By
From the Research Institute of Molecular Pathology, A-1030 Vienna, Austria
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Abstract |
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The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B
cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin µ (Igµ) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell
stage. The pre-BCR contains, in addition to Igµ, the surrogate light chains 5 and VpreB and
the signal transducing proteins Ig
and Ig
. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5
gene, which codes for the B cell-specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Ig
) gene and VH-to-DHJH recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes ~10- and
~50-fold, respectively. Here we demonstrate that complementation of these deficiencies in
pre-BCR components by expression of functionally rearranged Igµ and chimeric Igµ-Ig
transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (
/
) mice in
contrast to RAG2 (
/
) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igµ transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B
lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.
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Introduction |
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An important checkpoint in B cell development controls the transition from the pro-B (pre-BI) to the pre-B
(pre-BII) cell stage that is initiated upon completion of a
productive rearrangement at the immunoglobulin heavy
chain (IgH) locus. A consequence of expressing the membrane-bound Igµ protein is the transient formation of the
pre-B cell receptor (BCR).1 Signaling initiated by this receptor promotes allelic exclusion at the IgH locus, stimulates proliferative cell expansion, and induces differentiation
to small pre-BII cells undergoing Ig light chain gene rearrangements (for review see reference 1). In addition to the
Igµ protein, the pre-BCR consists of the two nonpolymorphic, surrogate light chains, 5 and VpreB, as well as
the signal transducing proteins Ig
and Ig
whose expression is initiated early in B lymphopoiesis (for review see
reference 2). B cell development is arrested at the pro-B
(pre-BI) cell stage in mice that lack one component of either the pre-BCR (mIgµ [reference 3],
5 [reference 4],
and Ig
[reference 5]) or of the V(D)J recombination machinery [RAG1; reference 6), RAG2 (7), DNA-dependent protein kinase (DNA-PK; reference 8)]. However,
expression of a functionally rearranged Igµ transgene is able
to complement the recombination defects of both severe
combined immunodeficiency (scid) and RAG mutant mice,
thus resulting in pre-BCR formation and subsequent progression to the small pre-BII cell stage (9). The early expression of a rearranged Igµ transgene significantly shortens the duration of pro-B cell development by directly inducing differentiation to small pre-BII cells (12). Likewise,
expression of a functionally rearranged
light chain gene is
capable of activating the pre-B cell transition in
5-deficient mice (13, 14).
The Ig and Ig
proteins form a disulfide-linked heterodimer that is associated through its transmembrane domain with the Ig molecule in the pre-BCR and BCR. This
heterodimer is not only essential for surface transport of Igs,
but also constitutes the signal transducing unit of these receptors (for review see references 2, 15). The Ig
and Ig
proteins both initiate signaling via immunoreceptor tyrosine-based activation motifs (ITAMs), which become
phosphorylated upon receptor engagement and recruit intracellular effectors such as protein-tyrosine kinases to the
receptor (2, 15). Apart from these motifs, the cytoplasmic
tails of Ig
and Ig
differ considerably in sequence, but yet
appear to fulfill redundant functions in B cell development.
Chimeric receptors, consisting of the Igµ protein fused to
the cytoplasmic domain of either the Ig
or Ig
protein,
are each independently sufficient to induce the pre-B cell
transition (16, 17) and to signal B cell maturation (18) in
transgenic mice.
