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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3624-3635
By
From Institut d'Embryologie Cellulaire et Moléculaire-CNRS UPR
9064, Nogent-sur-Marne, France; Unité de Recherche en
Hématopoïèse Moléculaire-INSERM U474,
Hôpital Henri Mondor, Créteil, France; and the Department
of Molecular and Cellular Biology, Howard Hughes Medical Institute,
Harvard University, Cambridge, MA.
It is now accepted from studies in animal models that hematopoietic
stem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form of
CD34+CD45+Lin
EARLY IN THE embryogenesis of higher
vertebrates, hematopoietic stem cells (HSCs) arise in situ in the
extraembryonic yolk sac mesoderm and produce locally a transient wave
of primitive nucleated red blood cells. Thereafter, migrating HSCs seed
the successively emerging hematopoietic organ rudiments of the embryo, where they give rise to multilineage differentiated blood cells. The
last blood-forming tissue anlage to be colonized is that of the bone
marrow, in which hematopoiesis becomes definitively stabilized at
postnatal stages. Consequently, the yolk sac was long considered as the
original and only provider of self-renewing stem cells for life-long
hematopoiesis.1,2 The demonstration in birds3,4 and in amphibians5,6 that HSCs responsible for definitive hematopoiesis do not emigrate from extraembryonic tissues but rather
originate within the splanchnopleural mesoderm of the embryo proper
prompted the search for an equivalent intraembryonic source of HSCs in
mammals. Indeed, the para-aortic splanchnopleura (p-SP) in the early
mouse embryo and derived aorta-gonad-mesonephros (AGM) territory were
found to harbor, in parallel with the yolk sac, pluripotential
hematopoietic cells before the onset of fetal liver
colonization.7-9 When dissected out before the
establishment of circulation, the p-SP but not the yolk sac gave rise
to multipotential hematopoietic progenitors in vitro.10 In
contrast, after circulation connected the yolk sac with the embryo,
stem cells endowed with T- and B-lymphoid potential as well as true
long-term repopulating activity were detected in both the p-SP and yolk
sac.10-13 Together, these data strongly suggested that,
independently of the yolk sac, a wave of HSCs arise within the
splanchnopleural mesoderm of the embryo between the presomitic and
liver colonization stages. Accordingly, transient clusters of
CD34+, c-Kit+, Flk-1+
hematopoietic cells were observed during that developmental period on
the ventral aspect of the mouse aorta and omphalomesenteric artery,14-16 reminiscent of the intra-aortic and
para-aortic blood cell foci at the origin of definitive hematopoiesis
in birds.17,18
The molecular mechanisms that locally influence the emergence and
primary expansion of HSCs from mesodermal precursors remain unclear,
although key regulators of these earliest developmental steps have now
been evidenced in mice. The importance of the c-kit signaling pathway
was first discovered through the severe hematopoietic stem cell defects
that occur in Sl and W mutant mice.19,20 Other potential
early hematopoietic factors have been identified by gene targeting in
mouse embryonic stem (ES) cells. c-myb knock-out causes a lethal
deficit in definitive multipotential progenitors,21 and a
complete block in definitive hematopoietic potential is observed in ES
cells null for the expression of the GATA-2, tal-1/SCL and AML-1
transcription factors.22-26 Inactivation of the flk-1 tyrosine kinase gene prevents yolk sac blood island formation and
endothelial development,27 but also precludes definitive hematopoiesis even when normal vascular structures are
present.16 It is not clear whether the primary
emergence or the survival and expansion of HSCs are disturbed in these
experiments. However, the low numbers of HSCs retrieved in both c-myb-
and GATA-2-null mutant embryos would suggest a role of these factors
in stem cell proliferation rather than in the commitment of primitive
mesoderm to hematopoiesis.21,22,28 On the other hand, the
lack of both primitive and definitive hematopoiesis resulting from
tal-1/SCL and flk-1 targeted mutations may suggest a common failure of
extraembryonic and intraembryonic mesodermal precursors to
differentiate into HSCs.29
In humans, hematopoiesis starts in the yolk sac at week 3 of
development and shifts to the liver around week 5.30,31 In an attempt to identify the primary hematogenous territory where definitive human HSCs originate, Huyhn et al32 evidenced
