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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1671-1679
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Medical University of South Carolina, Department of Cell
Biology and the Cardiovascular Developmental Biology Center,
Charleston, SC.
The process of vasculogenesis was characterized in the 6.5- to
9.5-day mouse embryo and in allantoic culture by analysis of spatial
and temporal expression patterns of the endothelial or hematopoietic
lineage-associated proteins, TAL1, Flk1, platelet/endothelial cell
adhision molecule (PECAM), CD34, VE-cadherin, and Tie2. The study
establishes that: (1) TAL1 and Flk1 are coexpressed in isolated mesodermal cells that give rise to endothelial cells and thus can be
defined as angioblasts; (2) hematopoietic cells of blood islands
express TAL1, but not Flk1; (3) vasculogenesis in the embryo proper is
initiated by mesoderm fated to give rise to the endocardium; (4) the
maturation/morphogenesis of blood vessels can be defined in terms of a
sequential pattern of expression in which TAL1 and Flk1 are expressed
first followed by PECAM, CD34, VE-cadherin, and later Tie2; and (5)
TAL1 expression is down-regulated in endothelial cells of mature vessels.
(Blood. 2000;95:1671-1679)
The first blood vessels to form in the embryo are
generated by vasculogenesis. Essential steps in this process are the
establishment of the endothelial cell lineage (angioblasts), the
assembly of angioblasts into cord-like vascular structures, the
formation of vascular lumens, and the organization of vascular
networks.1-3 New insight into vasculogenesis in mammals is
emerging from studies of transgenic mice.4-6 However, the
potential of these mice to provide insight into vasculogenic processes
is impaired by a lack of understanding of the normal morphologic and
biochemical aspects of murine vasculogenesis. Here the temporal and
spatial expressions of an array of proteins associated with the
endothelial and hematopoietic lineages were examined in the developing
mouse embryo, TAL1,7-9 Flk1,4,10,11
platelet/endothelial cell adhesion molecule (PECAM),12
CD34,13,14 VE-cadherin,15,16 and
Tie2.6,17,18 The analysis used a novel protocol that
renders the normally curved or lordotic mouse embryo into a planar
format. This procedure, combined with capability of the confocal
microscope that is able to represent all embryonic vessels in a single
image, facilitates analysis of vascular patterns and developmental
gradients. The data provide a number of new insights into the
processes of vasculogenesis and hematopoiesis that include a
more detailed understanding of the relationship between TAL1
and Flk1 expression in these lineages.
Antibodies
Whole-mount immunolabeling
Allantois culture and immunolabeling
Microscopy and image processing Embryos were analyzed using a Bio-Rad MRC 1024 Laser Scanning Confocal Microscope (Bio-Rad Microscopy Division, Cambridge, MA). Optical sectioning along the dorsoventral axis (Z axis) was performed and the images collapsed into a single focal plane using the manufacturer's software. Differential interference contrast images were generated using a research grade Leitz photomicroscope equipped with a Photometrics (Tucson, AZ) Quantix CCD camera. Images were processed using NIH Image 1.62 software (National Institutes of Health, Bethesda, MD) and Adobe Photoshop 5.0 (Adobe Systems, Inc., San Jose, CA).
