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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 695-705
Developmental Expression of Mouse Erythrocyte Protein 4.2 mRNA:
Evidence for Specific Expression in Erythroid Cells
By
Lingyun Zhu,
Samir B. Kahwash, and
Long-Sheng Chang
From the Division of Hematology-Oncology, Department of Pediatrics,
the Section of Clinical Pathology, Department of Laboratory Medicine,
and the Molecular Cellular & Developmental Biology Program, Children's
Hospital, The Ohio State University, Columbus, OH.
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ABSTRACT |
Erythrocyte protein 4.2 (P4.2) is an important component of the
erythrocyte membrane skeletal network with an undefined biologic function. Presently, very little is known about the expression of the
P4.2 gene during mouse embryonic development and in adult animals. By
using the Northern blot and in situ hybridization techniques, we have
examined the spatial and temporal expression of the P4.2 gene during
mouse development. We show that expression of the mouse P4.2 gene is
temporally regulated during embryogenesis and that the P4.2 mRNA
expression pattern coincides with the timing of erythropoietic activity
in hematopoietic organs. P4.2 transcripts are first detected in embryos
on day 7.5 of gestation and are localized exclusively in primitive
erythroid cells of yolk sac origin. These erythroid cells remain to be
the only source for P4.2 expression until the switch of the
hematopoietic producing site to fetal liver. In mid- and late-gestation
periods, P4.2 mRNA expression is restricted to the erythroid cells in
fetal liver and to circulating erythrocytes. Around and after birth, the site for P4.2 expression is switched from liver to spleen and bone
marrow, and P4.2 transcripts are only detected in cells of the
erythroid lineage. These results provide the evidence for specific P4.2
expression in erythroid cells. In addition, the timing and pattern of
expression of the P4.2 gene suggest the specific regulation of the P4.2
gene.
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INTRODUCTION |
THE ERYTHROCYTE protein 4.2 (P4.2) is one
of the major components in the erythrocyte membrane skeletal network
(for a review, see Becker and Benz1). P4.2 binds to the
cytoplasmic domain of the anion exchanger band 3 and interacts with
ankyrin in red blood cells (RBCs).2-5 Although the exact
function of P4.2 is not defined, patients with P4.2 deficiency in their
RBC membranes suffer from spherocytosis and various degrees of
hemolytic anemia, suggesting a role for P4.2 in maintaining the
stability and flexibility of RBCs (reviewed in Cohen et al6
and Yawata7). The results from recent biophysical and
electron microscopic experiments indicate that P4.2 may serve as an
accessory linking protein to strengthen the interaction between the
integral membrane protein, such as band 3, and the cytoskeletal
network.8-10
Molecular cloning of the human P4.2 cDNA from a reticulocyte cDNA
library shows that the amino acid sequence of P4.2 has significant homology with the transglutaminase family of cross-linking
enzymes.11,12 Two human P4.2 cDNA isoforms of about 2.4 and
2.5 kb, respectively, were isolated, consistent with the presence of
multiple species of P4.2 RNAs in human reticulocytes.12 The
two cDNA isoforms differ by a 90-bp in-frame insertion. As a result of
this difference, the long cDNA isoform encodes a protein of 721 amino
acids, whereas the short isoform encodes a protein of 691 amino acids.
Using genomic cloning and sequencing analysis, we showed that the two human P4.2 cDNA isoforms are generated by alternative
splicing.13 By using isoform-specific antibodies, the
protein products of the two P4.2 cDNA isoforms in human erythrocyte
membranes were identified.13 The relative molecular mass of
the protein encoded by the short P4.2 cDNA isoform on the sodium
dodecyl sulfate (SDS)-polyacrylamide gel is typically 72 kD, existing
as a major P4.2 species in human erythrocyte membranes. The relative
molecular mass of the long cDNA isoform-encoded protein is
approximately 75 kD. The ratio of the 72-kD to the 75-kD P4.2 protein
estimated from the Western blot analysis is about 15:1.13
When human reticulocyte cDNAs were amplified by polymerase chain
reaction (PCR) with primers flanking the two alternatively spliced
isoforms in the human P4.2 cDNA, two other smaller cDNA isoforms were
also detected.14,15 However, the protein products of these
small human P4.2 cDNA isoforms have not been identified. In addition,
the functional significance of all these different human P4.2 isoforms
is presently unknown.