high proliferative potential CD34+ clonogenic progenitors
within the embryo body deprived from its yolk sac and liver rudiment
around week 5 of gestation. Furthermore, upon immunostaining of 4- to
6-week human embryo sections, we characterized dense clusters of
CD34+ hematopoietic cells closely associated with the
ventral endothelium of the aorta33 that strongly evoked the
intravascular blood cell clumps previously observed in animal embryos
at an equivalent developmental stage.14,16,17,34,35 With
respect to spatio-temporal distribution, typical hematopoietic stem
cell surface phenotype (CD45+, CD34hi,
CD31+, CD38 The present work was aimed at defining the molecules involved in the
initial emergence and expansion of HSCs within the aortic wall of the
4- to 6-week human embryo. Transcripts expressed in HSCs from the aorta
were compared with those from the liver and bone marrow of the embryo
and fetus. The expression of surface receptors and transcription
factors already defined at the earliest steps of mouse hematopoietic
development was first investigated by hybridization on embryo sections
and reverse transcription-polymerase chain reaction
(RT-PCR) analysis on HSCs sorted from 12- to 29-week fetal
liver and bone marrow. This study showed important similarities between
successive generations of embryonic and fetal HSCs. We then searched
for novel genes expressed early by the unique HSC population arising
from the human p-SP. To that end, we sorted by flow cytometry rare
aorta-associated CD34+ cells from a single embryo to
construct a cDNA library from this primitive cell population. We then
performed a differential screening of that library with amplified cDNA
probes prepared from aorta-associated and embryonic liver HSCs. A novel
kinase was identified whose expression pattern suggests a role in the
development of both hematopoietic and endothelial cell lineages in
humans.
Human Tissues
Tissue Processing and Section Staining
In Situ Hybridization Probes.
Probes for the human tal-1/SCL, GATA-2, c-kit, flk-1/KDR, and KG-1
kinase genes were made from PCR fragments subcloned into the pGEM-T
vector (Promega, Madison, WI) and corresponding to the
following nucleotidic sequences in the Genbank database: tal-1/SCL: nt
4082-4979, accession no. M61108; GATA-2: nt 1861-2673, accession no.
M68891; c-kit: nt 4442-5077, accession no. X06182; flk-1/KDR: nt
1593-2411, accession no. X61656; and KG-1 kinase: nt 28-922, accession
no. D43636. The authenticity of the clones and their orientation were
determined by sequence analysis or by mapping the position of the
predicted restriction sites. A pGEM-3Z plasmid containing the coding
sequence of the hGATA-3 cDNA 5 In situ hybridization. In situ hybridization was performed on sections from paraffin-embedded human embryos. Before hybridization, slides were deparaffinized and treated with proteinase K as previously described.37 Sense and antisense riboprobes were transcribed from T3, T7, or SP6 flanking promoters of appropriate linearized vectors. The protocol used for synthesis and hybridization of 35S-labeled riboprobes was as previously described,37 whereas synthesis and hybridization of digoxygenin (DIG)-labeled probes was performed according to Myat et al.38 In case of subsequent staining with the anti-CD34 MoAb, sections were rinsed extensively in TBST and processed as described above. Each hybridization was performed at least three times on tissue sections corresponding to at least two distinct embryos. Isolation of Fetal HSCs Mononuclear cells from 12- to 29-week fetal liver and bone marrow (4 distinct samples of each tissue) were obtained by sedimentation at 100g over a lymphocyte separation medium (d = 1.077 g/mL; Eurobio, Les Ulis, France) for 30 minutes at room temperature. The cells recovered at the interface were washed in PBS containing 5% FCS and 2 to 4 × 107 cells were incubated for 30 minutes on ice with a mixture of fluorescein isothiocyanate (FITC)-conjugated anti-CD34 MoAb (HPCA-2; Becton Dickinson) and phycoerythrin (PE)-conjugated anti-CD38 MoAb (Immunotech, Marseille, France) diluted 1/10 in PBS, 5% FCS. The two populations of CD34+CD38+ and CD34+CD38 cells were sorted on a FACStar
Plus flow cytometer (Becton Dickinson) in the gates defined on Fig 3A.
Control labeling with irrelevant IgG1-PE and -FITC were used to
determine positivity for the CD34 and CD38 antigens. Dead cells and
debris were eliminated by using a high forward and orthogonal light
scatter window. After sorting, cells (104 to
105) were pelleted and processed for RNA extraction or kept
at  80°C.