Throughout this study, temporal and spatial aspects of vasculogenic and hematopoietic processes were immunologically evaluated in mouse embryos rendered into a planar format, a procedure that facilitates the analysis of vascular patterns and gradients of development (see Figure 1). Characterization of the angioblast and the hematopoietic cell phenotype Initial characterization of the angioblast was conducted in 8.3-dpc embryos, a stage when both established and forming vessels are present. Double immunofluorescence demonstrated that TAL1 and Flk1 colabeled endothelial cells of morphologically identifiable vessels (compare Figure 2, A and B) as well as dispersed mesodermal cells (Figure 2, C and D). To pursue the possibility that the dispersed TAL1+/Flk1+ cells represent the progenitors of endothelial cells, blood vessel development was followed in 6.5- to 7.0-dpc embryos. At 6.5 dpc, dispersed TAL1+/Flk1+ mesodermal cells were detected in extraembryonic regions (Figure 3, A and B, arrowheads). When the corresponding regions of 7.0- to 7.3-dpc embryos were examined, polygonal arrangements of small-caliber vessels (primary vascular networks) were evident in the regions previously populated by the TAL1+/Flk1+ cells (compare Figure 3, A and B, arrowheads, to Figure 4, A and B, braces). These data combined with previous work linking TAL1 expression with endothelial progenitor cells suggest that TAL1+/Flk1+ cells (angioblasts) are the precursors of endothelial cells.8,9
Intraembryonic vasculogenesis 6.5 to 8.0 dpc: TAL1, Flk1, and PECAM expression Intraembryonic vasculogenesis is initiated in the cranial region of 7.3-dpc embryos. Evident cranially were 2 populations of Flk1+ (see Figure 4B, doubleheaded arrow) and TAL1+ (data not shown) cells that were joined across the midline by a "string" of cells forming a crescent. The bilateral distribution of the TAL1+/Flk1+ cells coincides with regions of the embryo that are fated to give rise to the heart,23 suggesting that the TAL1+/Flk1+ cells are endocardial progenitors.Allantoic vasculogenesis: TAL1, Flk1, and PECAM expression Initial blood vessel formation in the allantois is indicated by the presence of a small number of dispersed TAL1+/Flk1+ cells at 7.0 dpc (see Figure 4A, bracket). By 7.3 to 7.5 dpc, TAL1+/Flk1+ cells are numerous (see Figure 4B and Figure 5A). At this stage, no organized blood vessels or vessel primordia could be detected (data not shown). By 8.3 dpc PECAM immunofluorescence indicated the presence of both vessel primordia and vascular networks in the allantois (Figure 5B).
TAL1, Flk1, PECAM, CD34, VE-cadherin, and Tie2 expression in intraembryonic vasculogenesis: 8.0 to 8.5 dpc Between 8.0 and 8.5 dpc, a rudimentary circulatory system is established. Figure 6 depicts the expression patterns of TAL1, Flk1, PECAM, CD34, VE-cadherin, and Tie2 in the vessels of 8.2 to 8.3 dpc (Figure 6, A-F) and 8.5 embryos (Figure 6, G-L). The expression of these proteins in prominent morphologic structures of the circulatory system such as the bilateral aortae, the endocardial primordia, and the primary vascular networks that form lateral to the embryonic axis, which are referred to as lateral vascular networks, are summarized in Table 1.
TAL1, Flk1, PECAM, CD34, VE-cadherin, and Tie2 expression in endocardial development: 8.2 to 8.5 dpc As described above, endocardiogenesis is initiated at 7.3 dpc (see Figure 4B). Between 8.2 and 8.5 dpc the bilateral heart fields are translocated to the midline forming the definitive endocardium (Figure 6 compare panel B, arrowheads, and panel H, arrow). At 8.2 to 8.3 dpc,
Flk1 expression was observed throughout the merging heart fields (see
Figure 6B, arrowheads). In contrast, although TAL1 expression was
associated with the caudal portions of the heart fields, those lying
along the anterior intestinal portal (see Figure 6A, more posterior
arrowheads), only weak staining was detected in the more cranial
portions of the fields (see Figure 6A, more anterior arrowheads). At
8.5 dpc the endocardium is characterized by strong Flk1
immunofluorescence (see Figure 6H, arrow) and the absence of detectable
TAL1 immunofluorescence (Figure 6G, arrow). Unlike TAL1,
immunofluorescence associated with PECAM, CD34, VE-cadherin, and Tie2
was readily detected on the endocardium (Figure 6, I-L, arrows). It is
concluded from these data that the TAL1+/Flk1+
cells observed in cranial regions at 7.3 dpc (see Figure 4B) and in heart fields at 8.2 dpc (see Figure 6, A and B) represent the progenitors of the TAL1 /Flk1+
endocardial endothelial cells seen at 8.5 dpc (see Figure 6, G and H, arrows).