Recently, we16 and others15,17 also cloned the
mouse P4.2 cDNA. Similar to the human protein, the mouse P4.2 amino
acid sequence shows homology with those of transglutaminases. Although there was a discrepancy on the estimated size of the mouse P4.2 RNA
reported by the three groups, only a single P4.2 cDNA that encodes a
protein of 691 amino acids (identical to the size of the major 72-kD
human P4.2 protein) was found in the mouse reticulocyte library. Using
genomic cloning and PCR analysis, we showed that the mouse reticulocyte
P4.2 RNA does not exhibit alternative splicing in the regions
identified in the human P4.2 RNA.16 Presently, the
significance for the difference in P4.2 isoform expression between
mouse and human is not known.
In addition to the P4.2 protein in erythrocytes, immunoreactive forms
of P4.2 with the molecular weight of 72 kD, or larger or smaller than
72 kD, have been detected in nonerythroid cells and
tissues.6,18-20 Presently, the exact structures of these proteins are not known. Immunologic cross-reactivity between the erythrocyte P4.2 protein and other cellular proteins has been reported.6,18,21 In light of the complexity found in other erythrocyte membrane proteins,22-35 whether these
immunoreactive analogs of erythrocyte P4.2 represent true P4.2 protein
isoforms or are just related proteins in nonerythroid cells remains to be determined.
Presently, very little is known about the expression of the P4.2 gene
during embryogenesis and in adult animals. Previous in vivo
pulse-labeling studies showed the asynchronous synthesis of membrane
proteins of the mature erythrocyte, with those of proteins 4.1 and 4.2 finishing last.36,37 By a two-phase liquid culture system
using erythroid burst-forming unit in normal human peripheral blood,
the P4.2 protein can be detected in cells equivalent to the stage of
early erythroblasts.7 In addition, changes in mRNA
expression and synthesis of membrane proteins, including  - and
 -spectrins, and ankyrin have been noted during murine erythroid
differentiation38,39 and erythropoiesis.40
In this report, we describe a comprehensive analysis of mouse P4.2 RNA
expression during development. Our results show that expression of the
mouse P4.2 gene is temporally regulated during embryogenesis and that
the P4.2 mRNA expression pattern matches the timing of erythropoietic
activity in hematopoietic organs. In addition, P4.2 expression is
detected only in the erythroid cell-producing organs and circulating
erythrocytes during mouse embryonic development and in adult animals.
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MATERIALS AND METHODS |
Northern blot analysis.
A mouse embryo Northern blot and a mouse multiple tissue Northern blot
were purchased from Clontech Laboratories, Inc (Palo Alto, CA). Each
lane in the blots contains 2 µg of poly A+ RNA from the
specific tissue as indicated. RNAs from various mouse tissues were also
isolated as described before16 and 50 µg of each tissue
RNA was used in Northern blot analysis.41 The mouse P4.2
cDNA clone pBS(+)/mP4.2-Ust containing the 2.2-kb EcoRI-fragment of the mouse P4.2 cDNA16 was
digested with EcoRI and BamHI enzymes. A 714-bp
BamHI-EcoRI cDNA fragment containing the 3 portion of
the P4.2-coding region was isolated, labeled with 32P by
the random priming technique (Amersham, Arlington Heights, IL), and used as the probe. The blots were hybridized and washed according to standard procedures41 as described
previously.16 The hybridization signal was visualized by
autoradiagraphy. Also, the same blots were stripped to remove the
previously hybridized radioactivity and rehybridized with a
32P-labeled -actin cDNA probe.42
Sample preparation.
FVB/N mice were purchased from Harlan Sprague Dawley, Inc
(Indianapolis, IN) and maintained in our animal facility. Embryos and
whole deciduas were obtained from the natural mating of FVB/N mice.
Gestational age was designated as embryonic day 0.5 (E0.5) on the day
the vaginal plug was found. Organs from mice at various times after
birth also were dissected. Samples were fixed in 10% neutral buffered
formalin (Surgipath, Richmond, IL) at 4°C overnight and
processed for paraffin embedding. Five-micron tissue sections were
prepared, mounted onto Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA), and stored at  80°C until
hybridization.