Insight into the transcriptional control of early B cell development has recently been gained by gene targeting in the
mouse. One of the critical transcription factors thus implicated
in early B lymphopoiesis is the B cell-specific activator protein (BSAP), which is encoded by the Pax5 gene (for review
see references 19, 20). Pax5 is expressed from the earliest B
lineage-committed precursor cell up to the mature B cell stage
(21), and, consistent with this expression pattern, is essential for B lineage commitment in the fetal liver (24). However, in adult bone marrow, Pax5 is required later for the
progression of B cell development beyond the early pro-B
(pre-BI) cell stage (24, 25). Interestingly, the VH-to-DHJH
recombination at the IgH locus is ~50-fold reduced in Pax5-deficient pre-BI cells (24). Moreover, the mb-1 (Ig) gene,
which has been identified as one of five direct BSAP (Pax5) targets, is expressed at an ~10-fold lower level in these
pre-BI cells, whereas Pax5 is not involved in the control of
5, VpreB, and B29 (Ig
) expression (24, 26). Hence, the
synthesis of two pre-BCR components, Igµ and Ig
, is affected in early B lymphocytes of Pax5 mutant mice.
Here we have tested the hypothesis that the inability to
express a pre-BCR might be the cause for the B cell developmental block in the bone marrow of Pax5-deficient
mice. For this purpose, we have introduced functionally
rearranged Igµ and chimeric Igµ-Ig transgenes into the
Pax5 mutant background. These transgenes were able to
neither advance B cell development to the small pre-BII cell stage nor to elicit normal signaling responses, although the pre-BCR was expressed on the Igµ transgenic, Pax5-deficient pre-BI cells. Moreover, expression of a bcl2 transgene was also incapable of rescuing the early developmental
block which is thus unlikely to result from the absence of a
survival signal in Pax5 mutant B lymphocytes. These data
therefore demonstrate that Pax5 fulfills an essential function
during pro-B cell development before the pre-BCR stage.
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Materials and Methods |
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Mice.
The different mouse strains were maintained on the hybrid C56BL/6 × 129/Sv background. The genotype of Pax5 mutant mice (25) was determined by PCR analysis as previously described (24). RAG2 mutant mice (7) were genotyped by PCR amplification with the following oligonucleotides: 5'-GCAACATGTTATCCAGTAGCCGGT-3' (primer 1), 5'-TTGGGAGGACACTCACTTGCCAGT-3' (primer 2), and 5'-GTATGCAGCCGCCGCATTGCATCA-3' (primer 3). A 605-bp PCR product was amplified from the wild-type RAG2 allele with primer pair 1 and 2 and a 1-kb DNA fragment from the mutant RAG2 allele with the pair 1 and 3. For simplicity, the mouse- human hybrid transgene mIgµ-IgPre-BI Cell Cultures.
Cell suspensions prepared from mouse bone marrow or fetal liver (at embryonic day 16.5 or 17.5) were plated at limiting dilutions on a semiconfluent layer ofAntibodies and Flow Cytometry.
The following mAbs were purified from hybridoma supernatants on protein G-sepharose columns (Pharmacia Biotech AB, Uppsala, Sweden) and conjugated with sulfo-NHS-biotin (Pierce Chemical Co., Rockford, IL) as recommended by the suppliers: anti-c-kit mAb (ACK4; reference 31), anti-µ mAb (M41.42; reference 32), anti-Intracellular Antibody Staining.
The cytoplasmic µ heavy chain protein was detected in bone marrow pre-BI cells as previously described (34). In brief, bone marrow cells were incubated with PE-coupled anti-B220 (RA3-6B2) and allophycocyanin (APC)- conjugated anti-c-kit (ACK45) antibodies at 4°C, washed twice with PBS, and then fixed with 2% paraformaldehyde (Fluka AG, Buchs, Switzerland) in PBS at room temperature for 20 min, followed by two washes with PBS. The fixed cells were subsequently permeabilized with 0.5% saponin (Sigma Chemical Co., St. Louis, MO) in 2% FCS/PBS and were simultaneously stained with FITC-conjugated anti-µ antibody (R6-60.2) for 40 min at 4°C, then washed twice in saponin buffer and once in 2% FCS/PBS before analysis on a FACSVantage® TSO flow cytometer (Becton Dickinson). Cultured bcl-2 transgenic, Pax5 (Western Blot Analysis.
