Circulating Blood Island-Derived Cells Contribute to Vasculogenesis in the Embryo Proper

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Developmental Biology 262 (2003) 162Ð172 www.elsevier.com/locate/ydbio

Circulating blood island-derived cells contribute to vasculogenesis in the embryo proper

Amanda C. LaRue,a Rusty Lansford,b and Christopher J. Drakea,*

a Cardiovascular Developmental Biology Center, Department of Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA b The California Institute of Technology, Division of Biology 139-74, 1200 E. California Blvd., Pasadena, CA 91125, USA Received for publication 16 December 2002, revised 2 June 2003, accepted 2 June 2003

Abstract While recent ﬁndings have established that cells derived from the bone marrow can contribute to vasculogenesis in the adult, it is unclear whether an analogous population of cells in the embryo can also contribute to vasculogenesis. Using a retroviral labeling strategy, we demonstrate that circulating blood island-derived cells contribute to the genesis of both extra- and intraembryonic blood vessels in the early quail embryo. This ﬁnding establishes that vasculogenesis in the embryo is a composite of two processes: the direct in situ formation of blood vessels from mesodermally derived angioblasts and the incorporation and differentiation of circulating endothelial cell progenitors into forming embryonic blood vessels. © 2003 Elsevier Inc. All rights reserved.

Keywords: Vasculogenesis; Circulating endothelial progenitor cell; Blood islands; Stem cell

Introduction strated that a population of circulating CD34ϩ mononuclear cells isolated from human peripheral blood were able to Two distinct neovascular processes mediate new blood participate in adult neovascularization in a mouse ischemia vessel formation, vasculogenesis and angiogenesis. Angio- model (Asahara et al., 1997). Studies evaluating the trans- genesis is the formation of blood vessels from endothelial differentiation potential of transplanted bone marrow cells cells of preexisting vessels, while vasculogenesis is the de further support the concept of adult vasculogenesis (Asa- novo formation of blood vessels from mesoderm. Vasculo- hara et al., 1999a; Ikpeazu et al., 2000; Shi et al., 1998, genesis was once thought to be restricted to only the earliest 1999). Together, these studies established that bone mar- stages of development, with subsequent vascular growth row-derived cells are a source of new endothelial cells that being mediated by angiogenesis. This view has changed participate in neovascular processes in the adult. with the demonstration that the initial vascularization of The ﬁnding that circulating endothelial progenitor cells developing organs such as the kidney (Robert et al., 1998; contribute to neovascularization in the adult led us to in- Tufro et al., 1999), lung (deMello et al., 1997; Gebb and vestigate whether a similar process is operative in the em- Shannon, 2000), and liver (Matsumoto et al., 2001) occurs bryo. Given the extensive vasculogenic activity associated by vasculogenesis. In addition, vasculogenic blood vessel with embryogenesis and the fact that the vertebrate embryo formation is now known to occur in the adult. The study of possesses structures analogous to the bone marrow, the Asahara et al. (1997) was seminal in bringing the concept of blood islands, it was postulated that blood island-derived adult vasculogenesis to the forefront. This group demon- circulating cells participate in embryonic vasculogenesis. Using a retroviral based lineage-tracing strategy, we demonstrate that circulating blood island-derived cells contrib- * Corresponding author. Fax: ϩ1-843-792-0664. ute to new blood vessels in both extra- and intraembryonic E-mail address: [email protected] (C.J. Drake). regions. These ﬁndings suggest that embryonic vasculogen-

