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Regular Articles |
From the Department of Biology*
and Molecular
Biology and Genetics Curriculum,
University of
North Carolina at Chapel Hill, Chapel Hill, North Carolina
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PECAM is expressed in the mouse embryo before the need for the inflammatory response, and it is used extensively as a developmental murine vascular marker.12,13 Recently, PECAM phosphorylation during embryonic vasculogenesis was documented,14 suggesting a possible signaling role for PECAM in blood vessel formation. PECAM interactions are also implicated in angiogenesis, the sprouting of new vessels from a pre-existing vascular tree.1 In postimplantation embryos PECAM is first detected in precursor cells of yolk sac blood islands at day 7.5 and subsequently in the developing embryonic vasculature.14,15 The expression of PECAM by yolk sac precursors suggests that PECAM is involved in the earliest steps of vasculogenesis, as the yolk sac is an early site of both vascular and hematopoietic development in mammals.16,17
Murine embryonic stem (ES) cells are derived from the inner cell mass of blastocysts and are totipotent. ES cells differentiated in culture undergo a programed differentiation that recapitulates normal developmental events in the yolk sac, including vascular and hematopoietic development.18-23 The endothelial cells and some hematopoietic cells in ES cell differentiation cultures express PECAM.24-26 It was recently reported that undifferentiated ES cells express PECAM.27-29 Because ES cells resemble the inner cell mass of the blastocyst, this suggested that PECAM may be expressed embryonically earlier than the described postimplantation stages.
To obtain a better understanding of the role of PECAM in early mouse development, we examined PECAM expression in ES cells during a time course of differentiation and in preimplantation and early postimplantation embryos. To our surprise, PECAM was expressed continuously during ES cell differentiation. In vivo, PECAM was expressed by blastocysts and in postimplantation yolk sacs, suggesting that PECAM functions outside the vascular/hematopoietic system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine ES cell line, IE6/2, was maintained in an
undifferentiated state and differentiated as described,25
except that in some cases ES cells were plated directly into tissue
culture dishes. Briefly, ES cells were expanded undifferentiated in
medium containing 5637-cell conditioned medium. For differentiation,
cells were cultured for 6 to 7 days to allow the gradual depletion of
LIF/DIA from the culture medium. These cultures were then dissociated
into clumps by treatment with Dispase (Boehringer Mannheim,
Indianapolis, IN) on day 0. The cell clumps were either plated directly
onto tissue culture dishes or were incubated in suspension in Petri
dishes for 3 days before transfer to tissue culture dishes. The culture
medium was DMEM-H, 20% fetal calf serum (lot tested for ability to
support differentiation), 150 µmol/L 
-thioglycerol, and 50
µg/ml gentamicin. ES cells and differentiation cultures were fixed
for 7 minutes with either cold 50% methanol/50% acetone (MA) or fresh
4% paraformaldehyde (PFA) and rinsed in phosphate-buffered saline
(PBS) before antibody staining.
Cell suspensions for immunostaining were treated with 0.2% collagenase (type II, Sigma, St. Louis, MO). After enzyme treatment, the cells were triturated and then washed with PBS to remove any remaining collagenase. The cell suspension was then either fixed with 4% PFA, then immunostained or first immunostained, then fixed. Both methods yielded identical staining patterns.
For ES rescue experiments, ES cells were treated with Dispase, then plated at a constant density onto multiple duplicate plates and grown in differentiation conditions. Cultures were dissociated with 0.2% collagenase on days 1 to 8 of differentiation, and a duplicate culture was fixed and immunostained for PECAM. After dissociation, cell counts were determined using a hemocytometer and a fixed number of cells was plated and grown in ES cell culture conditions. The cultures were maintained for 4 days, with daily medium replenishment, then fixed and scored for ES cell colonies. The PECAM staining pattern was visually analyzed.
Mouse embryos were obtained from timed matings, and day 0.5 was noon of the day the plug was observed. Blastocysts were isolated by flushing dissected uteri with PBS on day 3.5 followed by overnight fixation with 4% PFA at 4°C. Postimplantation embryos were dissected from the uterus and immediately fixed in cold 4% PFA. After 24 hours, the embryos were dehydrated through a methanol series and stored at -20°C in 100% methanol.
