The development of B lymphocytes from progenitor cells is dependent on the expression of a
pre-B cell-specific receptor made up by a µ heavy chain associated with the surrogate light
chains, immunoglobulin (Ig)
, and Ig
. A variant pre-B cell receptor can be formed in which
the µ heavy chain is exchanged for a truncated µ chain denoted Dµ. To investigate the role of
this receptor in the development of B cells, we have generated transgenic mice that express the
Dµ protein in cells of the B lineage. Analysis of these mice reveal that Dµ expression leads to a
partial block in B cell development at the early pre-B cell stage, probably by inhibiting VH to
DHJH rearrangement. Furthermore, we provide evidence that Dµ induces VL to JL rearrangements.
 |
Introduction |
During B cell differentiation, the genes encoding the
heavy and light chains of the immunoglobulin molecules are assembled from germline gene segments in an ordered fashion (1, 2). Initially, a DH segment is joined to a JH
segment in the heavy chain locus on both chromosomes.
Subsequently, a VH gene segment is rearranged to the DHJH
complex. If this renders a functional rearrangement, a µ heavy chain is expressed on the cell surface, together with
the surrogate light chains encoded by the genes
5 and
Vpre-B (3). This complex, denoted the pre-B cell receptor (pBCR),1 has been shown to be of vital importance
for maturation of B lymphocytes. Thus, in mice deficient
in this receptor, B cell differentiation is arrested at an early
stage (6). Moreover, the pBCR gives a signal to the cell
to stop further rearrangements on the heavy chain locus
(10) and to upregulate rearrangement of the light chain
gene segments (14, 15). Due to an inexact joining mechanism, the DH can be rearranged to the JH in three possible
reading frames (RFs). A majority of the DH segments carry their own promoter and an ATG translational initiation
codon. When the DH is rearranged to a JH in RF2, according to the nomenclature of Ichihara et al. (16), this DHJH
complex can be translated into a truncated µ chain, denoted Dµ (17). A well-documented underrepresentation of
RF2 in VHDHJH as well as DHJH joints (18) has been
suggested to be mediated by this protein expressed on the
cell surface (19). The mechanism by which this occurs is, however, unknown. It has been postulated that the Dµ
protein in association with the surrogate light chains (Dµ
pBCR) possess signalling properties similar to those given
by the pBCR, including signals mediating allelic exclusion
(21). If so, cells expressing the Dµ protein on the cell
surface would be arrested at an early developmental stage
due to the absence of a complete µ heavy chain.
To investigate the effect of Dµ expression on B cell differentiation, we generated mice transgenic for the Dµ protein under control of its endogenous promoter (Dµ-endo)
or, alternatively, under control of the pre-B cell and B cell-
specific mb-1 promoter (Dµ-mb-1; references 25).
 |
Materials and Methods |
Transgenic Constructs.
The Dµ transgenic constructs were
created by PCR amplification of DHJH rearrangements, using
DNA from large pre-B cells as template. The primers (Fig. 1 A, a
and b) hybridize to sequences 0.42 kb 5
of the DH segment and
0.62 kb 3
of JH4, respectively. The PCR products were sequenced, and a fragment consisting of DFL16.1 joined to JH4 in
RF2 was cut with NotI and EcoRI, and cloned into pBluescript.
A second construct in which the endogenous promoter was replaced with the mb-1 promoter, was generated by PCR amplification using primers c and b (Fig. 1 A), and the fragment was cut
with BamHI/EcoRI and cloned into pBluescript containing the
mb-1 promoter. The 0.3-kb fragment containing the mb-1 promoter was isolated by PCR according to the published sequence
(27). Next, the plasmids were cut with Xba1 and Xho1, and ligated with a 9.8-kb XbaI-XhoI fragment from the plasmid p21-H22 (10), provided by Dr. T. Leandersson (University of Lund,
Lund, Sweden), containing the complete membrane heavy chain
constant region. The plasmids containing the final constructs
were digested with NotI and XhoI, the inserts were gel purified
and injected into fertilized oocytes of F1(C57BL/6 × CBA)
mice. The injected zygotes were transplanted into oviducts of
pseudopregnant female mice. Tail DNA from offspring was digested with BamHI, and a 0.3-kb probe comprising the Cµ1 exon was used for Southern blot, detecting a band of 6.2/6.5 kb in transgenic mice (Fig. 1 B).