Fig. 1. YFP expression is observed in extra- and intraembryonic regions 15 h after injection of retroviral tracer into quail blood island primordia. Shown in (A) is an LSCM image of a 13-somite stage quail embryo that was injected with retrovirus at the 2-somite stage and cultured for 15 h. Numerous YFPϩ cells are evident in areas where blood vessels are found within the extraembryonic (i.e., yolk sac) and intraembryonic (i.e., lateral networks) portions of the embryo and absent in avascular regions, i.e., Henson’s node (star). (B) A higher magnification image of the boxed area in (A) showing the distribution of YFPϩ cells (green) within the QH1 immunolabeled (red) blood vessels of the sinus venosus. Scale bars: (A) 200 ␮m; (B) 50 ␮m. esis is a composite of both in situ vasculogenesis and vas- viral vector was assembled with the G envelope protein of culogenesis mediated by circulating cells. vesicular stomatitis virus (VSV-G). Pseudotyping, or the exchange of surface antigen among both DNA and RNA virus, allows the host range of viruses to be altered to possess Materials and methods a broad host range (Zavada, 1982). In addition, this allows the virus to be concentrated 1000-fold with minimal loss of bio- Animals logical activity (Burns et al., 1993). In test experiments, the VSV-G pseudotyped retrovirus efficiently (Ͼ99%) infected Fertile Japanese quail (Coturnix coturnix japonica) eggs avian cells throughout embryonic development (data not were obtained from Manchester Farms (Dalzel, SC). Fertile shown). The infected cells appear to follow their normal mi- specific pathogen-free (SPF) chicken (Gallus domesticus) gratory routes and undergo normal differentiation. The retro- eggs were obtained from Hy-Vac (Adel, IA). virus used in these studies was constructed by fusing cDNA encoding human histone H2B to the gene encoding the Yel- Tracing vectors low Fluorescent Protein (YFP) and cloning the fusion gene into the pCLNCX vector (Kanda et al., 1998). H2B-YFP Replication-defective pseudotyped retrovirus that expresses expression was driven by the chick ␤-actin promoter/CMV human histone H2B-yellow fluorescent fusion protein enhancer transcription unit EF-1␣ promoter (Mizushima and (H2B-YFP) was obtained from Dr. Rusty Lansford (California Nagata, 1990). The viral titer was between 5 ϫ 108 and 1 ϫ Institute of Technology, Pasadena, CA). The H2B-YFP retro- 109 pfu/ml. 164 A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172

0.01% azide, the vitelline membrane was removed. Em- bryos were permeabilized in absolute methanol at Ϫ20¡C for 1 h, rehydrated through a graded series of ethanol/water solutions, and then blocked overnight at 4¡C using 3% BSA/PBS. The embryos were washed and immunolabeled overnight at 4¡C using QH1 hybridoma supernatant, a quail- specific endothelial cell marker (QH1 hybridoma supernatant developed by F. Dieterlen was obtained from the De- velopmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA) and/or rabbit anti-recombinant TAL1 IgG (obtained from Dr. Steven Brandt, Vanderbilt University, diluted to 1 ␮g/ ml). After washing, secondary fluorochrome-conjugated antibodies (Jackson Immunological Research Labs Inc., West Grove, PA) were added at 10 ␮g/ml and incubated for 6 h. The labeled embryos were mounted under a #1 coverslip using anti-bleaching mounting medium [5% n-propyl gallate, 0.25% 1,4 diaza-bicyclo-(2,2,2) octane, and 0.0025% Fig. 2. Retroviral injection of quail blood island primordia results in three phenylendiamine in glycerol (Giloh and Sedat, 1982)]. populations of YFPϩ cells. When retrovirally injected embryos were immunolabeled with antibodies to QH1, a quail-specific endothelial cell Laser scanning confocal (LSCM) and differential ϩ marker, at the 12-somite stage and imaged using LSCM, numerous YFP interference contrast (DIC) microscopy (green) cells were associated with the QH1ϩ (red) intraembryonic blood vessels of the lateral networks. Arrows indicate YFPϩ cells within the lumen of the QH1ϩ vessels. Arrowheads show elongated cells that appear LSCM to be intercalated into the vascular endothelium. Asterisk indicates YFPϩ Embryos were analyzed by using a BioRad MRC-1024 cells within the avascular mesoderm. Scale bar, 25 ␮m. laser scanning confocal microscope and Adobe PhotoShop 7.0 image processing software. Using previously described Embryo microinjection and culture procedures, the embryos were scanned in a plane parallel to