For preparing bone marrow sections, the femurs from mice at various
times after birth were paraffin-embedded, decalcified with Cal-EX
decalcifying solution (Fisher Scientific), and processed for tissue
sections as described before.
Probe preparation.
Both antisense and sense riboprobes derived from the mouse P4.2 cDNA
were prepared for in situ hybridization studies. Briefly, the same
714-bp BamHI-EcoRI mouse P4.2 cDNA fragment containing the 3 P4.2-coding region as that used in Northern blot analysis was
isolated from plasmid pBS(+)/mP4.2-Ust and cloned into pBS(+) (Stratagene, La Jolla, CA). For preparing the P4.2
antisense riboprobe, the resulting transcription plasmid was linearized
with BamHI and used in the in vitro transcription
reaction41 with T7 RNA polymerase in the presence of
33P-UTP (Amersham). For preparing the P4.2 sense riboprobe,
the transcription plasmid was digested with EcoRI and the
linearized plasmid DNA was transcribed with T3 RNA polymerase as
described above.
In situ hybridization.
Slides containing embryo or tissue sections were allowed to warm to
room temperature and then dewaxed with xylene. Mounted sections were
immersed in 0.2 N HCl for 5 minutes, incubated in 1 µg/mL proteinase
K for 5 minutes at room temperature, and then treated with 0.25%
(vol/vol) acetic anhydride in 0.1 mol/L triethanolamine-HCl, pH 8.0, for 5 minutes. For hybridization, the slides were first incubated with
the prehybridization solution (Novagen, Madison, WI) for 1 hour at 50°C and then hybridized overnight with either the P4.2
antisense or sense riboprobe at 50°C. Subsequently, the slides were
sequentially washed in 2× SSC at 50°C for 30 minutes, in RNase
buffer (0.5 mol/L NaCl, 10 mmol/L dithiothreitol [DTT], and 20 µg/mL RNase A) at 37°C for 30 minutes in buffer containing 0.1×
SSC, 50% formamide, and 10 mmol/L DTT at 50°C for 30 minutes, and
finally in buffer containing 1× SSC, 14 mmol/L DTT, and 0.07% sodium
pyrophosphate at 50°C for 30 minutes. For exposure to
autoradiographic emulsion, the slides were dipped into Kodak NTB-2
emulsion (Eastman Kodak, Rochester, NY) exposed in the
dark at 4°C for 1 to 2 weeks, and then developed with Kodak D-19
developer and Fixer. For counterstaining, the slides were treated with
0.1% crystal violet. Autoradiographic images of the embryo and tissue
sections were visualized under a Zeiss Axiophot microscope and
photographed with Kodak Technical Pan films.
For comparison of histologic cell types, adjacent sections were dewaxed
and processed for standard hematoxylin-eosin staining.
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RESULTS |
Expression of the P4.2 mRNA in adult mouse tissues and during mouse
embryogenesis.
To assess the expression of the P4.2 gene, we first analyzed poly
A+ RNAs from various adult mouse tissues by Northern blot
analysis using a 714-bp mouse P4.2 cDNA fragment containing the 3
portion of the P4.2-coding region as the probe. A single 3.5-kb P4.2
transcript was detected at relatively high levels in spleen (Fig
1A), analogous to our previous findings
using a 2.2-kb P4.2 cDNA probe containing the entire coding
region.16 In contrast, little or no P4.2 hybridization was
seen in other tissues examined. Upon longer exposure of the autoradiogram, a very small amount of the P4.2 message was detected in
heart, whereas no hybridization signal was seen in brain, lung, liver,
skeletal muscle, kidney, or testis. As the control, hybridization of
the same RNA blot with the probe for actins showed comparable intensity
of a 2.1-kb mRNA for cytoskeletal actin and/or a 1.7-kb muscle
actin mRNA43,44 (eg, in heart and skeletal muscle) in each
sample (Fig 1A), indicating the intactness and relatively similar
quantity of mRNAs in each sample.