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Fig. 1.
(A) Schematic outline of the transgenic constructs
used. The Dµ-endo construct
included a 0.3-kb fragment containing the endogenous promoter (open box), a DFLJH4 rearrangement in RF2, the IgH
enhancer (E), and the complete
Cµ sequence. Arrowheads, the location of PCR primers used to
generate the constructs. The
probe used for Southern blot
analysis is depicted in the figure
together with the size of the
BamHI fragment detected in
transgenic mice (6.5 kb). The
construct used to create Dµ-
mb-1 transgenic mice was derived from the Dµ-endo construct by replacing the region
upstream of the ATG with a 0.3-kb fragment containing the mb-1
promoter (shaded box). B,
BamHI; N, NotI; R, EcoRI; Xb,
XbaI; Xh, XhoI. (B) Southern
blot analysis of genomic tail
DNA digested with BamHI and
hybridized with Cµ exon 1 as a
probe. The 6.5-kb Dµ transgenic band and the ~10-kb band representing the endogenous Cµ locus are indicated. Lane 1, C57/BL6; lane 2, Dµ-endo,
founder 13; lane 3, Dµ-mb-1, founder 23. The transgenic band in lane 3 is 0.3 kb shorter than in lane 2, due to the insertion of a BamHI site 3 of the
mb1 promoter in the construct. (C) Northern blot analysis of total RNA from bone marrow and spleen cells hybridized with a probe complementary to
the DHJH joint used in the constructs. Blots from transgenic (tg) mice and littermate controls (lm) are shown. Lanes 1, 2, 5, and 6: Dµ-mb-1 line 26. Lanes 3, 4, 7, and 8: Dµ-endo line 11. The 1.9-kb Dµ transcript and the 18 S ribosomal RNA are indicated in the figure. GAPDH was used as a control
for quantification. (D) Expression of the Dµ protein in transgenic mice. Proteins extracted from bone marrow or spleen cells were analyzed by Western
blot. A band of ~69 kD was detected in transgenic mice using an anti-IgM antibody. Blots representing transgenic (tg) mice and littermate controls (lm)
are shown. Dµ-mb-1, lanes 1, 2, 5, and 6; Dµ-endo, lanes 3, 4, 7, and 8. (E ) VHDHJH and DHJH rearrangements in Dµ-endo transgenic mice.
B220+CD43+ pre-B cells were isolated by cell sorting from transgenic mice (tg) and from littermate controls (lm). Semiquantitative PCR (28) was performed using a primer hybridizing to a sequence downstream of JH2, together with primers complementary to either recombination sequences 5 of all D
regions or to members of the J558 family. As the target sequence for the JH primer is not included in the transgenic construct, there is no amplification
of the transgene itself. Limited PCR amplification of a nonrearranging locus ( 5) was used to normalize the DNA content in the reactions. PCR products were hybridized with a probe complementary to the JH1 and JH2 exons, or to the 5 gene. The upper and lower panels show rearrangements to the
JH1 and the JH2 gene segments, respectively.
|
|
PCR Amplifications and Southern Blot Analysis.
PCR amplifications performed to isolate the gene fragments used for the transgenic vectors were carried out over 30 cycles (1 min at 95°C, 1 min
at 55°C, and 2 min at 72°C) in a programmable thermal controller
(PTC-100; MJ Research Inc., Watertown, MA), using DynaZyme II DNA polymerase (Finnzymes Oy, Espoo, Finland) with
the supplied buffer. The following primers were used for PCR
amplification: (a) 5
-CGCGCGGCCGCTCAAAGCACAATGCCTGG-3
, (b) 5
-GGAATTCCTTCTAATATTCCATACACATA-3
, and (c) 5
-AAGGATCCATGACAACTGAAACTCAACC-3
.