Whole cell extracts of in vitro cultured pre-BI cells were prepared by lysis in 0.25 M Tris, pH 7.5, and 0.1% Triton X-100, followed by removal of insoluble material by centrifugation. Total protein (10 µg) was separated by 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then incubated with a rabbit polyclonal anti-IgRNase Protection Analysis.
A mouse terminal deoxynucleotidyl transferase (TdT) riboprobe was generated by inserting a 244-bp cDNA fragment of the mouse TdT mRNA (35) into the HindIII and EcoRI sites of pSP64. This cDNA fragment was amplified from RNA of 70Z/3 cells by reverse transcriptase PCR using the following primers: 5'-GCGGAATTCAAGGTGGATGCTCTCGACCAT-3' and 5'-GCGAAGCTTCGTGGTTGTCCAGCATCATCT-3'. Total RNA was prepared from cultured pre-BI cells and analyzed by RNase protection assay exactly as previously described (24). ![]() |
Results |
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Based on the expression of cell
surface markers, we have recently demonstrated that B cell
development is arrested in the bone marrow of Pax5 mutant mice (24) at a similar pro-B (pre-BI) cell stage as in
mice that are deficient in one of the components of the
pre-BCR (µMT [reference 36], 5 [reference 36], and Ig
[reference 5]) or the V(D)J rearrangement machinery (RAG1 [reference 9] and RAG2 [reference 7]). Moreover, Pax5-deficient pre-BI cells are essentially unable to synthesize the
Igµ protein, an important constituent of the pre-BCR, due
to an ~50-fold reduction of the VH-to-DHJH recombination frequency at the IgH locus (24). The inability to form
a functional pre-BCR could therefore explain the early B
cell developmental block observed in Pax5 mutant mice. This hypothesis makes the clear prediction that expression
of a functionally rearranged Igµ transgene in Pax5 mutant
mice should result in the formation of the pre-BCR, thus
traversing this important checkpoint and advancing B cell
development to the pre-BII cell stage. To test this hypothesis, we have introduced a rearranged murine Igµ transgene, which directs expression of a membrane-bound Igµ
protein under the control of a VH gene promoter (28) into Pax5 (
/
) mice. As the chosen Igµ transgene has not yet
been used for similar experiments, we have also tested its
ability to guide B cell development to the pre-BII cell stage
in RAG2-deficient mice. The transition from the pre-BI to
the pre-BII cell stage is known to be accompanied by the
downregulation of the early markers CD43 and c-kit, by
the initiation of CD2 and CD25 expression, and by an increase in the total B cell number (9, 10, 37). B lymphocytes
from RAG2 (
/
) bone marrow lacking or containing the
Igµ transgene were compared by flow cytometric analysis
(Fig. 1), demonstrating that the synthesis of CD43 and c-kit
was indeed downregulated, the expression of CD2 and
CD25 was initiated, and the number of B220+ cells was increased by about twofold in the presence of the transgene. In marked contrast, the B lymphocyte number and cell surface phenotype did not change in the bone marrow of
Pax5 (
/
) mice irrespective of the presence or absence of
the Igµ transgene (Fig. 1). Hence, the Pax5 and RAG2
gene mutations clearly differ, as the presence of a rearranged Igµ transgene is unable to rescue the early B cell developmental block in Pax5-deficient mice in contrast to RAG2-deficient mice.
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Possible trivial explanations for the failure of the Igµ
transgene to induce the pre-B cell transition could be that
Pax5 itself is involved in the transcriptional control of the
transgene or that B cell development is arrested before the
initiation of transgene expression in Pax5 mutant mice. To
investigate these possibilities, we have analyzed the presence of cytoplasmic Igµ protein in c-kit+ B220+ pre-BI
cells of Pax5-deficient bone marrow (Fig. 2 A). No cytoplasmic Igµ protein could be detected by intracellular
staining in Pax5 (/
) pre-BI cells in agreement with the
fact that the VH-to-DHJH recombination is drastically reduced in these cells (24). In contrast, the Igµ protein was
expressed in the majority of Pax5 (
/
) pre-BI cells carrying the transgene. We therefore conclude that early expression of a rearranged Igµ transgene is not sufficient to trigger
the pre-B cell transition in Pax5 mutant mice.