Reverse Transcription-Polymerase Chain Reaction
Total RNA was prepared from ES cells and differentiation cultures by the method of Chomczynski and Sacchi.30 PolyA+ RNA was prepared from approximately 65 blastocysts using guanidinium thiocyanate disruption followed by oligo(dT)-cellulose chromatography (Quick Prep Micro, Pharmacia, Piscataway, NJ). Either this polyA+ RNA or 5 µg of total RNA from cultured cells was reverse transcribed using random primers and Superscript II (Life Technologies, Gaithersburg, MD), as recommended by the manufacturer. The resulting cDNAs were amplified by the polymerase chain reaction (PCR). Cycling conditions were: 94°C, 45 seconds; 55°C, 1 minute, 15 seconds; 72°C, 2 minutes for 35 cycles, followed by a 15-minute incubation at 72°C. The primers used were PECAM 5.1, nucleotides 1704–1723, 5'-CAAGCGGTCGTGAATGACAC-3'; PECAM 3.1, nucleotides 2278–2298, 5'-CACTGCCTTGACTGTCTTAAG-3'; ß-actin 1, nucleotides 950–971, 5'-TACTGCTCTGGCTCCTAGCACC-3'; and ß-actin 2, nucleotides 1076–1096, 5'-CCGGACTCATCGTACTCCTGC-3'. Products of PCR reactions were analyzed on 7% polyacrylamide gels.
Immunolocalization
Antibody staining was performed as described.24,25 The following antibodies were used, with the fixation methods and working dilutions in parentheses: purified rat anti-mouse PECAM antibody Mec 13.3 (PFA or MA, 0.5 µg/ml), purified rat anti-mouse CD34 antibody RAM34 (PFA, 1 µg/ml) (PharMingen, San Diego, CA), purified rat anti-mouse PECAM antibody EA-3 (MA, 1.5 µg/ml), affinity-purified rabbit anti-mouse PECAM antibody (PFA, 1:1000) (both kind gifts of Beat Imhof), and affinity-purified mouse anti-mouse SSEA-1 antibody MC-480 (PFA, 29 ng/ml) (Developmental Studies Hybridoma Bank, Iowa City, IA). The anti-PECAM antibody Mec 13.3 was also biotinylated for use in double staining (PFA, 0.5 µg/ml). After blocking with 5% fetal calf serum for 1 hour, cultures were incubated with primary antibodies at 37°C for 1 hour. Blastocysts were incubated in 5 µg/ml Mec 13.3 or 15 µg/ml EA-3 overnight at 4°C.
After washing in 5% fetal calf serum, cultures were incubated with
secondary antibodies for 45 to 60 minutes at 37°C. Detection
of rat monoclonal antibodies was with
B-phycoerythrin-conjugated goat anti-rat IgG (1:1000),
B-phycoerythrin-conjugated donkey anti-rat IgG (1:1000), or
FITC-conjugated donkey anti-rat IgG (15 µg/ml), SSEA-1
was visualized using B-phycoerythrin-conjugated goat
anti-mouse IgM (1:500) (Jackson Immunoresearch, West Grove, PA).
Biotinylated Mec 13.3 and the PECAM antiserum were visualized using
R-PE-labeled streptavidin (0.5 µg/ml) and FITC-conjugated goat
anti-rabbit secondary antibody (2 µg/ml), respectively (Southern
Biotechnology Associates, Inc., Birmingham, AL). Blastocysts were
incubated with a 1:100 dilution of the B-phycoerythrin-conjugated goat
anti-mouse secondary antibody overnight at 4°C. As controls, cultures
and embryos were also incubated with secondary antibody alone and with
an isotype-matched rat anti-mouse IgE FcR antibody (B3B4, PharMingen)
detected with an anti-rat secondary antibody. Purified mouse IgM, 
subtype, was used in conjunction with the anti-mouse IgM secondary
antibody as a control for SSEA-1 staining. Staining was
visualized with an Olympus IX50 microscope equipped with phase contrast
and epifluorescence optics and an Olympus PM-30 camera.