Detailed methods for preparing, microinjecting, and cul- turing quail embryos have been described previously (Drake et al., 1992a; Packard and Jacobson, 1979). Briefly, primitive streak to two-somite-stage embryos were removed from the yolk by using a paper ring and placed ventral side up on an agar bed in Dulbecco’s phosphate-buffered saline (DPBS; Gibco BRL/Life Technologies, Baltimore, MD). Microinjection was accomplished by using a transmitted light stereomicroscope, a micromanipulator, and an injection pump. Nanoliter-sized aliquots of reagent (approxi- mately 1 ng/nl in 10 or 20 nl) were delivered into the extracellular matrix separating the splanchnic mesoderm and the endoderm. After microinjection, embryos were washed three times in DPBS. For heart extirpation, the bilateral heart fields were excised at the six-somite stage by using a fine carbon steel blade (Electron Microscopy Sci- ences, Fort Washington, PA). All embryos were cultured (0.5Ð22 h) in DMEM (Gibco) containing 10% chicken se- Fig. 3. LSCM shows YFPϩ cells incorporated into intraembryonic vessels. rum (Gibco) and 1% penicillin streptomycin/L-glutamine Shown is a LSCM image from a retrovirally injected embryo immunolabeled (Gibco) in a humidified CO2/air mixture (5%/100%) at with antibodies to QH1 at the 12-somite stage. The image was generated by 37¡C. optically sectioning the intraembryonic blood vessels from the lateral networks along their longitudinal axis and then compositing 2 serial Z-sections (total ϳ ␮ Embryo fixation and immunolabeling thickness of 4 m) acquired midway through the vessel. The level of the sections chosen passes through the vascular endothelium and the lumen of the vessel at its largest diameter. Arrowhead shows YFPϩ cells incorporated into Embryos were rinsed in DPBS and fixed in 3% parafor- the QH1ϩ vascular endothelium. Avascular space and lumen of the blood maldehyde for 45 min. After washing in PBS containing vessel are labeled. Scale bars, 25 ␮m. A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172 165 the embryonic plate (Drake et al., 1997). Individual optical ogy of the cells was examined by using LSCM and differ- planes were sequentially analyzed for TAL1 and QH-1 ential interference contrast microscopy (DIC). Fig. 3 shows immunolabeled cells. The final images were projections of two YFPϩ cells (arrowhead) within the QH1ϩ vascular ϳ20 optical planes collapsed using BioRad software to epithelium. In contrast to the round nuclei of YFPϩ cells produce a single virtual image representing the TAL1 or observed within the lumen of the vessel (Fig. 2), the nuclei QH-1 immunolabeling of all blood vessels in the optical of YFPϩ cells within the vascular endothelium had an field. elongated shape. The YFPϩ cells incorporated into the vascular endothelium were also imaged by using epifluores- DIC cence and DIC microscopy (Fig. 4). A high magnification Embryos were evaluated by using a Leica DMR light image of a segment of a blood vessel from a retrovirally microscope equipped with epifluorescence capabilities injected embryo immunolabeled with QH1 (red) revealed (Vashaw Scientific, Inc., Norcross, GA). Images were pro- three YFPϩ cells (green) that coexpress QH1 (Fig. 4A, cessed by using NIH Image 1.62 (National Institutes of arrows). When the same field was examined by using DIC Health) and Adobe Photoshop 7.0 software (Adobe Sys- microscopy, the nuclei of the three YFPϩ cells (Fig. 4B, tems, Inc., San Jose, CA). arrows) were indistinguishable from those of adjacent endothelial cells. Both the YFPϩ/QH1ϩ cells and the YFPϪ/ QH1ϩ cells exhibited nuclei that were elongated in shape. Results Similarly, comparison of the morphology of endothelial cells in control embryos (Fig. 4D, arrows) with the YFPϩ Blood island-derived YFPϩ cells can be detected within cells in endothelium of experimentally manipulated em- the embryo proper bryos (Fig. 4C, arrows) revealed that, at least at the gross level, the cells shared morphological characteristics. To evaluate whether cells of blood island origin participate in intraembryonic vasculogenesis, the extraembryonic YFPϩ endothelial cells express the transcription factor mesoderm that is the primordia of the blood islands (prim- SCL/TAL1 itive streak to 2-somite stage) was microinjected with 20 nl of H2B-YFP-expressing retrovirus (n ϭ 30). Examination SCL/TAL1 is a helixÐloopÐhelix transcription factor that of embryos 15 h postinjection (11Ð13 somite stage) using is expressed in both angioblasts and newly differentiated laser scanning confocal microscopy (LSCM) revealed endothelial cells and is immunohistochemically