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| Fig 1.
P4.2 mRNA expression during mouse development. Northern
blot analysis of RNAs from adult mouse tissues (A) and embryos at various embryonic ages (B) was conducted as described in the Materials and Methods. A 714-bp mouse P4.2 cDNA fragment containing the 3
portion of the P4.2-coding region was used as the probe. The P4.2-hybridization signal was visualized by autoradiography (top). A
longer exposure of the blot in (A) is shown in the middle. As the
control, the same blots were stripped and rehybridized with a
32P-labeled -actin cDNA probe (bottom).
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Subsequently, we performed a similar Northern blot analysis of poly
A+ RNAs from mouse embryos at different stages of
development using the same P4.2 cDNA probe. As shown in Fig 1B, no P4.2
hybridization was detected in mRNAs from E6.5 embryos. The expected
3.5-kb P4.2 mRNA was seen in E10.5 embryos and its intensity increased
in the embryos at later ages (E14.5 and E16.5). Similarly, when the actin probe was used as the control, the 2.1-kb cytoskeletal actin band
of about equal intensity was detected in all embryo mRNAs (Fig 1D). In
addition, the 1.7-kb muscle actin message was detected in E14.5 and
E16.5 embryos, consistent with its timely expression during
myogenesis.45 These results indicate that expression of the
mouse P4.2 gene is temporally regulated during embryogenesis and that
P4.2 transcripts are primarily detected in hematopoietic tissues such
as the spleen in the adult animal.
P4.2 gene expression during early gestation embryos.
To determine if the mouse P4.2 mRNA expression pattern coincided with
particular developmental events, in situ hybridization analysis on
sections derived from embryos corresponding to various developmental
stages was performed. Because the 714-bp mouse P4.2 cDNA probe
containing the 3 portion of the coding region specifically detected
the 3.5-kb P4.2 message in Northern blot analysis, the same cDNA
fragment was cloned into pBS(+) and the resulting transcription plasmid
was used to synthesize both the antisense and sense riboprobes for in
situ hybridization. In agreement with the results from Northern blot
analysis, no P4.2-specific labeling was detected in sections of embryos
at E6.5 days when hybridized with the P4.2 antisense riboprobe (data
not shown). A P4.2 hybridization signal, albeit of weak intensity, was
first detected in E7.5 embryos (Fig 2). By
comparing the dark-field image with the bright-field embryo structure,
P4.2-specific labeling was found to be localized in primitive erythroid
cells, such as in the early heart region, of E7.5 embryos (Fig 2A and
B). At this embryonic stage, the primitive erythroid cells are large
nucleated RBCs and derived from the yolk sac, which is the initial site
of erythropoiesis.46-48 The yolk sac consists of an
endodermal epithelium and underlying mesoderm within which blood
islands and vessels develop. Blood islands are the only source of
embryonic erythroid cells until E11.5 days. These primitive erythroid
cells are transported into the embryo for circulation via blood
vessels. P4.2 expression was also detected in the blood islands and
vessels of yolk sac at E7.5 and E8.5 days (Fig 2C and D), consistent
with that detected in primitive erythroid cells in the early heart
region. In contrast, no hybridization signal was seen when P4.2 sense
riboprobe was used (data not shown). Similar hybridizations were
performed with the sense probe for sections of embryos or tissues at
various time points as described below and were found to be similarly
negative.
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| Fig 2.
P4.2 gene expression during early gestation embryos. In
situ hybridization analysis was used to detect P4.2 expression in mouse
embryos at E7.5 (A and B) and E8.5 days (C and D). Sagittal sections of
embryos within the deciduas were hybridized with the P4.2 antisense
riboprobe. (A and C) Bright-field illumination; (B and D) dark-field
illumination. Upon examination of multiple embryo sections,
P4.2-specific labeling was detected in erythroid cells in the early
heart region and blood vessels (indicated by arrowheads in A and B).
NT, neural tube. The blood islands (BI) of the visceral yolk sac (YS)
in (C) and (D) also show P4.2 hybridization. Micrographs were taken
with a 20× (A and B) or 40× (C and D) objective.