RT-PCR analysis of total cellular RNA. Total RNA was isolated from cell pellets by the RNAzol method (Tel-Test Inc, Friendswood, TX), in 200 µL/pellet, according to the instructions of the manufacturer. RNAs were dissolved in 20 µL H2O containing 200 ng Random Primers (Promega) and heated at 70°C for 5 minutes. For first-strand cDNA synthesis, 10 µL of a mixture containing 6 µL of 5× SuperScript buffer (GIBCO-BRL, Paisley, Scotland), 1.5 µL of 10 mmol/L dNTPs (Boehringer Mannheim, Mannheim, Germany), 0.5 µL of RNA guard (Pharmacia, Uppsala, Sweden), 1 µL of 10 mmol/L dithiothreitol (DTT; GIBCO), and 1 µL of SuperScript II reverse transcriptase (GIBCO) were added and the reaction was performed at 37°C for 1 hour. One thirtieth of the reaction was used for each subsequent PCR analysis, performed in a 50 µL final volume containing 5 µL of Gene Amp 10× PCR Buffer II (Perkin Elmer, Norwalk, CT), 2.5 mmol/L MgCl2, 200 µmol/L dNTPs, and 1 U Taq polymerase (GIBCO). Approximately 1.5 pmol (50 ng) of each gene-specific primer (Table 1) was added and 35 cycles of PCR (94°C for 1 minute, 55°C for 1 minute, and 72°C for 1.5 minutes) were performed, with a final extension step of 7 minutes. Aliquots of PCR products were agarose gel electrophoresed, transferred onto N+ nylon membranes (Amersham, Amersham, United Kingdom), and hybridized with specific 32P-labeled internal cDNA probes as described below.
Isolation of Aortic and Hepatic Embryonic HSCs The aorta and liver were microdissected and dissociated by incubation for 1 hour at 37°C in 50 µL collagenase/dispase (Boehringer) 0.25% (vol/vol) in PBS without Ca2+ and Mg2+. After washing, the cell suspension was double-labeled for 30 minutes on ice with FITC-HPCA-2 anti-CD34 and either PE-anti-CD45 (DAKO) or PE-anti-CD38 MoAbs and sorted as above. After sorting, the cells (<60) were directly collected in thin-walled PCR tubes (Perkin Elmer) containing PBS, pelleted, resuspended in 4 µL ice-cold cell lysis buffer, and immediately processed for 3 cDNA synthesis.
Synthesis of Total cDNA and Southern Blot Analysis The procedure for first-strand cDNA synthesis and amplification from low numbers of cells sorted by fluorescence-activated cell sorting (FACS) was derived from the protocol of Brady and Iscove39 and performed exactly as described by Dulac and Axel.40 Aliquots of amplified cDNAs were run on 1.5% agarose gels in Tris-borate buffer, denatured in NaOH/NaCl, and transferred onto Hybond N+ nylon membranes (Amersham). After prehybridization for 1 hour at 65°C in 0.5 mol/L sodium phosphate buffer (pH 7.3) containing 1% bovine serum albumin (BSA) and 4% sodium dodecyl sulfate (SDS), hybridization was performed in the same buffer overnight at 65°C by adding 106 cpm/mL of 32P-labeled cDNA probe (Random Primer Labeling Kit; Stratagene, La Jolla, CA). After two washes at 65°C in 0.5× SSC, 0.1% SDS, membranes were autoradiographed on a Kodak X-OMAT film (Eastman Kodak, Rochester, NY) for 30 minutes to 24 hours.Probes.
Probes were excised from plasmids containing the appropriate
3 Construction of a cDNA Library From Purified Aortic HSCs and Differential Screening The procedure for cDNA library construction and differential screening was exactly as previously described.40 Briefly, 10 µL of cDNAs from CD34+CD45+ aorta-associated cells was submitted to an additional polymerization step (94°C for 5 minutes, 42°C for 5 minutes, and 72°C for 30 minutes), phenol/chloroform-extracted, and EcoRI-digested. After agarose gel purification, 50 ng of cDNAs was ligated into the ZAP II vector
(Stratagene) and packaged according to the instructions of the
manufacturer. The resulting library consisted of 9 × 105 plague forming units, with insert sizes
comprised between 500 and 800 bp. Probes for differential screening
were obtained by reamplifying for 10 cycles in the presence of 100 µCi [32P]  -dCTP, 1 µL cDNAs from
CD34+CD45+ 5-week aortic HSCs (probe 1),
same-stage CD34+CD45+ liver HSCs (probe 2), and
6.5-week CD34+CD38 liver HSCs (probe 3).