Quantification of IgH chain rearrangements were performed
according to Costa et al. (28). In brief, DNA was prepared by lysing 4-16 × 104 CD43+, B220+, IgM
cells in 200 µl lysis buffer
(50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1%
SDS, and 60 µg/ml proteinase K), incubating them at 55°C for 2 h,
and then precipitating the DNA with isopropanol. The DNA was
dissolved in water, and used for PCR at a concentration of ~5-20
ng/reaction. PCR amplifications were carried out for 30 cycles
(30 s at 95°C, 2 min at 55°C, and 1 min and 45 s at 72°C) in a
programmable thermal controller (PTC-100; MJ Research Inc.),
using DynaZyme II DNA polymerase (Finnzymes Oy) with the supplied buffer. Limited PCR amplification of a nonrearranging locus (
5) was used to normalize the DNA content in the reactions. The following primers, at a concentration of 100 ng/reaction, were used for PCR amplification: JH: 5
-GGCTCCCAATGACCCTTTCTG, DH: 5
-GTCAAGGGATCTACTACTGTG,
VJ558: 5
-TCCTCCAGCACAGCCTACATG, 5
5: 5
-CAAGTCTGACCCCTTGGTCACTC, 3
5: 5
-TGTGAGGCATCCACTGGTCAGATA. One-tenth of the 50 µl PCR reaction
was run on a 1.7% agarose gel, blotted onto Zeta-Probe GT blotting membranes (BioRad, Hercules, CA) and hybridized with a
510-bp probe spanning the JH1 and JH2 exons (for detection of
DHJH and VHDHJH rearrangements) or a 690-bp probe (29; provided by Dr. L. Mårtensson, University of Lund, Lund, Sweden)
for detection of
5. Intensity of the bands was determined using a
Phosphor Imager (Molecular Dynamics, Sunnyvale, CA).
Northern Blot Analysis.
Total RNA from spleen and BM was
isolated using Ultraspec RNA isolation system (Biotecx, Houston, TX). The RNA was electrophoresed in a 1.2% agarose/
formaldehyde gel, transferred to Zeta-Probe GT blotting membranes (BioRad), and hybridized according to the manufacturer's
recommendations. A 0.8-kb fragment from the transgene construct, spanning the DHJH complex, was used as a probe to detect Dµ transcripts.
transcripts were detected using a 0.4-kb fragment comprising the 3
portion of the C
gene, and a 0.9-kb
probe containing part of the mouse mb-1 gene (25; provided by
Dr. Michael Reth, Institute for Biology III, Freiburg, Germany)
was used to determine the amount of B cell-derived RNA in the
samples.
Western Blot Analysis.
Proteins were prepared by lysing 3-10 × 106 cells from bone marrow, or from spleen, in 100 µl sample
buffer (135 mM Tris-HCl, pH 6.8, 2.5% SDS, 10% glycerol, 10%
2-mercaptoethanol, and bromophenolblue indicator [BFB]) and
applying the suspension to a 5-15% SDS-PAGE gradient gel.
Fractionated proteins were electroblotted onto Immobilon-P transfer membranes (Millipore Corp., Bedford, MA) using a
Trans-Blot cell (BioRad). Immunodetection of the Dµ chain was
carried out using a horseradish peroxidase-labeled anti-IgM antibody (Southern Biotechnology Associates, Birmingham, AL) and
an ECL Western blotting kit (Amersham Corp., Arlington
Heights, IL) according to the manufacturer's protocol.
Flow Cytometry Analysis and Cell Sorting.
Bone marrow cells
were flushed out of the femurs with HBSS. Spleen cells were obtained by homogenization of the organ in the same medium.
Cells were collected by centrifugation, resuspended in FACS medium (3% fetal calf serum and 0.1% sodium azide in PBS), counted, and 106 cells/25 µl were incubated with antibodies.
The antibodies used were: biotin-coupled anti-B220 RA3.6.B2
(30), FITC-labeled anti-IgM (Southern Biotechnology Associates), FITC-labeled anti-CD43 (PharMingen, San Diego, CA),
biotin-coupled anti-heat stable antigen (HSA) (PharMingen), and
streptavidin PE-labeled anti-BP-1 (PharMingen). PE- and Cy-chrome-conjugated streptavidin were obtained from PharMingen. Stained cells were analyzed on a FACSCalibur® (Becton
Dickinson, Mountain View, CA). For cell sorting, bone marrow
cells were stained with the same reagents and separated on a
FACStar Plus® (Becton Dickinson).
 |
Results and Discussion |
Dµ Expression Leads to Arrest in VH to DH JH Rearrangements.