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After 8.5 days of gestation, the embryo undergoes accelerated
organogensis. By E9 days, the four-chambered heart is developed and the
circulation is established.48 Analysis of embryos at gestation ages from E8.5 to E10.5 days showed that P4.2-specific labeling in the heart region remains to be seen. P4.2 hybridization signal with increasing intensity was detected only in the heart and
blood vessels of E10.5 embryos (Fig 3A and
B). Specifically, P4.2 expression in the
heart region was confined to circulating erythroid cells but was not
seen in cardiomyocytes (Fig 3C and D). Upon higher magnification of
E10.5 embryo sections, P4.2 hybridization signal with weak intensity
was observed in the liver (Fig 3E and F), localized notably in
erythroid cells in the sinusoids and vessels (indicated by arrowheads).
The remaining organs, such as brain, showed no hybridization. These
results indicate that the erythroid cells of yolk sac origin are the
only source for P4.2 expression during early embryonic development.
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| Fig 3.
P4.2 gene expression in E10.5 embryos. Bright-field and
dark-field views are displayed of hybridization of sagittal sections of
embryos at E10.5 days with the P4.2 antisense riboprobe (A and B).
Higher-power views of the heart (C and D) or liver (E and F) region are
also shown. P4.2 hybridization signal was observed in the erythroid
cells in the sinusoids and vessels of the liver (representatives are
marked with arrowheads). (A, C, and E) Bright-field illumination; (B,
D, and F) dark-field illumination. H, heart; L, liver. Micrographs were
taken with a 10× (A and B) or 20× (C, D, E, and F) objective.
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P4.2 gene expression during mid- and late-gestation embryos.
Starting from E12.5 days, there is a switch in the hematopoietic
producing sites, from the yolk sac to the fetal liver. Upon the
colonization of the multipotential stem cells from the yolk sac to the
liver structure at E10.5 days, the fetal liver begins to take over and
becomes the almost exclusive hematopoietic organ from E12.5 to E16.5
days. In contrast to primitive erythroid cells derived from the yolk
sac, definitive erythrocytes produced from the fetal liver are smaller
and enucleated. As soon as the hematopoietic activity appears in fetal
liver, P4.2 expression could be seen in this organ. As shown in Fig
4, P4.2 hybridization signal could be
detected in the liver, heart, and blood vessels of E12.5 embryos (Fig
4A and B). Other tissues remained negative for P4.2 hybridization. Similar to those observed in embryos at earlier ages (Figs 2 and 3),
P4.2-specific labeling in the heart was detected only in erythroid cells inside the heart chambers (Fig 4C and D). In addition, some P4.2
hybridization signal was seen in circulating erythroid cells in blood
vessels (Fig 4E and F). It should be mentioned that, at this stage,
only about 1% of erythroid cells in circulation are enucleated and
derived from the fetal liver.46-48
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| Fig 4.
P4.2 gene expression in E12.5 embryos. Sagittal sections
of embryos at E12.5 days were hybridized with the P4.2 antisense riboprobe and photographed under bright-field (A) or dark-field (B)
illumination. P4.2 hybridization signal was detected in the heart,
liver, and blood vessels. H, heart; L, liver; DA, dorsal aorta.
Higher-power views of the heart ([C] bright-field illumination and
[D] dark-field illumination) or dorsal aorta ([E] bright-field illumination and [F] dark-field illumination) region are also shown.
Arrowhead points to P4.2-specific labeling in erythroid cells in the
dorsal aorta (E and F). Micrographs were taken with a 10× (A and B)
or 20× (C, D, E, and F) objective.
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As compared with that in E10.5 embryos (Fig 3), P4.2 expression in the
liver was greatly enhanced in E12.5 embryos (Fig 5A and
B). By E14.5 days, the liver showed the
strongest P4.2 hybridization signal among all organs (Fig 5C and D).
P4.2-specific labeling was also detected in the heart and blood
vessels, but their intensity was greatly reduced (compare Fig 5 with
Fig 4), consistent with the dramatic increase of fetal liver-derived,
enucleated erythrocytes in circulation. In contrast, as the main source
of T-lymphocyte differentiation, the thymus showed no P4.2
hybridization in E14.5 embryos (Fig 5C and D). This lymphogenic organ
remained negative for P4.2 expression even after birth (data not
shown).
