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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 359-361
Embryonic Hemoglobins Are Expressed in Definitive Cells
By
H.Y. Luo,
X.L. Liang,
C. Frye,
M. Wonio,
G.D.V. Hankins,
D.H.K. Chui, and
B.P. Alter
From the Divisions of Pediatric Hematology/Oncology and
Maternal/Fetal Medicine, University of Texas Medical Branch,
Galveston, Texas; and the Department of Pathology and Molecular
Medicine, Faculty of Health Sciences, McMaster University, Hamilton,
Ontario, Canada.
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ABSTRACT |
Human embryonic  and  globin chains are synthesized in yolk
sac-derived primitive erythroid cells, and decrease rapidly during
definitive erythropoiesis. Examination of and globin expression
at the cellular level using dual-color immunofluorescence staining with
specific monoclonal antibodies showed that embryonic globin proteins
are present in definitive erythroid cells. More than half of fetal
erythrocytes were positive for and ~5% for globin.
Approximately one third of newborn red blood cells were -positive
and less than 1% -positive. Adult erythrocytes did not have
embryonic globins. Erythroblasts that developed in liquid cultures also
contained embryonic globin in amounts which declined with ontogenic
age, and the proportion of positive cells in vitro was less than in the
comparable erythrocytes that developed in vivo. Thus, embryonic globin
chains are synthesized in definitive erythroid cells and decrease with
ontogeny. Modulation of embryonic globin gene expression is not solely
due to a switch from primitive to definitive erythropoiesis.
© 1999 by The American Society of Hematology.
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ARTICLE |
YOLK SAC-DERIVED primitive
erythroid cells remain nucleated and definitive erythroid cells
terminally enucleate. Hemoglobin (Hb) expression evolves simultaneously
from embryonic Hbs Gower 1 (2 2), Gower 2 ( 22), and Portland 1 (2 2) to fetal Hb F (22) and then to adult Hb A
(2 2).1-4 Small amounts of
embryonic globins have been detected in hemolysates from definitive
fetal and newborn erythrocytes; usually exceeded
.5-10 Fetal, cord, and adult erythroblasts and
reticulocytes had small amounts of embryonic globin mRNA, with more than .11-14 The only adults who expressed globin had
-thalassemia trait because of the  SEA/ deletion of
20.5 kb including  , 1, and 2, which leaves the gene
intact.15-17 Single cell immunocytology has confirmed the absence of embryonic-positive erythrocytes in normal adults, as well
as in children with juvenile chronic myelogenous leukemia (JCML) in
which Hb F is increased.16,18 Lysates from cultured fetal
erythroblasts have also been shown to contain small amounts of
embryonic globin, with usually exceeding .10,19,20 A few cells from JCML cultures were embryonic positive, with more + than + cells.18
None of those studies systematically examined embryonic globin
expression in normal definitive erythrocytes and erythroblasts at all
ontogenic stages. To address this, we used multicolor single-cell immunofluorescence with monoclonal antibodies (MoAbs) specific for and globin chains7,17,21 to examine erythrocytes derived in vivo and erythroblasts developed in vitro in
cytokine-induced liquid cultures from fetal, newborn, and adult
blood.22 We show that the proportion of embryonic globin
positive cells decreases during ontogeny, and globin expression
exceeds .
Heparinized blood was obtained from normal adults, term deliveries, and
fetuses at termination of pregnancy for nonhematologic indications. All
procedures were approved by the Institutional Review Board of the
University of Texas Medical Branch. Mononuclear cells (MNC) were
cultured in a serum-free modification of a two phase liquid
system22 by using BIT (StemCell Technologies, Inc, Vancouver, British Columbia, Canada). Phase 1 (days 0 to 7) contained 106 MNC/mL and stem cell factor (SCF, 4 ng/mL; Boehringer
Mannheim, Corp, Indianapolis, IN), and Phase 2 (days 7-14) had 2 × 105 cells/mL, 4 ng/mL SCF, 2 U/mL erythropoietin (Ep;
Amgen, Inc, Thousand Oaks, CA), 20 u/mL interleukin-3 (IL-3), and 100 u/mL IL-6 (both from Boehringer Mannheim). Anti- and anti- MoAbs were developed previously.7,17 Blood smears or culture
cytospin slides were dried overnight and fixed with acetone:methanol
(8:2) for 10 minutes (erythrocytes) or acetone:methanol:ethanol (3:1:1) for 20 minutes (cultured erythroblasts). Cells were stained for 30 minutes at 37°C with MoAbs labeled with fluorescein isothiocyanate (FITC; anti-) or Texas Red (TR; anti-) (Molecular Probes, Inc, Eugene, OR). The stained slides were thrice washed with phosphate buffered saline and mounted with Vectoshield (Vector, Burlingame, CA).