localized to YFPϩ cells in both extra- and intraembryonic regions (Fig. the nucleus of both cell types (Drake et al., 1997; Drake and 1A). To relate the distribution of YFPϩ cells to the devel- Fleming, 2000; Kallianpur et al., 1994). To evaluate oping blood vessels, retrovirally labeled embryos were im- whether the YFPϩ cells also expressed TAL1, in addition to munolabeled with antibodies to QH1, a carbohydrate that is possessing morphological characteristics of endothelial expressed on the surface of quail endothelial cells (Drake et cells, retrovirally injected embryos were immunolabeled al., 1997; Pardanaud et al., 1987; Peault et al., 1983). Fig. with antibodies to QH1 and TAL1. Fig. 5A is a LSCM 1B is a high magnification composite of a YFP image image of QH1-labeled blood vessels from the region of a (green) and the corresponding QH1 image (red) of blood 12-somite-stage embryo located lateral to the axis and be- vessel in the area of the sinus venosus (boxed area in Fig. tween somites 3 and 8. Fig. 5B depicts TAL1 expression in 1A). the same optical field. Elongated TAL1-positive nuclei are evident at the margin of the vessel. When the pattern of YFPϩ cells are present as endothelial cells, circulating QH1, TAL1, and YFP expression was superimposed (Fig. cells, and cells within the splanchnic mesoderm 5D), a subpopulation of YFPϩ cells was found that coex- pressed both QH1 and TAL1 (arrowheads). The expression Three populations of YFPϩ cells were detected within of TAL1 in YFPϩ endothelial cells is consistent with the the embryo proper (intraembryonic regions) 15 h postinjec- pattern of gene expression that defines vasculogenesis tion using LSCM. Each of these populations was evident in (Drake et al., 1997, 2000; Drake and Fleming, 2000). areas lateral to the axis at the level of the 3rd through 8th somites. The first population of YFPϩ cells (Fig. 2, arrow- YFPϩ endothelial cells within the embryo proper are heads) was closely associated with the vascular endothelium derived from circulating cells (QH1ϩ cells). A second population of YFPϩ/QH1Ϫ cells was evident within the lumen of the blood vessels (Fig. 2, The experiments above establish the presence of YFPϩ arrows). A third and less prominent population of YFPϩ/ endothelial cells in the blood vessels of the embryo proper QH1Ϫ cells was found within the avascular splanchnic me- (intraembryonic regions). While these experiments suggest soderm (Fig. 2, asterisks). that these cells were derived from circulating cells of ex- To evaluate whether the YFPϩ cells associated with the traembryonic origin, there are other possibilities that could vascular endothelium were endothelial cells, the morphol- account for the presence of YFPϩ endothelial cells within 166 A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172 the intraembryonic vasculature. These possibilities include: vessels of the embryo proper at 4.5 h after circulation is (1) YFPϩ cell migration/viron diffusion, (2) viron diffusion initiated argues for an extraembryonic origin of YFPϩ cells via circulation, and/or (3) viral recombination. (Fig. 7E, upper panel). To confirm that circulation is required for transport of YFPϩ cell migration and viron diffusion YFPϩ cells from the extraembryonic blood islands to the To evaluate whether YFPϩ cells at the site of injection embryo proper, the distribution of YFPϩ cells was evalu- migrated into the embryo and/or whether free viron diffused ated in embryos in which the heart was extirpated. To throughout the tissue and subsequently infected cells of the achieve this, the blood island primordia of primitive embryo proper, the distance of YFPϩ cells from the site of streak-1 somite stage were retrovirally labeled and the em- injection was analyzed as a function of time. These exper- bryos cultured to the 6-somite stage. At this time, the bilat- iments are schematically outlined in Fig. 6. Extraembryonic eral heart-forming fields were extirpated and the embryos blood island primordia of primitive streak stage embryos cultured for an additional 15 h. In embryos in which the were retrovirally injected and the embryos cultured for extirpation completely ablated cardiac development, (6 out specific time periods (0.5, 1, 2, 4, 5, 6, and 10 h) to allow for of 8 embryos), no YFPϩ cells were detected within the viron incorporation and cell migration and/or viron diffu- embryo proper. In contrast, YFPϩ cells were detected in- sion. At the end of the culture period, the extraembryonic traembryonically in the remaining two embryos (embryos in tissue was separated from the intraembryonic tissue. The