and wild-type
animals using specific antibodies for anti-B220, anti-CD19,
anti-IL-7R To further characterize the defect in B-cell lymphopoiesis, BM cells of L6 and L8 transgenic and nontransgenic animals were subjected to in vitro pre-B colony formation assays. After 7 days of culture on methylcellulose we obtained 2 to 3 (L6, L8) and 12 (wild type) pre-B colonies, indicating that B-cell differentiation was not only reduced in vivo but also in vitro and suggesting an intrinsic defect within the lymphoid compartment. As fetal B-cell development has been described to differ from the adult setting, we analyzed the B-cell population in day 16.5 to day 18.5 fetal livers by FACS and found that, as in the adult BM, the B-cell compartment was affected by the ectopic expression of TAL-1 protein at least up the pro-B cell stage (data not shown). To analyze TAL-1 effects on the expression of genes previously
described as being important for B-cell differentiation, we sorted
B220+ CD19+ IL-7R Ectopic hTAL-1 expression in B cells leads to a defect in isotype class switch recombination We then studied the effects of ectopic hTAL-1 expression on splenic B-cell maturation. Morphology of the spleen of transgenic animals revealed a disorganization of the germinal center (Figure 5A). Absolute splenocyte numbers of transgenic and wild-type littermates were similar, and FACS analysis showed a 30% to 50% reduction of B220+ CD19+ and B220+ IgM+ cells in the spleen (Figure 5B and data not shown). To assess antigen-dependent maturation further, we analyzed the ability of transgenic splenocytes to undergo isotype class switch recombination on activation with T-cell-independent antigen. Because lipopolysaccharide (LPS) induces murine B cells to switch from µ to 3 and 2b, we used LPS activation on sorted transgenic and
nontransgenic B splenocytes. After exclusion of B220
cells, splenocytes derived from the high copy number transgenic mice
showed a 4.5-fold reduction in immunoglobulin G2b (IgG2b) and IgG3
isotypes (Figure 5C and data not shown) after 72 hours, indicating an
effect of ectopic hTAL-1 expression on antigen-dependent B-cell
differentiation. B220+ splenocytes derived from the low
copy number transgenic mice showed a reduction in IgG3 of only
1.9-fold, thus implying that TAL-1 induces an isotype switch
recombination defect in a dose-dependent manner (data not
shown).
Because E2A is involved in class switch recombination and heterodimerizes with TAL-1, we performed electrophoretic mobility shift assays on nuclear extracts of LPS-activated B splenocytes of transgenic L6, heterozygous L8, and wild-type littermates. This analysis showed specific binding of a TAL-1/E2A complex to tal-1 oligonucleotides, which is supershifted by an hTAL-1-specific antibody (Figure 5D). The same complex, however much weaker, is present in splenocytes of L8 animals. Thus, a complex containing TAL-1/E2A is present in activated B splenocytes of transgenic mice, and its expression correlates with the transgene copy number in each line. Together with previously published data,25-28 these results indicate that TAL-1 protein may sequester E2A in these cells. NOD/SCID mice receiving transgenic BM transplants display features of htal-1 transgenic mice B-cell differentiation has been shown to depend on the interaction of lymphoid precursor cells with the surrounding BM stroma and microenvironment. We, therefore, aimed to exclude that the reduction in the B-cell compartment was secondary to an extrinsic, cell nonautonomous phenomenon by performing transplantation experiments of transgenic and nontransgenic BM into sublethally irradiated NOD/SCID mice.At weeks 6 and 7 after transplantation, blood was collected and FACS
analysis performed. The majority (75%) of NOD/SCID mice, injected
either with transgenic or nontransgenic BM, successfully engrafted. In
NOD/SCID mice receiving transgenic BM transplants, the Sca-1 antigen
persisted on 30% to 50% of myeloid cells, indicating their transgenic
origin. It was found that 11% versus less than 1% of peripheral blood
lymphocytes of NOD/SCID mice receiving nontransgenic versus transgenic
BM were B220+ IgM+ cells (Figure
6A). Analysis of the BM of all animals
receiving transplants at 8 weeks after transplantation showed that the
population of B220high CD19+ cells in
transgenic grafts was severely reduced compared with wild-type grafts.
NOD/SCID mice not receiving transplants normally show an accumulation
of pro-B (B220low) cells (Figure 6B, first scatter). These
cells disappeared in NOD/SCID mice receiving transplants regardless of
the transgenic or nontransgenic origin of the BM as shown in the second
and third scatter of Figure 6B. This absence of pro-B-cell
accumulation in the transgenic transplants indicates that a possible
block in B-cell differentiation may be prior to this stage.
Thus, this data shows that in contrast to nontransgenic transplants htal-1 grafts failed to reestablish early and late primary B-cell lymphopoiesis and that the effects of ectopic hTAL-1 expression on myelopoiesis and lymphopoiesis are intrinsic to the cell populations and not secondary to an epiphenomenon of the transgenic microenvironment.