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The mb-1 gene coding for Ig was recently shown to be
a direct BSAP (Pax5) target whose expression is reduced
~10-fold in Pax5-deficient pre-BI cells compared with
wild-type cells (26). In addition to the Igµ protein, Ig
is
therefore a second component of the pre-BCR that is expressed under the control of Pax5. As the heterodimer consisting of the proteins Ig
and Ig
constitutes the signal
transducing unit of the pre-BCR (2), it is conceivable that
the reduced Ig
expression in Pax5-deficient pre-BI cells prevents the formation of a functional pre-BCR even in
the presence of a rearranged Igµ transgene. To address this
question, we have introduced a chimeric Igµ-Ig
transgene
(16) into the Pax5 (
/
) background. The Igµ component
of this transgene codes for a membrane-bound Ig with two
transmembrane mutations (Y587V, S588V) which prevent its normal association with the Ig
-Ig
dimer (27). The
cytoplasmic domain of the fusion protein is encoded by Ig
and directly mediates signaling independent of the presence
of endogenous Ig
or Ig
proteins (27). Furthermore, the
chimeric Igµ-Ig
receptor was shown to efficiently activate transition to the pre-BII cell stage and to induce allelic
exclusion at the IgH locus in RAG-deficient mice (16).
Hence, signaling of this chimeric receptor should be independent of the reduced expression levels of both Ig
and Igµ proteins that are observed in Pax5-deficient mice.
Nevertheless, the chimeric Igµ-Ig
gene was unable to advance B cell development in the bone marrow of Pax5
mutant mice, since its presence neither altered the expression
of cell surface markers nor increased the number of B220+
cells (Fig. 3 A). However, the Igµ-Ig
fusion protein was
expressed in pre-BI cells regardless of the Pax5 genotype
(Fig. 3 B). Together, these in vivo data indicate that expression of the pre-BCR is not sufficient to rescue the early B
cell developmental block in Pax5-deficient mice. Hence, the
Pax5 mutation appears to arrest B lymphopoiesis at an early
stage that is not responsive to pre-BCR signaling.
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The survival of B cell precursors is controlled by differential expression of the antiapoptotic genes bcl-2 and bcl-xL
during B lymphopoiesis (38, 39). Interestingly, the bcl-xL
but not the bcl-2 gene is consistently expressed at a 10-fold
lower level in Pax5-deficient pre-BI cells compared with
wild-type cells, although this downregulation was shown
to be an indirect consequence of the absence of Pax5 (26).
In agreement with this finding, the pre-BI cells of Pax5
mutant bone marrow proved to be ultrasensitive to growth
factor withdrawal, as they rapidly undergo apoptosis ex
vivo in the absence of survival signals emanating from the IL-7 receptor (data not shown). In this context it is interesting to note that the expression of a bcl-2 transgene was
previously shown to promote B cell development in scid
mice (40) that also exhibit a defect in V(D)J recombination
of Ig genes (for review see reference 41). Hence, we investigated the possibility that sustained cell survival may also
rescue the early developmental block in Pax5-deficient
bone marrow. For this purpose, the same Eµ-bcl-2-36
transgenic mouse, carrying a human bcl-2 cDNA under the
control of the IgH Eµ enhancer (29), was crossed with
Pax5 mutant mice. Expression of the bcl-2 transgene in
Pax5 (/
) pre-BI cells was demonstrated by cytoplasmic
staining with an anti-human Bcl-2 antibody as well as by
its ability to completely block apoptosis upon IL-7 withdrawal (data not shown). Nevertheless, the bcl-2 transgene
was unable to advance B cell development to the pre-BII cell stage, as no CD43
B220+ B lymphocytes were observed in the bone marrow of bcl-2 transgenic, Pax5 (
/
)
mice (Fig. 2 B). Instead, deregulated bcl-2 expression led to
a modest increase in CD43+ B220+ pre-BI cells similar to
the situation observed in RAG2 mutant mice carrying a bcl-2
transgene (42). Therefore, these data demonstrate that
blocking apoptosis is not sufficient to promote B cell development in Pax5 mutant mice.