TAL1, Flk1, PECAM, CD34, VE-cadherin, and Tie2 expression in the dorsal aortae: 8.2 to 8.5 dpc The dorsal aorta is derived from the fusion of bilateral primordia, the dorsal aortae. At 8.3 dpc both cranial and caudal portions of the dorsal aortae exhibited intense PECAM staining (see Figure 6C and Figure 7C, arrowheads), whereas the more intermediate portion stained less intensely (Figure 7C, bracket, and 7E). This immunostaining pattern coincided with morphogenetic features of the developing aortae. Intense PECAM staining was associated with segments that, based on physical sections, had a defined lumen, whereas less intense staining was detected in segments composed of primary vascular networks. It is concluded that the aortae form in a bidirectional manner and that vascular networks are an essential component of aortic morphogenesis. Similar to PECAM, immunostaining for TAL1, Flk1, CD34, and VE-cadherin was localized to the aortic primordia of 8.2- and 8.5-dpc embryos. In contrast to these proteins, Tie2 immunofluorescence was absent at 8.2 dpc (see Figure 6F); however, expression was detected at 8.5 dpc (see Figure 6L). This observation suggests that Tie2 expression correlates with a discrete step in vessel maturation.
TAL1, Flk1, PECAM, CD34, VE-cadherin, and Tie2 expression in the lateral vascular networks: 8.2 to 8.5 dpc Between 8.2 and 8.5 dpc the lateral vascular networks are formed. These networks extend from a region just lateral to the aortae to an ill-defined boundary where they connect with the extraembryonic vasculature (see Figure 4C, dashed lines). Isolated TAL1+/Flk1+ cells can be detected within the lateral regions as early as 7.6 dpc (see Figure 4C); by 8.2 dpc the first networks are apparent (see Figure 6, A and B, asterisk) and by 8.5 dpc the lateral vascular networks are clearly discernible (see Figure 6, G and H, asterisk). Double immunofluorescence experiments revealed that TAL1 and Flk1 are coexpressed in cells of both the forming and established lateral vascular networks (data not shown). In contrast to the expression of TAL1 and Flk1, PECAM expression was conspicuously absent in these vessels at both 8.2 dpc (compare PECAM in Figure 6C to that of TAL1 in Figure 6A and Flk1 in Figure 6B) and 8.5 dpc (compare PECAM in Figure 6I to that of TAL1 in Figure 6G and Flk1 in Figure 6H). The immunostaining patterns of CD34 (see Figure 6, D and J) and VE-cadherin (see Figure 6, E and K) at 8.2 and 8.5 dpc were similar to that of PECAM, with expression associated with the forming aortae (bracket) but absent in the lateral vascular networks (asterisk).
TAL1 is down-regulated as part of endothelial cell maturation The diminution of TAL1 expression associated with endocardial development (see Figure 6, A and B) suggested a relationship between the level of TAL1 expression and the state of endothelial cell maturation. To investigate this possibility, TAL1 expression was followed during aortic development. Although strong TAL1 immunofluorescence was associated with the aortae of 8.2- and 8.4-dpc embryos (see Figure 6, A and B), by 9.0 dpc no expression was detected. In Figure 8, expression of TAL1, Flk1, and PECAM in the aortae of 9.0-dpc embryos was examined in triple immunofluorescence studies. Figure 8A shows a segment of aortae and the associated intersomitic and intervertebral vessels immunostained with PECAM antibodies. In contrast to the uniform expression of PECAM on endothelial cells, TAL1 immunofluorescence was confined to a population of uniformly round cells (see Figure 8B). Analysis of optical sections demonstrated that these cells were confined to the vascular lumen, suggesting that they are associated with the hematopoietic rather than the endothelial lineage. When the TAL1 and PECAM immunostaining patterns are superimposed (see Figure 8C), the lack of detectable TAL1 expression in endothelial cells was evident; this is most apparent in the vessel segment indicated by the arrow. Flk1 expression was examined to determine if a correlation exists between the level of TAL1 expression and that of Flk1 (see Figure 8D). Clear Flk1 immunofluorescence was associated with endothelial cells (compare Figure 8A and D); this expression (arrowheads) was most evident in the vessel segment denoted by the arrow. Comparison of TAL1 and Flk1 expression (Figure 8, B and D) establishes that mature endothelial cells are TAL1/Flk1+. The ability to
detect Flk1 protein in endothelial cells lacking TAL1 expression
suggests that the expressions of these proteins are independently
regulated.