An average of 6,000 recombinant phages were plated and two differential
screenings were performed in parallel. After a 6-hour prehybridization
step at 65°C in 0.5 mol/L sodium phosphate buffer, pH 7.3, containing 1% bovine serum albumin and 4% SDS, one half of replica
filters (Hybond N+; Amersham) was hybridized with probes 1 and 2 and the second half was hybridized with probes 1 and 3 (107 cpm/mL, overnight at 65°C). One
hundred candidate clones that exhibited specific hybridization or much
brighter intensity with the probe 1 were isolated. Phage inserts were
amplified by PCR using the T3 and T7 primers and the PCR products were
rehybridized with the three cDNA probes on three independent Southern
blots. Only clones specifically hybridizing to probe 1 were further
processed for phagemid rescue as instructed by the manufacturer
(Stratagene) and sequenced.
DNA Sequencing and Sequence Analysis DNA sequencing was performed using the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer) on the ABJ model 377 DNA Sequencer (Applied Biosystems, Foster City, CA). Sequence comparisons with all available nucleic acids were performed using the National Center for Biotechnology Informations' Basic Local Alignment Search Tools [BLASTN, available at the National Center for Biotechnology Information (NCBI) website].
Expression of Early Hematopoiesis-Regulating Factors in CD34+ Embryonic and Fetal Human HSCs Expression in aorta-associated CD34+ cells. In situ hybridization of digoxygenin- or 35S-labeled riboprobes on 5-week embryo cross-sections was first used to detect the expression by aorta-associated CD34+ cells of known hematopoietic transcription factors and surface receptors. To label both endothelial and hematopoietic cells in the region of interest, CD34 immunostaining was performed as a control either on adjacent sections, when radiolabeled probes were used, or directly on the same slide when labeled with a digoxygenin-labeled probe. As shown in Fig 1, the cell clusters associated with the endothelial floor of the 5-week aorta express mRNAs encoding the tal-1/SCL, c-myb, GATA-2, and GATA-3 transcription factors. We noticed a higher expression of the GATA-3 transcript in CD34+ cells that were closely adjacent to the aortic endothelium, as well as in mesodermal cells underlying the floor of the aorta (Fig 1E and F). Circulating CD34+GATA-2+ cells visible in the lumen of the aorta (Fig 1D) might reflect the ongoing process of liver colonization, which was shown by the presence of c-myb+ cells scattered in the epithelial framework of the hepatic rudiment (not shown).
Expression in fetal liver and bone marrow HSCs.
The presence of hematopoiesis-associated transcripts was also examined
in HSCs that later populate regular fetal blood-forming tissues, ie,
liver and bone marrow. Larger and heterogenous hematopoietic populations are present in these organs. For this reason, the study was
performed by RT-PCR on CD34+ cell subsets sorted by FACS
from 12- to 29-week fetal liver and bone marrow. Because the expression
of the CD38 cell surface molecule defines an early step of human HSC
activation and commitment, CD38+ and
CD38
Cloning of Novel Sequences From CD34+
Aorta-Associated Blood Precursor Cells
Preparation of 3
Construction and differential screening of a library of embryonic
aorta-associated HSC 3 Expression of KG-1-kinase mRNA in the human embryo. Upon in situ hybridization of a radiolabeled KG-1 kinase probe, a faint specific signal was observed within aortic CD34+ blood cell foci in a 5-week embryo (Fig 6C). Unexpectedly, the whole endothelial network of the embryo in veins, arteries, and capillaries also specifically expressed the KG-1 kinase messenger (Fig 6B), thus displaying an expression pattern similar to that of the CD34 antigen (Fig 6A). This observation prompted us to investigate more closely the ontogeny of KG-1 kinase expression in the human embryo.
The ontogeny of the hematopoietic system is not limited to prenatal
stages, since most blood cells are permanently renewed during the whole
life of the organism. It is therefore likely that the identification of
novel factors governing the emergence and amplification of HSCs in the
embryo could permit critical improvements in the experimental and
clinical manipulation of adult HSCs. We first reasoned that
blood-forming tissue rudiments should drive the active expansion of
ingressing stem cells at the stage of hematopoiesis incipience.