Five founder transgenic mouse lines were established expressing Dµ under the control of the endogenous
DH promoter, and four lines were established with Dµ expression controlled by the mb-1 promoter (25-27; Fig. 1
A). Each of the founder mice were crossed to C57BL/6
mice and were analyzed for integration of the transgene by
genomic Southern blots using a probe hybridizing to the
Cµ1 exon (Fig. 1 B). Transcription of transgenic Dµ in
splenic and bone marrow cells from transgenic mice was
demonstrated by Northern blot analysis (Fig. 1 C). Western
blot analysis of cell lysates was used to confirm the expression of transgenic Dµ protein. Thus, Dµ protein was
readily detected in bone marrow cells and in spleen cells
from transgenic, but not from littermate mice (Fig. 1 D). To directly test if the expression of Dµ protein would affect the VHDHJH rearrangement process, the relative
amount of complete VHDHJH and of incomplete DHJH rearrangements was estimated in B220+CD43+ early B cell
progenitors using a semiquantitative PCR assay (28). As illustrated in Fig. 1 E, the relative amount of complete VHDHJH rearrangements was found to be severely reduced in
the transgenic mice compared to littermate controls. In
contrast, DJH rearrangements were more abundant in the
transgenic mice. Together, these data provide evidence for
that expression of the Dµ protein mediates inhibition of
VH to DJH rearrangements.
Partial B Cell Depletion in Dµ Transgenic Mice.
To study
the effect of the transgenically expressed Dµ protein on the
B cell compartment, newborn liver, bone marrow, and spleen cells from two transgenic lines were analyzed by
flow cytometry. Analysis of B lymphocytes from newborn
mice revealed an ~3- and 15-fold reduction of IgM positive cells in the liver (Table 1, Fig. 2). In 3-wk-old mice, B
cell numbers were reduced about fourfold in transgenic
mice compared to littermate controls (Table 1). In adult
spleen, the number of total B cells was approximately twofold lower in the transgenic mice (Table 1), whereas the T cells numbers were apparently unchanged (data not
shown). At all time points analyzed, no significant difference in the proportion of CD5+ and CD5
B cells in the
peritoneum was observed, indicating that the generation of
B-1 cells and of conventional B cells were similarly affected by the transgenic Dµ expression (data not shown).

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Fig. 2.
Flow cytometric analysis of newborn
liver and bone marrow cells from Dµ-endo transgenic mice, Dµ-mb-1 transgenic mice, and littermate controls. Cells were stained with anti-B220-PE
and anti-IgM-FITC and analyzed on FACScan®.
The lymphocyte population was gated according to
standard forward- and side-scatter values. The numbers above the framed areas represent the percentage of B220+IgM and B220+IgM+ cells out of the
total number of gated lymphocytes.
|
|
Impairment of B Cell Differentiation in Dµ Transgenic Mice
Occurs at the Pro/Pre B Cell Stage.
To identify the stage at
which the B cell development was affected by the transgenic expression of Dµ, bone marrow cells from adult
mice were analyzed by flow cytometry. The number of
immature and mature B cells (B220+IgM+) was diminished
approximately two- to threefold in transgenic mice,
whereas the B220+IgM
population, including most B cell
progenitors, was similar or only slightly reduced compared
to littermate controls (Fig. 2).
The stages of B cell differentiation have been subdivided
into fractions (A-F) on the basis of expression of the cell
surface markers B220, CD43, HSA, BP-1, and IgM (31).
Analysis of bone marrow cells using these markers revealed
that the B220+CD43+ early progenitors were slightly increased in transgenic mice, whereas B220+CD43
cells
were fourfold reduced compared to littermate controls
(Fig. 3). These results suggested that the observed block in
B cell development induced by Dµ expression occurs before the transition of late pro-B cells to the pre-B cell
stage.

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Fig. 3.
Flow cytometric analysis of bone marrow cells from 3-wk-old
Dµ-endo transgenic mice, Dµ-mb-1 transgenic mice, and littermate controls. (A) FACS® profiles of cells stained with anti-CD43 and anti-B220 and analyzed on FACScan®. The lymphocyte population was gated
according to standard forward- and side-scatter values. The numbers
above the framed areas represent the percentage of B220+CD43 and
B220+CD43+ cells out of the total number of gated lymphocytes. (B)
Numbers of B220+CD43 and B220+CD43+ cells per femur displayed as
histogram plots.
|
|
To further dissect at what point in early B cell differentiation Dµ expression exerts its effect, BP-1, CD43, and
HSA expression was used to subdivide B cell progenitor
cell populations (31). As shown in Fig. 4, the CD43intBP1+
cell population (late pro-B cells) was slightly increased in transgenic mice compared to littermate controls. However,
although the number of CD43intBP1+ cells with low expression of HSA was almost twofold higher in the transgenic versus wild-type mice, the number of CD43intBP1+
cells with high level expression of HSA was similar. Thus,
the partial block in B cell development induced in the Dµ
transgenic mice occurs at the transition of the fraction C to
the fraction C
in the nomenclature of Hardy et al. (31),
i.e., at the developmental stage where µ chain expression
on the cell surface is required for further differentiation.