Expression of a functionally rearranged Igµ chain
has previously been shown to alter the IL-7 responsiveness
of precursor B cells in wild-type and RAG2 mutant mice
(10). The proliferative response to IL-7 was considerably
decreased in bone marrow cells of Igµ transgenic mice,
thus preventing the establishment of long-term pre-BI cell
cultures (10). One possible reason for this phenomenon may be the downregulation of c-kit expression in response
to pre-BCR activation (33, 37), which eliminates an essential costimulatory signal for IL-7-dependent proliferation
of B lymphoid precursor cells (43). To further study the
function of the pre-BCR, we have established pre-BI cell
cultures from bone marrow of Pax5-deficient mice carrying an Igµ(-Ig) transgene. These pre-BI cells were cultured in the presence of stromal ST2 cells and IL-7, and
their long-term proliferation potential was assessed after 1 mo
of in vitro culture. Surprisingly, Pax5-deficient pre-BI cells
could be efficiently established and maintained even in the
presence of transgenic Igµ or chimeric Igµ-Ig
proteins
(Table 1). In contrast, no pre-BI cell cultures with long-term proliferation capacity were obtained from homozygous or heterozygous RAG2 mutant mice carrying an Igµ
transgene, as previously described (10). Thus, these data indicate that expression of the Igµ protein does not interfere
with the proliferation potential of Pax5-deficient pre-BI
cells in contrast to control B lymphocytes.
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Given the possibility to grow Igµ transgenic, Pax5 (/
)
precursor cells, we next investigated whether these cells
could assemble the pre-BCR on their surface. As shown by
flow cytometric analysis, Pax5-deficient pre-BI cells containing or lacking the Igµ transgene expressed a similar
level of the surrogate light chain
5 on their surface (Fig. 4
A). In contrast, the Igµ protein was only found on the transgenic pre-BI cells. Furthermore, staining with a monoclonal antibody (SL156), which recognizes a conformational epitope
present on the surrogate light chain-Igµ complex of the pre-BCR (33), demonstrated that the Igµ protein was part of the
pre-BCR (Fig. 4 A). Three conclusions can be drawn from
these data. First, the pre-BCR is stably expressed on the surface of Igµ transgenic, Pax5 (
/
) pre-BI cells despite the
fact that the pre-BCR is only transiently expressed and rapidly internalized on wild-type precursor B cells (33, 44,
45). Second, the surrogate light chains are expressed at normal levels on transgenic, Pax5 (
/
) pre-BI cells, although
their expression is usually downregulated in response to
pre-BCR signaling (9, 10, 37, 38, 44). Third, the Ig
protein is known to be essential for cell surface transport of Igs
(46, 47), and yet the 10-fold lower mb-1 expression in
Pax5-deficient pre-BI cells (26) seems to provide sufficient Ig
protein for pre-BCR formation.
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The expression of the TdT gene is rapidly downregulated during the pre-B cell transition in response to expression of a functionally rearranged Igµ protein (37, 38, 48). The TdT gene is therefore considered to be a downstream target in the signaling cascade initiated by the pre-BCR (48). As shown by RNase protection analysis, the level of TdT transcripts was similar in Pax5-deficient pre-BI cells regardless of the presence of the Igµ transgene (Fig. 4 B, lanes 2 and 3). In summary, the different results obtained with cultured pre-BI cells all demonstrate that the pre-BCR is unable to elicit its normal signaling response in the absence of Pax5 function.