TAL1/Flk1 Based on this study, we propose a working definition of the phenotype of angioblasts, embryonic endothelial cells, and extraembryonic hematopoietic stem cells. Angioblasts are dispersed (not associated with an organized vessel) TAL1+/Flk1+ mesodermal cells. That such cells represent angioblasts and not hemangioblasts is indicated by the fact that blood vessels lacking blood cells (see Figure 4, A and B, braces) form in extraembryonic regions where dispersed TAL1+/Flk1+ cells were detected earlier in development (see Figure 3B, arrowheads) and the fact that blood vessels in the embryo proper, which are known to form without associated hematopoietic development, also arise from TAL1+/Flk1+ cells (see Figure 4 and Figure 6, A-F).2,3 Additionally, that TAL1+/Flk1+ cells represent angioblasts, which are defined as the progenitors of endothelial cells, is supported by the observation that endothelial cells of early embryonic blood vessel are TAL1+/Flk1+. Our definition of endothelial cells is conditional because the data indicate that the endothelial cells of more mature blood vessels are TAL1/Flk1+ (see Figure 8).
That mature endothelial cells do not express TAL1 is also
indicated by work demonstrating that embryonic endothelial cells of
zebra fish express messenger RNA (mRNA) for Flk1 but not
TAL1.7 Based on these data we define endothelial cells
comprising the most primitive vessels of 7.0- to 8.5-dpc embryos as
TAL1+/Flk1+ cells, whereas those of more mature
vessels are TAL1/Flk1+ cells. Based on
our analysis of protein expression in the 6.5-dpc embryo, we have
defined extraembryonic hematopoietic stem cells as
TAL1+/Flk1. This definition is supported
by in situ RNA hybridization studies that show a population of blood
island cells in zebra fish embryos that are
TAL1+/Flk1,7 and the finding
that Flk/embryonic stem cells implanted into
normal mouse embryos can generate at least primitive components of the
hematopoietic lineage.24
PECAM, CD34, and VE-cadherin The immunolocalization of PECAM, CD34, and VE-cadherin observed in this study demonstrated similar temporal and spatial expression patterns. At 8.0 dpc expression of each was intense on aortic endothelial cells, moderate on primordial endothelial cells, and absent on angioblasts. These patterns suggest that there is a correlation between protein expression and the state of endothelial cell maturation/vessel morphogenesis. Previous studies examining the expression patterns of these proteins in vivo are limited to CD34.13,14,19 Our observations of CD34 expression were similar to those previously reported but differ as regards expression by angioblasts.Tie2 A striking aspect of Tie2 expression was the rapid up-regulation in immunofluorescence intensity seen between 8.3 and 8.5 dpc. In contrast to the other proteins examined, virtually no Tie2 immunostaining was detected on the aortae of 8.2-dpc embryos (compare Figure 6F with Figure 6, A-E). However, by 8.5 dpc distinct Tie2 expression was detected along the entire length of the aortae (see Figure 6L). The "late" expression of Tie2 is in accord with the findings of others that suggest that Tie2 functions in later events of vascular morphogenesis.17 The immunolocalization of Tie2 to the developing aortae and endocardium represents, to our knowledge, the first time that Tie2 expression has been described at the protein level.Allantoic vasculogenesis Our in vitro data established that blood vessel formation in the allantois occurs by vasculogenesis and that TAL1+/Flk1+ cells represent the precursors of endothelial cells. When allantoides containing TAL1+/Flk1+ cells but no morphologically distinct vessels were placed in tissue culture (see Figure 5A), networks of PECAM+ vessels were observed after 12 to 24 hours (see