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| Fig 5.
Expression of the P4.2 transcript during liver
development. Shown are bright-field (A, C, E, and G) and dark-field (B,
D, F, and H) views of hybridization to the P4.2 antisense riboprobe. (A
and B) The liver from an E12.5 (A and B) embryo section; (C and D) the
abdominal region containing the liver from an E14.5 (C and D) or E16.5
(E and F) embryo; (G and H) a section of liver from the mouse at 4 weeks after birth. T, thymus; H, heart; L, liver; M, metanephros.
Micrographs were taken with a 20× (A, B, G, and H) or 2.5× (C, D,
E, and F) objective.
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In E16.5 embryos, P4.2-specific labeling remained strong in erythroid
cells of the hepatic mesoderm (Fig 5E and F). A weak P4.2 hybridization
signal was detected in the heart and blood vessels, because at this
time about 99% of circulating erythrocytes are enucleated. Little or
no P4.2-specific labeling was seen in metanephros, which later develop
into kidneys (Fig 5E and F). After E16.5 days, hepatocytes begin to
speed up in proliferation and differentiation with the biliary duct as
center, developing a mature lobular format; simultaneously, the
hematopoietic cells in the liver begin to recede in hematopoietic
function and their proliferation decreases, with hepatocytes gradually
replacing the hematopoietic cells. In correlation, P4.2 hybridization
signal was greatly reduced in the liver after E16.5 days (Fig 5), being almost undetectable after birth (Fig 5G and H). These results indicate
that P4.2 expression is restricted to hematopoietic cells in the fetal
liver during mid- and late-gestation embryos.
P4.2 gene expression in spleen and bone marrow.
Because around and after birth, the spleen together with the bone
marrow become the major hematopoietic organs in mice, we examined P4.2
expression in these hematopoietic organs by in situ hybridization of
tissue sections, as described above. In newborn mice, P4.2-specific
labeling was detected in specific areas of the spleen (data not shown).
Significant P4.2 expression was seen in the emerging red pulp, which
later makes up the largest part of the organ and is the center for
erythropoiesis and megakaryocyte production. As the components of the
spleen become more distinguished, increasing P4.2 hybridization signal
was still confined to the red pulp on postnatal day 7 and day 15 (Fig
6A and B). No P4.2-specific labeling was
seen in the white pulp, which consists of germinal centers for
lymphocytes, plasma cells, and macrophages. In older animals, P4.2
expression remains localized exclusively in the red pulps (data not
shown). Upon higher magnification of areas containing red pulps from
spleen sections of 15-day-old mice, P4.2-specific labeling was detected
in erythroid cells, particularly at higher intensity in areas with more
mature erythroid cells (lightly stained by crystal violet; Fig 6C and
D). In contrast, P4.2 hybridization signal was not detected in
megakaryocytes (easily identified as giant cells with a polylobulated
nucleus surrounded by a large cytoplasm; Fig 6C and D). Histologic
identification of all these cell types was confirmed by standard
hematoxylin-eosin staining of adjacent tissue sections.
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| Fig 6.
P4.2 gene expression in the mouse spleen and bone marrow.
Shown are in situ hybridization analysis of serial spleen sections from
a 15-day-old mouse (A through D) using the P4.2 antisense riboprobe. A
representative region of red pulp (RP) or white pulp (WP) in the spleen
is indicated in (A) and (B). Higher-power views of a red pulp region
are shown in (C) and (D). The lightly stained areas (labeled with
"E") enrich more mature erythroid cells and show substantial P4.2
hybridization. Arrowheads indicate isolated megakaryocytes. Sections
containing bone marrows from a 7-day-old mouse (E and F) were also
hybridized with the P4.2 antisense riboprobe. A representative
erythroid colony in the bone marrow is labeled with "E."
Micrographs were taken with a 20× (A and B) or 40× (C through F)
objective. (A, C, and E) Bright-field illumination; (B, D, and F)
dark-field illumination.