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Fig. 4.
Flow cytometric analysis of bone marrow cells from 3-wk-old
Dµ-endo transgenic mice, Dµ-mb-1 transgenic mice, and littermate controls. (A) FACS® profiles of cells stained with anti-CD43, anti-BP-1,
and anti-HSA, and analyzed on FACScan®. The lymphocyte population
was gated according to standard forward and side-scatter values. Cells expressing BP-1 and intermediate levels of CD43 (indicated as a framed
population) were analyzed for HSA expression and are displayed as a separate histogram. (B) Numbers of CD43+, BP-1+, HSAhigh and CD43+,
BP-1+, HSAlow cells per femur are displayed as histogram plots.
|
|
Our results provide support for the hypothesis that the
Dµ pBCR can mediate a block in B cell development,
probably by inhibiting further VH to DHJH rearrangement
(32, 33) similar to the pBCR (10). This mechanism appears, however, to allow leakage of cells that can complete
VHDHJH rearrangement in the presence of Dµ expression. This is not surprising in view of the observed production of
endogenous rearrangements in transgenic mice expressing a
IgH chain (10). It is predicted from the proposed action
of Dµ expression that most or all of these mature B cells
should contain only one complete VHDHJH rearrangement.
Experiments addressing this issue are presently ongoing.
Dµ Induces Light Chain Rearrangements.
In addition to
inhibiting VH to DHJH rearrangements, Dµ expression has
been suggested to mediate induction of VL to JL rearrangements (21, 32). If so, progenitor B cells of the Dµ transgenic mice would be expected to rearrange the light chain
locus despite the arrest in B cell development and the possible impairment of VH to DHJH rearrangements. To test
this hypothesis, we analyzed the levels of expression of
chain messenger RNA in bone marrow cells. The levels of
transcripts were found to be similar in transgenic mice
compared to littermate controls (Fig. 5). Since in transgenic mice there is a three- to four-fold reduction in the B cell
progenitors that normally produce L chain transcripts, these
results suggest that Dµ expression induces VL to JL in progenitors that normally would contain the light chain loci in
germline configuration. It appears, thus, that Dµ can replace the complete µH chain in terms of mediating induction of VL to JL rearrangements.

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Fig. 5.
Northern blot analysis of total RNA from bone marrow and spleen cells hybridized
with a probe complementary to
the C . Blots from transgenic (tg)
mice and wild-type littermates
(lm) are shown. Lanes 1, 2, 5,
and 6: Dµ-mb-1 line 26. Lanes
3, 4, 7, and 8: Dµ-endo line 11. The transcript is indicated in
the figure. mb-1 was used as a
control for quantification.
|
|
We conclude from these results that the Dµ pBCR can
mediate a block in B cell development, probably by inhibiting VH to DHJH rearrangements, as well as inducing VL to
JL rearrangements. In contrast, Dµ cannot substitute for the
requirement of µH chain expression for pre-B cell transition. These observations are in agreement with previous
reports demonstrating that Dµ counterselection is mediated through the transmembrane domain of the membrane
Dµ protein (33) and is dependent on the expression of Ig
(23) and Syk (22). It has been suggested that the inability of
Dµ to mediate pre-B cell transition would be due to a failure to pair with L chains (24). This explanation seems unlikely, however, because counterselection of the Dµ protein-encoding RF2 appears to occur before the stage of L
chain expression (34). An alternative explanation may be
that the Dµ pBCR and the pBCR generate qualitatively different signals (12). Further analysis of the Dµ transgenic mice will be able to directly assess this possibility.
Received for publication 4 September 1997 and in revised form 18 December 1997.
We thank Drs. A. Cumano, J. Demengeot, B. Eriksson, and P. Perreira for discussions and for reviewing the
manuscript.
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