This work was designed to assess the effects of ectopic hTAL-1
expression on hematopoietic cells normally switching off
tal-1 gene expression. For these purposes, we generated 2 htal-1 transgenic models: L6 and L8
Ly-6E.1-htal-1 lines expressing different levels of the
full-length hTAL-1 transcription factor. Additionally, we examined the
L3 Ly-6E.1- Transgenic mice presented hematopoietic and nonhematopoietic disorders.
Apart from infertility, the most characteristic abnormality was the
presence of kinked tails in 100% of high-copy and 70% of low-copy
animals, whereas they were absent in the By using real-time RT-PCR we showed a correlation between transgene copy numbers and mRNA expression level in the different animal lines. We could also establish a correlation between htal-1 mRNA and protein levels in different organs except for the kidneys that expressed high levels of transgene mRNA but no protein. This discrepancy is in accordance with previously published observations on lacZ reporter gene expression under the control of Ly-6E.1 showing high mRNA but no protein expression in the kidneys18 and could be due to tight control of translation or protein degradation in this organ. Despite the essential role of TAL-1 in HSCs,11,12 we did
not detect any quantitative modification of the Sca-1+,
c-kit+, thy-1low, or Lin Examination of the myeloid pool revealed an increase in myeloid precursors and the persistence of the Sca-1 antigen on granulocytes. This phenotype was dose dependent, indicating that its generation may need a threshold of hTAL-1 expression in myeloid cells. This finding suggests that Sca-1 gene expression might be repressed during terminal myeloid differentiation and that the repressor might be sequestered by hTAL-1. Alternatively, high hTAL-1 protein expression might directly maintain Sca-1 gene expression throughout myeloid differentiation by a direct positive mechanism. In this latter case, hTAL-1 itself would be normally sequestered and titrated. Importantly, in the Ly-6E.1-htal-1 mouse model, as in single
transgenic CD2/tal-1 and
sil/tal-1,1,6 ectopic hTAL-1 did not induce
T-cell leukemia in any of the transgenic lines generated in more than
150 offspring during their first year of life. This finding is in
contrast to data published on single transgenic lck/ tal-1
and on double transgenic tal-1/ckII In the Ly-6E.1-htal-1 transgenic model we observed a
decrease of the immature DN, ISP CD8+ cells, and DP subsets
that was not because of increased apoptosis (data not shown). However,
compared with nontransgenic mice the number of mature SP cells remained
unchanged, indicating an increased differentiation capacity from the DP
to the SP cell stage or a proliferation ability of the transgenic SP
cells. T-cell-specific E box complexes are largely composed of E2A/HEB
heterodimers; however, their DNA-binding activity is decreased on
transition from the DP to the SP cell subset.29 Thus,
ectopic expression of TAL-1 could perturb this balance and induce an
increase in the differentiation rate of the DP cells. A recent study
using standard RT-PCR followed by PhosphorImaging quantification
(Amersham Pharmacia Biotech, Buckinghamshire, England) showed a
significant reduction of pT The most striking characteristic of Ly6E.1-htal-1 and
Ly6E.1- The B-cell deficiency observed may be due to a differentiation block,
similar to the one described in various gene inactivation studies such
as Pax-5 We also found disorganization of the germinal center and a defect in isotype class switch recombination of mature B cells. Similar morphologic features have been found in mice lacking OBF-1, a transcription factor involved in class switching.34 As E2A is required in class switch recombination,35 we performed electrophoretic mobility shift assays on nuclear extracts of LPS-activated B splenocytes and showed that the heterodimer TAL-1/E2A was present in these cells and that its level correlated with the transgene copy number. Additionally, the class switch recombination defect observed was dependent on the expression level of TAL-1 protein, indicating that it was most likely due to titration of E2A. This finding is in contrast to the reduction in the B-cell compartment, which was dose independent and thus suggests other possible mechanisms than E protein titration on early B-cell differentiation. Taken together, ectopic TAL-1 expression sheds further light on the critical role of E proteins in B-cell development. In conclusion, we have shown that overexpression of hTAL-1 alone in a transgenic scenario does not only lead to an imbalance in thymocyte maturation but also to a block in B-lineage differentiation. These results indicate why the tal-1 gene extinction observed during B and T lymphopoiesis is necessary for the normal development of these hematopoietic lineages.
We thank Laure Coulombel for advice on FACS analysis. We thank Françoise Pflumio for her helpful assistance with NOD/SCID experiments and for her interesting discussions and Anne Dubart for her pertinent remarks. We also thank Maïté Mitjavilla for providing us with antibodies and Rita Cologon and Evelyne Souil for their help in histopathology, Isabelle Bouchaert and Nicolas Lebrun for FACS sorting, Danielle Mathieu and Karen Pulford for providing us with TAL-1 monoclonal antibodies, and Franck Letourneur and Eric Delabesse for their help with setting up the real-time PCR. |
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Abstract
Introduction