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To examine what types of cells in bone marrows expressed P4.2
transcripts, we conducted in situ hybridization of bone marrow sections
from mice at various times after birth. Similar to that observed in the
spleen, P4.2-specific labeling was detected only in erythroid cells and
colonies in the bone marrow from 7-day-old mice (Fig 6E
and F). Similar results were obtained in bone marrow of older animals
(data not shown). In addition, no P4.2 hybridization signal was seen in
cells of nonerythroid lineage, including megakaryocytes. These results
indicate that P4.2 message was specifically expressed in cells of the
erythroid lineage in postnatal hematopoietic organs.
| |
DISCUSSION |
We describe here the temporal and spatial expression pattern of the
erythrocyte protein 4.2 gene during mouse embryonic development. We
show that expression of the mouse P4.2 gene is temporally regulated during embryogenesis and that the P4.2 mRNA expression pattern coincides with the timing of erythropoietic activity in hematopoietic organs, suggesting a role for this gene in the differentiation of
erythroid cells.
Erythropoiesis is a complicated differentiation process during which
pluripotent hematopoietic stem cells are orchestrated to become the
differentiated erythrocytes.46,47,49 In mice, this process
is performed in various hematopoietic organs at different stages of
development: yolk sac (E7.5-11.5 days), fetal liver (E12.5-16.5 days),
and bone marrow and spleen (around and after birth). Our in situ
hybridization results show that P4.2 expression follows the ebb and
flow of the erythropoietic activity in each hematopoietic organ at
specific developmental stages.
Consistent with the detection of the P4.2 protein in cultured erythroid
cells corresponding to the stage of early erythroblasts,50 P4.2 transcript was first detected in nucleated erythroid precursors derived from the yolk sac at E7.5 days. During embryonic stage, nucleated erythroid cells derived from the yolk sac progress from pre-type 1 to type 3 erythroblasts during E10.5 to 14.5 days, whereas
fetal liver cells, primarily pro- and basophilic
erythroblasts at E12.5 to 14.5 days, differentiate predominantly into
erythrocytes in the peripheral blood by E16.5 days.40,49 By
comparing with the hybridization signal of circulating erythroid cells
in the heart and vessels of E10.5 embryos, P4.2 expression appears to be increased at E12.5 embryos and then decreased at 14.5 days, indicating the up-and-down change of P4.2 mRNA levels after the differentiation of yolk sac-derived erythroblasts. Similarly, the
up-and-down expression pattern of P4.2 is also observed in the liver
from E10.5 days to after birth. By comparing P4.2 expression in
circulating erythroid cells in the heart with that in fetal liver of
E16.5 embryos, mature erythrocytes released from the fetal liver show
much less P4.2 expression. Analogously, we note that circulating
erythrocytes derived from the spleen and bone marrow after birth gave
rise to very weak P4.2 hybridization signal.
The P4.2 gene is mapped to human chromosome 15 at bands
q15-2113,51 and to mouse chromosome 2.52 The
chromosomal location of the mouse P4.2 gene was found to be near the
mouse pallid (pa) mutation. Pallid was found in a mouse
caught in the wild,53,54 which shows dilution of coat
color,55 increased bleeding time, and abnormal lysosomal
enzyme secretion.56,57 Pallid is one of several
independent mouse mutations that are models of the platelet storage
pool disease.58 In addition, Pallid mice have been
proposed as a model of genetic 1-antitrypsin
deficiency.59 Using Southern and Northern blot analyses,
White et al52 previously suggested that pa is a
mutation in the P4.2 gene. However, patients with P4.2 deficiency do
not have the platelet storage pool deficiency seen in pa/pa
mice and the mutant mice do not have the hemolysis and spherocytosis
seen in the patients. Recently, Gwynn et al60 conducted
additional analyses of P4.2 in pallid mice and concluded that
P4.2 and pa are distinct loci and changes in P4.2 in
pallid mice are not responsible for the pallid
mutation.60
In agreement with our previous findings,16 the Northern
blot analysis detected the expression of a single 3.5-kb mouse P4.2 message in the spleen. Little or no P4.2 expression was found in other
tissues examined when either commercially available RNAs or RNAs that
we isolated from various mouse tissues were used. These results are
different from those reported by White et al.52 They
previously reported the detection of P4.2 transcripts in tissues other
than spleen, including kidney, heart, brain, and liver, and showed that
the heart expressed a larger mRNA than the brain, liver, and kidney in
normal mice. Also, the kidney in pallid mice, an affected organ
by the pallid mutation, expressed a smaller
transcript.52 Intriguingly, we detected no P4.2 transcript in the brain, lung, liver, skeletal muscle, kidney, and testis of adult
mice. Similar results were also obtained when a 2.2-kb mouse P4.2 cDNA
containing the entire coding region was used as the probe (our
unpublished results). In addition, this P4.2 expression pattern was further confirmed by the in situ hybridization analysis, which detects an erythroid tissue-restricted expression for the P4.2
gene. Presently, the reason for the discrepancy is not known. It is
possible that the probe used may affect the specificity of detection,
because we noted that the 3 untranslated region of the mouse P4.2 cDNA
appears to cross-react with other genomic sequences. Alternatively, the
stringency for hybridization and washing conditions in the analysis may
need to be considered.