Postimplantation embryos were stained as described by Kispert and Herrmann31 with some modifications. Most incubations were performed using embryos placed in nets (Costar 3477) that fit into the wells of 12-well dishes along with 2 ml of each reagent. All incubations were at room temperature on a rocking platform unless otherwise noted. PFA-fixed embryos that were stored in 100% methanol at -20°C were rehydrated in PBS for 30 minutes. Free aldehyde groups were blocked in 1 mol/L glycine, pH 7–8, for 30 minutes, and embryos were then washed in PBS for 30 minutes. Embryos were next incubated in 6% H2O2 in PBS for 25 minutes to block endogenous peroxidases, and washed in three changes of PBT (PBS plus 0.1% Tween 20) for 5 minutes each. Embryos were incubated in 10 µg/ml proteinase K in PBS for 3 to 5 minutes (depending on size), and washed in 2 mg/ml glycine. Embryos were washed in three changes of PBT for 5 minutes each, then in PBTN (PBT plus 5% heat-inactivated fetal calf serum) for 30 minutes. Primary antibody was rat anti-mouse PECAM (Mec 13.3, Pharmingen) diluted to 1:200 in PBTN, and embryos were incubated overnight at 4°C with rocking. The next day, embryos were washed in PBT three times for 5 minutes each, then three times for 30 minutes each, then washed in PBTN for 30 minutes. Secondary antibody was goat anti-rat horseradish peroxidase (Accurate, IgG (H+L)), diluted to 1:200 in PBTN, and embryos were incubated overnight at 4°C with rocking. The next day embryos were washed in PBT three times for 5 minutes each, four times for 25 minutes each, then transferred to a glass watch dish. Embryos were incubated in a mixture of 0.3 mg/ml 3,3'-diaminobenzidine and 3 mg/ml NiSO4 in PBT in the dark for 10 minutes, then incubated with the same mixture plus H2O2 at 1:1000 dilution. After development (3 minutes for smaller embryos, 5 minutes for larger embryos), the reaction was stopped by washing in PBS 3 times for 5 minutes each. Embryos were photographed using a Nikon SMZ-U stereomicroscope. Embryos were embedded in paraffin, and 8-µm sections were cut and mounted using DPX mounting medium (Fluka). Sections were photographed using a Nikon Optiphot-2 microscope outfitted with DIC optics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To verify that ES cells expressed PECAM,27-29
ES
cells were fixed either 3 or 6 days after plating and stained with an
anti-PECAM antibody. As expected, a majority of the ES cells expressed
PECAM, and expression was localized to cell-cell boundaries (Figure 1)
. To further characterize the stained
ES cells, cultures were enzymatically dissociated and double stained
for PECAM and SSEA-1 (Figure 2)
, since
SSEA-1 has been used as a marker of undifferentiated and partially
differentiated embryonic cells.32-34
The majority of the
cells stained for both antibodies (Figure 2, C and F)
, although rare
cells stained for either PECAM or SSEA-1. This finding shows that the
PECAM+ cells in ES cell cultures also express SSEA-1, which
suggests that the cells are either undifferentiated ES cells or
partially differentiated ES cell derivatives.
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To determine the relationship between the PECAM expression seen on
ES cells and in vascular endothelium, RNA analysis was done on a time
course of ES cell differentiation. Reverse transcription-PCR of RNA
from both ES cells and differentiation cultures from days 1 to 7 showed
that a band of the predicted size was amplified with PECAM primers from
all of the cultures (Figure 3)
. This
finding suggested that PECAM was expressed throughout the
differentiation time course.
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. PECAM was expressed throughout the time course. The widespread
staining pattern seen in ES cells was restricted in day 2 cultures, and
large areas of unstained cells were interspersed between small
clumps of PECAM+ cells, in addition to ES-like balls of
PECAM+ cells (Figure 4A
and data not shown). On day 4
of differentiation the pattern was similar to day 2, except that
the ES-like PECAM+ balls decreased and the clumps of
PECAM+ cells became more well defined (Figures 4B
and
6B
). On day 6, cords of PECAM+ cells were evident that
were morphologically distinct from PECAM+ blood vessels
that are present at later days (compare Figures 4C and 4G
). On day 8
the PECAM+ cells were organized into blood vessels and
small lacunae began to form (Figures 4G and 6B)
. By day 10 the blood
vessels increased in size and formed blood islands in some areas,
although areas of vascular plexus were also evident (Figure 4H)
. The
blood islands and vascular plexus persisted through day 12 (Figure 4I)
,
then became less well defined with further time in culture (data
not shown). In all cases, the PECAM staining was localized to cell-cell
boundaries.
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Because the PECAM staining seen on days 0–6 of the time course
was not associated with patent blood vessels, a second vascular marker
was used in conjunction with PECAM to determine the expression
profile of non-vessel PECAM+ cells. CD34 is a surface
marker of vascular endothelium that is expressed during embryonic
development.35
Unlike PECAM, CD34 is not expressed on
undifferentiated ES cells (Figure 5F)
.
Both PECAM and CD34 are expressed in the vessels of a differentiated
culture at day 10. (Figure 5, C and H)
. In contrast, at day 6 of
differentiation, some of the newly formed vessels are stained for both
PECAM and CD34 (arrowhead in Figure 5, B and G
), while the tips of
other vessels are PECAM-positive but CD34-negative (arrow in Figure 5, B and G
).