Cells were examined with a Nikon fluorescence microscope (Melville, NY)
with a dual color bandpass filter. Fluorescence images were captured
with a Vysis SmartCapture FISH Imaging System (Vysis, Downers Grove, IL).
Fetal definitive erythrocytes contain variable amounts of embryonic globin. The proportion with strong green fluorescence (+) was 53 ± 4% in eight samples at 15 to 22 weeks' gestation, while only 5% ± 2% of the fetal erythrocytes were red or yellow, ie, + or
+/+ (Fig 1A). Cultured fetal
erythroblasts from four experiments were 18% ± 4% + and only 1%
+ or +/+ (Fig 1D). The proportion of embryonic positive cells
was lower in cultured erythroblasts than in erythrocytes, perhaps
because the progenitors that differentiated in culture were
ontogenically older than the progenitors which had led to erythrocytes
in vivo. Both embryonic globin chains are clearly present in definitive
cells after the first trimester, although there is a difference in
their regulation.
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| Fig 1.
Embryonic globin protein detection with dual color
immunocytofluorescence staining. Samples were from 22-week-old fetus,
term cord, and adult blood. Blood smears or cytospins were stained with
MoAbs specific for and globins conjugated with FITC and TR,
respectively. (A) Fetal, (B) cord, and (C) adult erythrocytes. (D)
Fetal, (E) cord, and (F) adult cultured erythroblasts.
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In six cord blood samples there were 34 ± 6% + erythrocytes,
significantly fewer than in fetal samples. Five of six cord samples had
0.6 ± 0.5% + or +/+ erythrocytes (Fig 1B). In cultures from
three cord blood samples 4% ± 1% of the erythroblasts were + and
rare cells were + (Fig 1E). Thus, embryonic globins are clearly
present in erythroid cells at term birth. There were no embryonic-positive adult erythrocytes (Fig 1C), and cultured adult erythroblasts positive for or comprised 0.2% of the cells in only 1 of 7 experiments (Fig 1F).
The ontogenic decline in the proportion of embryonic-positive cells is
documented in Fig 2. The differences
between the percent + cells in fetal, cord, and adult erythrocytes
and erythroblasts are significant at P < .001. The data for
the expression of globin are also compelling. The amount of
embryonic globin per cell appears qualitatively to decrease during
ontogeny.
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| Fig 2.
The percent of cells that were positive for embryonic
globins in erythrocytes and cultured erythroblasts. Left, + cells.
Right, + cells. RBC, red blood cells; Culture, day 14 of culture;
F, fetal; C, cord; A, adult. Note the difference in the y-axis scales
for the two embryonic globins. There is an ontogenic decline in the
percent positive cells, both in vivo and in culture.
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The and non- globin gene clusters may be regulated differently.
The non- cluster consists of , G, A, ,  , and
genes and has embryonic, fetal, and adult stages. The locus
control region (LCR) 5 to 20 kb upstream from has open chromatin
only in erythroid cells, and gene regulation is autonomous. In
transgenic mice, is expressed only in primitive erythroid cells and
undergoes developmental silencing in definitive cells. With a yeast
artificial chromosome (YAC) construct of , , and genes, all
primitive cells contained RNA, although many also had RNA,
which is consistent with sequential or simultaneous transcription of
the globin genes. In definitive cells, developmental stage-specific transacting factors may affect the interaction of the LCR with or , but not with the gene.23-26 Our single cell
data suggest that silencing may be leaky, because a few definitive
cells in fetal and newborn blood were strongly positive for globin protein.
The cluster consists of a DNAse I hypersensitive region (HS-40),
which is 40 kb upstream, followed by 2, 1, 2, 1, 2, 1, and  genes. Unlike the LCR, the HS has open
chromatin in both erythroid and nonerythroid cells. Developmental
silencing of is regulated by synergy between the 5' promoter
and 3' flanking sequences, with an additional autonomous component.
There are both transcriptional and posttranscriptional regulators of normal and -globin gene expression.23,27-30
We have documented embryonic globin chains in definitive cells, a
developmental decline in the proportion of positive cells, and an
apparent decrease in the amount per cell. expression decreases
more rapidly than , perhaps related to transcription autonomy,
and is modulated at both transcriptional and posttranscriptional levels. One reason for these differences may be that there is a
clear fetal stage for non- genes (ie, ), but there is not one
for genes. The persistence of -gene expression in fetal definitive cells may be comparable to the appearance of globin chains.
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FOOTNOTES |
Submitted November 23, 1998; accepted March 5, 1999.
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.
Address reprint requests to B.P. Alter, MD, Division of Pediatric
Hematology/Oncology, Children's Hospital C3.270, 301 University Blvd,
University of Texas Medical Branch, Galveston, TX 77555-0361;
e-mail:balter{at}utmb.edu.
| |
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