Our Northern blot analysis also detected a very small amount of the
same 3.5-kb P4.2 transcript in the heart. The results from in situ
hybridization analysis showed that, during embryonic development, P4.2
expression in the heart was detected only in erythroid cells inside the
heart chambers but not in cardiomyocytes. Similarly, we did not detect
any P4.2 expression in the heart muscle at various ages after birth. We
only observed very weak P4.2 hybridization signal in circulating
erythrocytes in the heart and blood vessels. Thus, it is likely that
the low level of P4.2 expression detected in this organ in Northern
blot analysis may represent residual P4.2 mRNA in circulating
erythrocytes.
Immunoreactive forms of the erythrocyte P4.2 protein have been detected
in nonerythroid tissues such as brain, kidney, and platelets and cell
lines including HeLa and HT-29.6,18-20 However, the results
presented in this report show that P4.2 mRNA expression is restricted
to cells of the erythroid lineage. No P4.2 message was detected in
nonerythroid organs or megakaryocytes. In addition, by the reverse
transcription-PCR61 we have not been able to detect any
P4.2-specific cDNA product using RNAs isolated from HeLa or HT-29
cells. Taken together with the observation that the mouse P4.2 gene
only express a 3.5-kb message,15-17 these results indicate
that the immunoreactive forms of P4.2 detected in nonerythroid tissues
or cells are likely not P4.2 protein isoforms; yet, they may be
P4.2-related proteins. It should be noted that proteins related to the
erythrocyte protein 4.1, another erythrocyte membrane protein, have
been identified in nonerythroid cells.32,35 Alternatively,
the immunoreactive forms of P4.2 detected in nonerythroid cells may
represent differences in P4.2 expression among different species or due
to immortalization/transformation (eg, in certain cell lines). Further
characterization of the molecular nature of these P4.2-immunoreactive
analogs will be needed to show their identity. In addition, we are
presently producing mouse P4.2-specific antibodies to examine the P4.2
protein expression.
Nevertheless, the erythroid cell-restricted P4.2 expression pattern in
mice suggests the existence of specific regulation of the P4.2 gene.
Our transcription mapping and sequencing analyses show that the
upstream regulatory regions of both the mouse and human P4.2 genes
contain multiple transcription factor-binding sites, including those of
the erythroid transcription factors GATA-1, TAL-1, and EKLF (Karacay
and Chang, manuscript in preparation). It has been
suggested that these transcription factors, by regulating the cell-type
specific expression of genes, can control the differentiation status of
the hematopoietic cells.62,63 Experiments are in progress
to examine how the P4.2 gene is regulated in the erythroid environment.
| |
FOOTNOTES |
Submitted May 8, 1997;
accepted September 19, 1997.
Supported by research grants from American Heart Association and
Children's Hospital Research Foundation.
Address reprint requests to Long-Sheng Chang, PhD, Department of
Pediatrics, Children's Hospital, The Ohio State University, W230, 700 Children's Dr, Columbus, OH 43205-2696.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs Vincent V. Hamparian and Philip T. Nowicki for critical reading of the manuscript and to members of the
Chang Laboratory for helpful discussions.
| |
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Abstract
Introduction
Abstract