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and a smaller number of round cells adjacent to the blood islands
(data not shown) are CD34/PECAM double positive. Because CD34 also
stains a subset of hematopoietic cells within the developing yolk sac
in vivo,36
these CD34+ cells are
most likely hematopoietic cells. ES Colony Recovery from Differentiating Cultures
Although the PECAM+ cells seen in clumps or cords on
day 6 of differentiation are distinguished from mature blood island
endothelial cells by lack of expression of CD34, they are not
distinguished from ES cells by the markers used in our study. To
further delineate the relationship between the PECAM+
clumps of cells and ES colony-forming potential, a time course of
differentiation was analyzed for both PECAM staining pattern and ES
colony recovery (Figure 6)
. As expected,
the number of ES colonies recovered from plating differentiation
culture cells in ES culture conditions decreased with differentiation
time (Figure 6A)
. A significant drop in the percentage was seen between
days 3 and 4, and from day 4 to day 8 the percentage of ES colonies
recovered remained fairly constant. In contrast, the PECAM+
clumps were first evident on day 2 of duplicate culture plates, and
their presence remained fairly constant through day 6 (Figure 6B)
,
although they assumed more "vessel-like" arrangements with time
(compare Figures 4A and 4G
). Significantly, the appearance of vessels
between days 6 and 7 was accompanied by a decrease in the relative area
covered by the PECAM+ clumps, suggesting a possible
precursor relationship between the PECAM+ clumps and blood
islands. Moreover, the functional assay for ES colony forming potential
has a different temporal pattern than the clumps of PECAM+
cells, suggesting that the PECAM+ cells are not ES cells.
PECAM Expression in Mouse Blastocysts
To determine whether the PECAM expression seen in ES cell cultures
recapitulates expression during embryonic development, RNA from
blastocysts was analyzed by reverse transcription-PCR for PECAM
expression (Figure 7)
. PolyA+
RNA from a pool of blastocysts produced a band of the appropriate size
when amplified with PECAM primers. Individual blastocysts were then
analyzed by immunofluorescence to localize PECAM expression (Figure 8)
. Two different PECAM antibodies showed
similar patterns, with staining localized to cell-cell junctions. The
patterns suggested that the inner cell mass, from which ES cells are
derived, may selectively express PECAM protein, but the background of
trophoblast cell reactivity made definitive localization difficult.
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In a previous study PECAM expression was detected in angioblasts
on day 7.5 in the developing yolk sac.14
Because PECAM is
expressed before overt vascularization during ES cell differentiation,
we examined embryos from early postimplantation stages to confirm that
PECAM was expressed before the development of mature endothelial
cells. Both whole mount in situ hybridization with a PECAM
probe and immunofluorescence of sectioned embryos with an anti-PECAM
antibody failed to detect PECAM expression in egg cylinder to early
primitive streak stage embryos (data not shown). Both PECAM RNA and
PECAM protein were detected in the developing yolk sac of mid-streak
stage embryos (Figure 9
and data not
shown). Sectioning of the antibody-stained embryos showed that PECAM
was expressed in clumps of mesodermal cells before the detection of
mature endothelial cells (Figure 9, B–D)
. The PECAM+ cells
in the yolk sac in vivo resemble in morphology and temporal
sequence the PECAM+ cell clumps seen during ES cell
differentiation, suggesting that an in vivo counterpart of
the PECAM+ clumps of cells does exist.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Undifferentiated ES cells express PECAM,27-29 and we confirmed this finding using double immunofluorescence analysis with PECAM and SSEA-1, a marker of undifferentiated and partially differentiated embryonic cells.32-34 PECAM expression in ES cells is not an artifact of tissue culture, since cells of the mouse blastocyst also express PECAM. This early expression of the PECAM gene during development is also suggested by the report of a PECAM promoter-driven cre transgene.37 The ubiquitous excision of the loxP-flanked VCAM-1 target allele indicates that the PECAM gene is expressed early in development, consistent with the blastocyst expression that we have observed. Moreover, PECAM is localized to cell-cell borders in ES cells and blastocysts, suggesting that it may play a role in cell adhesion or transmembrane signaling at this early stage.38-42 However, it is unlikely that PECAM is involved in intercellular migration at this early stage of development, since no cellular migration into the blastocele cavity is known to occur at this time.