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Discussion |
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The transcription factor Pax5 (BSAP) is involved in the
control of VH-to-DHJH recombination and in the transcriptional regulation of the mb-1 gene, which results in reduced
expression of the two pre-BCR components, Igµ and Ig,
in Pax5-deficient pre-BI cells (24, 26). Here we have demonstrated that complementation of these deficiencies by the
expression of Igµ and Igµ-Ig
transgenes is not sufficient to
initiate the pre-B cell transition in Pax5 mutant mice.
Hence, the inability to form a pre-BCR cannot be the
cause of the early B cell developmental block in mice lacking Pax5. Instead, the absence of Pax5 arrests B cell development by a different mechanism compared with mice
which lack a component of the pre-BCR (mIgµ [reference
3],
5 [reference 4], or Ig
[reference 5]) or of the V(D)J
recombination machinery (RAG1 [reference 6], RAG2
[reference 7], or DNA-PK [reference 8]). Consistent with
this conclusion, the lack of Pax5 or RAG2 function has
opposite effects on the in vitro differentiation potential of B
lymphocytes. Pre-BI cells of RAG2 mutant mice efficiently differentiate ex vivo to the mature B cell stage upon stimulation with IL-4 and anti-CD40 antibodies, which by-passes in vitro the requirement of Ig gene rearrangements
for further development (49). In contrast, Pax5 mutant
pre-BI cells entirely fail to differentiate under the same in
vitro conditions, further demonstrating a strict dependency
of early B lymphopoiesis on Pax5 (Nutt, S.L., unpublished
data).
It has been notoriously difficult to demonstrate expression of the pre-BCR on the surface of precursor B cells
(33, 44), which reflects both a slow, inefficient cell surface
transport and rapid, tyrosine phosphorylation-dependent
internalization of this receptor (2, 45). Quite in contrast,
we have now observed stable expression of the pre-BCR
on the surface of Igµ transgenic, Pax5-deficient pre-BI
cells. Interestingly, the constitutive cell surface expression
of the pre-BCR correlates with the absence of normal signaling responses. The transgenic, Pax5-deficient pre-BI
cells neither lost their long-term proliferation potential in
the presence of IL-7 and stromal cells nor did they downregulate expression of the TdT or surrogate light chain
genes, which are normal responses to pre-BCR signaling in
wild-type precursor B cells (9, 10, 37, 38, 44, 48). Therefore, it is conceivable that Pax5 may either regulate the expression of an essential component of the signal transduction cascade or act in the nucleus as the critical mediator of
pre-BCR signaling. Stimulation of the BCR is known to
result in the phosphorylation and association of the Ig-
Ig
heterodimer with the protein-tyrosine kinases Lyn,
Fyn, Blk, Btk, and Syk (50). Moreover, the Syk kinase
has been shown to play an important role in pre-BCR signaling (54, 55). However, none of these tyrosine kinase
genes is expressed under the control of Pax5, as shown by a
comprehensive analysis of putative BSAP (Pax5) target
genes (26). Hence, there is at present no evidence that
Pax5 is involved in the expression of cytoplasmic signal transducers. Moreover, an exclusive role of Pax5 in mediating signal transduction of the pre-BCR seems unlikely
for several reasons. First, Pax5 expression is already initiated
at B lineage commitment long before the pre-BCR stage
and thereafter is maintained at a rather constant level
throughout B lymphopoiesis (21). Second, all our attempts have so far failed to demonstrate any alteration in
the posttranslational modification pattern of BSAP (Pax5)
in response to signal transduction (M. Busslinger, unpublished data). Third, the developmental arrest in Pax5 mutant mice is tight (25) rather than leaky as it would be expected, in analogy to the syk (
/
) mouse (54, 55), for a
mutation in a downstream component of the signal transduction pathway. Last but not least, a role for Pax5 in the
regulation of
5, VpreB, or TdT has recently been excluded
(26), although the expression of these genes is downregulated in response to pre-BCR signaling.