Figure 5, C and D). This observation and the work of Downs and colleagues clearly establish that initial neovascularization of the allantois occurs by vasculogenesis.43 Further, we speculate that the TAL1+/Flk1+ cells present at the initiation of culture, or similar cells generated during the culture period, represent the precursors of allantoic endothelial cells.Endocardiogenesis The first vasculogenic activity in the embryo proper is associated with the development of the endocardium. At presomite stages (7.3 dpc) 2 bilateral fields of TAL1+/Flk1+ mesodermal cells were evident in cranial regions of the embryo (see Figure 4B). That these TAL1+/Flk1+ cells represent the progenitors of endocardium is supported by fate mapping studies23 and the fact that the expression patterns of TAL1 and Flk1 are virtually superimposable with the pattern of Csx/Nkx-2.5 mRNA, a putative marker of myocardial progenitors.44,45 Taken together these data define the primordial heart fields in terms of each of the constituent cell populations (endocardial and myocardial). A second and potentially important observation regarding endocardiogenesis is the down-regulation TAL1 expression in more mature endocardial endothelial cells (compare Figure 6A and Figure 6G). The significance of this observation will only become clear when the function of TAL1 is more clearly defined. Our immunolocalization of Tie2 to the developing endocardium at 8.5 dpc (Figure 6L, insert) links the temporal expression of this receptor with the abnormal cardiac phenotype observed in Tie2 null mice.6Vascular morphogenesis in the dorsal aortae and lateral vascular networks An unexpected finding of this study was the restricted pattern of protein expression observed in the lateral vascular networks (compare Figure 6, A and B, to Figure 6, C-F, and Figure 6, G and H, to Figure 6, I-L). Although the absence of PECAM, CD34, and VE-cadherin expression (Figure 6, C-E) at 8.2 dpc could be attributed to angioblasts being the predominant cell type (Figure 6, A and B), the lack of staining at 8.5 dpc is not as easily explained. Specifically endothelial cells of vascular structures such as those depicted in Figure 7D (arrows) would "normally" express these proteins. Although the basis for this difference is unknown, data from avians suggests that vascular morphogenesis in this region may be unique. In quail embryos, this region progressively becomes avascular despite the initial presence of endothelial cells.46-48 Although we have no definitive evidence as to the fate of cells in this region, we did observe angioblasts that appeared to be in the process of integrating into existing vessels (see Figure 7D, arrowheads).
The authors express thanks to the following for their generous gift of antibodies: Dr Stephen J. Brandt (Vanderbilt University and Veterans Affairs Medical Center, Nashville, TN) for TAL1, Drs Elisabetta Dejana and Maria Lampugnani (Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy) for VE-cadherin, Dr Lawrence A. Lasky (Genentech, Inc, San Francisco, CA) for CD34, Dr Andre Schuh (University of Toronto, Toronto, Ontario, Canada) for Flk1, and Dr Steven Stacker (Ludwig Institute for Cancer Research, Victoria, Australia) for Tie2. Additionally, the authors would like to thank Drs Charles Little, Scott Argraves, and Vladimir Mironov (Medical University of South Carolina, Charleston SC) for their insights and critical comments.
Submitted April 30, 1999; accepted October 26, 1999.
Supported by NIH R01 HL 57375 grant to C.J.D.
Reprints: Christopher Drake, Department of Cell Biology, Medical University of South Carolina, PO Box 250508, 173 Ashley Avenue, Charleston, SC 29425; email: drakec{at}musc.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Abstract
Introduction