To our surprise, PECAM was expressed continuously throughout ES cell differentiation. Although many vascular markers are also expressed in other lineages, continuous expression of a single marker from uncommitted cells through differentiated endothelium has not been observed. This result also differs from the findings of Vittet et al,27 who describe a PECAM-negative stage as ES cells begin to differentiate. However, Ling and Neben29 documented expression of PECAM (named ER-MP12) throughout ES cell differentiation using FACS analysis. In our study using immunofluorescence, PECAM was initially expressed in all or most ES cells, but after several days of differentiation most cells did not express PECAM. The remaining PECAM+ cells were found in small clumps that were morphologically distinct from the blood islands that form at later times.
The expression of other vascular markers also distinguishes early PECAM+ cells from later PECAM+ endothelial cells. The early PECAM+ cells do not express CD34, which is expressed by all blood island endothelial cells and a subset of hematopoietic cells (Ref. 36 ; this report). The early PECAM+ cells also do not express ICAM-2, which is expressed by all patent vessels and blood islands that are PECAM+ at later stages (M. Harmaty, M. Inamdar, and V. L. Bautch, unpublished observations). Preliminary results suggest that many of the PECAM+ cells found at early stages of ES cell differentiation express SSEA-1 (S. Redick and V. L. Bautch, unpublished observations), a marker of undifferentiated and partially differentiated embryonic cells.33,43 SSEA-1 is not expressed by PECAM+ endothelial cells in blood islands. Thus the expression of PECAM during ES cell differentiation may characterize an endothelial precursor that subsequently matures to a blood island endothelial cell that expresses CD34 and ICAM-2 in addition to PECAM. This model is supported by time course analysis of ES colony recovery and PECAM staining that shows distinct temporal patterns for PECAM+ clumps and ES colony potential, suggesting that at least a subset of PECAM+ cells present before vessel formation are not ES cells. A precursor relationship is also suggested by a decrease in PECAM+ cell clumps that accompanies vessel formation during ES cell differentiation. Likewise, analysis of mutant ES cells that do not express VEGF shows that relatively large numbers of the early PECAM+ cells accumulate, concomitant with severely reduced numbers of blood islands (V. L. Bautch, S. Redick, A. Scalia, P. Carmeliet, and R. Rapoport, manuscript in preparation).
The in vivo analysis was consistent with reported analyses14,15 that first detected PECAM expressing cells in the developing yolk sac of day 7.0 to 7.5 embryos, at or just before blood island formation. Our detailed analysis confirms that clumps of yolk sac mesodermal cells express PECAM before the development of morphologically distinct endothelial and hematopoietic cells. This finding suggests that yolk sac vascular/hematopoietic precursors express PECAM. Some PECAM+ cells are also found in the developing allantois, which is consistent with a recent report that the murine allantois has high vascular potential.44 Postimplantation embryonic PECAM expression is first seen slightly later in the lateral mesoderm where the dorsal aorta will form (14, 15 ; V. L. Bautch, unpublished observations). Taken together, these expression patterns suggest that PECAM expression after the blastocyst stage is confined to cells and/or precursors of the vascular/hematopoietic lineages. Moreover, if a common precursor of both lineages called the hemangioblast exists, it is likely to express PECAM.
Because the PECAM+ nonvascular cells found just before blood island formation in ES cell differentiation are similar in morphology and temporal appearance to yolk sac blood island precursor cells, it is possible that they are the in vitro counterpart of the yolk sac precursor cells. Several recent reports provide evidence that differentiating ES cells at early stages provide an environment conducive to vascular/hematopoietic development. Blast colonies that apparently form from single vascular/hematopoietic precursor cells peak early during in vitro differentiation of ES cells.45 Up to half of the cells in early embryoid bodies are positive for flk-1, a marker of early vascular and hematopoietic cells.46 Differentiation of ES cells mutant for flk-1 revealed an ability to form primitive erythroid cells that was not seen when the same cells were analyzed in chimeras.47 One explanation of the data were that ES cell differentiation provided yolk sac-derived signals to mesodermal cells without a concomitant requirement for migration, and these signals promoted hematopoietic differentiation. Thus ES cell differentiation appears to recapitulate the early yolk sac environment and early vascular development. Fortunately, the accessibility of the PECAM+ ES-derived cells should allow further characterization of mammalian vascular development with lineage markers as well as functional studies of purified cells.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grants HL43174 (to V.L.B.), RCDA HL02908 (to V.L.B.), and NRSA HL09499 (to S.D.R.).
Accepted for publication December 28, 1998.
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