The mouse scid mutation affects the XRCC7 gene coding for the catalytic subunit of the DNA-PK, which is essential for V(D)J recombination and double-stranded DNA break repair (8, 41). The phenotype of the scid mouse is known to be leaky in contrast to the RAG mutations, as scid B lymphocytes are still able to generate DH-to-JH and VH-to-DHJH rearrangements at a very low frequency (41). Hence, the scid and Pax5 mutations appear to be comparable with regard to their low efficiency of Ig gene rearrangements and early B cell developmental block. However, expression of a bcl-2 transgene in scid mice results in the accumulation of almost normal numbers of B lymphocytes that express many markers of mature B cells (40, 56). Due to the increased life span, the early progenitor cells present in the bcl-2 transgenic scid bone marrow seem to have a higher probability to generate productive DH-to-JH rearrangements in reading frame 2 and thus to express the truncated Dµ protein that promotes maturation to later B cell stages (56). Interestingly, we have previously shown that DH-to-JH rearrangements occur relatively frequently in reading frame 2 in pre-BI cells of Pax5 mutant bone marrow (24). Nevertheless, expression of the same bcl-2 transgene fails to promote B cell development in Pax5-deficient mice, thus further demonstrating that the early arrest of B lymphopoiesis is neither caused by a rearrangement defect nor by the lack of a survival signal.
The inability of Igµ and bcl-2 transgenes to advance B cell development and the constitutive cell surface expression of the pre-BCR strongly argue that B lymphopoiesis is arrested in Pax5 mutant mice at an early stage that is not responsive to pre-BCR signaling in the absence of Pax5 function. Consistent with this notion, the cell surface marker BP-1, which is specifically expressed on late pro-B (pre-BI) cells (57), is absent on bone marrow cells of Pax5-deficient mice (24). Therefore, it appears that Pax5 controls a critical step between initial B lineage commitment and the pre-BCR stage of adult B lymphopoiesis. In this context it is interesting to note that the interruption of Ras signaling also arrests early B cell development well before the pre-BCR stage (58). Our analysis of Pax5-deficient pre-BI cells has recently demonstrated a pleiotropic role of the transcription factor BSAP (Pax5) in gene regulation during early B lymphopoiesis (26). Hence, it will be a challenge for the future to identify the critical, and thus far unknown, BSAP target gene(s) that mediates the Pax5-dependent control of early B cell development.
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Footnotes |
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Address correspondence to Meinrad Busslinger, Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. Phone: 43-1-797-30-452; Fax: 43-1-798-71-53; E-mail: busslinger{at}nt.imp.univie.ac.at
Received for publication 8 April 1998 and in revised form 9 June 1998.
Claire Thévenin's present address is Department of Medical and Molecular Parasitology, New York University Medical Center, New York, NY 10016.We thank F. Alt (Harvard Medical School, Boston, MA) for providing the RAG2-deficient mouse; T. Imanishi-Kari (Tufts University, Boston, MA) for the transgenic M54 mouse; S. Cory (Walter and Eliza Hall
Institute, Melbourne, Australia) for the Eµ-bcl2-36 strain; M. Nussenzweig (The Rockefeller University,
New York) for the mIgµ-Ig (YS:VV) mouse and anti-Ig
antiserum; T. Rolink (Basel Institute for Immunology, Switzerland) for monoclonal antibodies; and P. Steinlein for help with flow cytometric analysis.
This work was supported by the Research Institute of Molecular Pathology, and in part by a grant from the Austrian Industrial Research Promotion Fund.
Abbreviations used in this paper APC, allophycocyanin; BCR, B cell receptor; BSAP, B cell-specific activator protein; DNA-PK, DNA-dependent protein kinase; IgH, immunoglobulin heavy chain; RAG, recombination activating gene; scid, severe combined immune deficiency; TdT, terminal deoxynucleotidyl transferase.
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