Therefore, these could be candidate sites for the identification of
stromal cells stimulating early progenitors. Although that approach
allowed us to describe the cellular environment of emerging
hematopoiesis in the human early fetal bone marrow, no significant
expansion of progenitors could be detected at these early stages in the
medullary cavities, where blood cells even appeared to develop in the
absence of phenotypically identifiable stem cells.45
Unexpectedly, the highest local concentration of blood cell progenitors
was detected in the ventral wall of the aorta, under the form of
several hundred endothelium-adherent CD34+Lin Expression of early hematopoiesis-regulating factors. We show here that CD34+ aorta-associated cells coexpress the tal-1/SCL, c-myb, GATA-2, and GATA-3 mRNAs. These transcription factors were previously identified in animal models as key players in the onset of definitive hematopoiesis. Although their expression within intravascular clumps of hematopoietic cells present at the equivalent stage of mouse development has not been yet reported, the tal-1/SCL and GATA-2 (but not GATA-3) transcripts were found in lymphohematopoietic progenitors generated in mouse day-7 embryo cultures.46 tal-1, GATA-2, and c-myb are also expressed by hematopoietic progenitors emerging in vitro from ES cells in the course of their differentiation into embryoid bodies.47 Moreover, tal-1, GATA-2, GATA-3, and c-myb were shown to be continuously transcribed in Xenopus from the stage of early neurula in both the ventral blood islands and dorsal lateral plate, which are equivalent to the murine yolk sac and p-SP, respectively.48 Altogether these data suggest that hematopoietic commitment from either extraembryonic or intraembryonic mesoderm involves a similar early transcriptional program from lower vertebrates to humans.
Cloning of a novel early marker of hematopoietic and endothelial development. A cDNA library differential screening method permitted us to clone a sequence already isolated from undifferentiated KG-1 cells in a systematic attempt to clone unidentified human genes.42 The sequence of this gene includes a serine/threonine kinase consensus motif together with a presumptive prenyl group-binding site42 similar to that encountered in the Ras family of protein kinases. KG-1 kinase shows a striking expression in vascular endothelial cells in the whole embryo. In the late 3-week yolk sac, the earliest stage tested so far, the KG-1 kinase mRNA is already detected in the blood islands. This novel hematopoietic factor thus adds to the list of markers shared by cells of these two lineages from the early stages of development, such as MB1 in birds,64 CD34 in mouse and human,14,33 and, to a lesser extent, flk-1/KDR.16 The shared expression of the KG-1 kinase by endothelial and hematopoietic cells in both intraembryonic and extraembryonic territories of active primary hematopoiesis may be related to the derivation of these two cell lineages from common hemangioblastic precursors. Interestingly, the closely related expression of the KDR tyrosine and KG-1 serine/threonine kinase messengers in the early developing human embryo may imply that both act in concert. On the other hand, the expression pattern of the KG-1 kinase-encoding gene is reminiscent of that of cloche, whose mutation in zebrafish dramatically perturbs both hematopoietic and endothelial differentiation.65 Because cloche acts upstream of flk-1 and GATA-2,66 it would be of interest to compare the expression patterns of KG-1 kinase, KDR, and GATA-2 mRNAs in the early human embryo. The identification of the gene encoding the mouse KG-1 kinase gene will help to determine its expression from the stage of gastrulation and to analyze its role in the establishment of the endothelial and hematopoietic cell lineages through targeted mutation in ES cells.
The authors are indebted to P. Vaigot for expert cell sorting by flow cytometry and to C. Debacker for excellent technical assistance throughout. We are grateful to H. San Clemente, F. Viala, F. Beaujean, and S. Gournet for their help in the design and preparation of figures and to M. Scaglia who expertly typed the manuscript. We also thank C. Carrière and Prof E. Aubeny for procurement of first trimester embryos and Drs M. Catala, F. Narcy, A.-L. Delezoide, and Prof C. Nessmann for providing fetal tissues. We are thankful for the generous hospitality of R. Axel that was essential for the further construction of the PCR-based cDNA library from embryonic HSCs.
Submitted May 18, 1998;
accepted July 10, 1998.
Address reprint requests to Marie-Claude Labastie, PhD, Institut d'Embryologie Cellulaire et Moléculaire, CNRS UPR 9064, 49bis, avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France; e-mail: labastie{at}infobiogen.fr.
1.
Zon LI:
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14.
Wood HB, May G, Healy L, Enver T, Morriss-Kay GM:
CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis.
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Top
Abstract
Introduction
-actin (nt 1042-1741: accession
no. 