which cardiac function was only partially disrupted). These two fragments (extra- and intraembryonic) were then cul- findings suggest that the circulation acts as the conduit for tured separately for an additional 16Ð22 h to allow suffi- YFPϩ cells from the extraembryonic blood islands to the cient time for viral protein expression. The time period was embryo proper. determined based on an evaluation of the minimum time required from viral infection to nuclear YFP fluorescent Viral recombination protein expression, which was shown to be 6.5 h (data not Despite the fact that the retrovirus used was designed to shown). Analysis of the cultured intraembryonic fragments be replication defective and tested for replication compe- revealed a complete lack of YFP expression (Fig. 6A and tency using cell lines, the possibility existed that in an in B). In contrast, YFPϩ cells were evident in the correspond- vivo setting, a three-way recombination event could have ing extraembryonic tissues (Fig. 6C and D). Microinjected, occurred. As the quail eggs routinely used in this study are uncut embryos that were cultured for 16Ð22 h had numer- not tested for pathogens, the presence of a wildtype virus in ous YFPϩ cells in both the extra- and intraembryonic por- the egg could result in the generation of a replication com- tions (Fig. 6E and F). As no gradient of YFPϩ cells was petent virus (Ory et al., 1996). To address this possibility, observed from the site of injection toward the axis of the primitive streak-staged embryos from specific pathogen- embryo, the findings of this experiment suggest that the free (SPF) chicken eggs were retrovirally injected in the YFPϩ cells/endothelial cells are neither the result of YFPϩ area of the blood islands and cultured 15 h as described cell migration nor viron diffusion, but rather are the result of above. Examination of SPF chicken embryos shows that, circulating YFPϩ cells of extraembryonic origin. This is like retrovirally labeled quail embryos, blood island-derived also supported by the fact that YFPϩ cells within the em- fluorescent cells were incorporated into the intraembryonic bryo are not located in regions previously described as blood vessels of the embryo proper (Fig. 8). While incor- avascular, i.e., those adjacent to Henson’s Node (Fig. 1A, poration into the vasculature of quail embryos was demon- star). strated by the colocalization of QH1 immunolabeled cells and virally infected fluorescent cells (Fig. 8A, arrows), Viron transport via the circulation incorporation of fluorescent cells into blood vessels of SPF While the above findings establish that infected cells do chicken embryos was evaluated by using a combination of not simply migrate through the mesoderm or that virons fluorescence and DIC imaging (Fig. 8B, arrows), as QH1 diffuse and infect intraembryonic endothelial cells, they do does not react in the chicken. The use of SPF embryos in not rule out the possibility that free virons gain access to conjunction with retroviral vectors eliminated the possibil- intraembryonic endothelial cells once the circulation is es- ity that replication competent virus was the source of la- tablished. To evaluate this possibility, the set of experiments beled cells within the embryo proper. outlined in Fig. 7 was conducted. Our observations of cardiovascular development reveal that extraembryonic derived blood cells first gain access to the embryo proper Discussion when heartbeat is initiated at the 10-somite stage. Based on this fact and the fact that the minimal time from infection to Our findings establish that the blood islands are a source YFPϩ expression was determined to be 6.5 h (data not of circulating cells that contribute to the formation of new shown), primitive streak-stage embryos were infected, cul- blood vessels in both the extra- and intraembryonic regions tured to the 12-somite stage, and examined by using LSCM. of the embryo. Using a retroviral labeling strategy in con- The clear presence of YFPϩ cells incorporated into blood junction with confocal, DIC and epifluorescence micros- A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172 167

Fig. 4. DIC demonstrates incorporation of YFPϩ cells into the intraembryonic vasculature. (A) A high magnification fluorescent image of YFPϩ cells (green, arrows) within a segment of a QH1ϩ intraembryonic blood vessel (red) from the lateral networks. (B) The same field taken using DIC to reveal the nuclear shape of the constituent endothelial cells. (C) An overlay of the fluorescent and DIC images that shows the morphology of the YFPϩ cells intercalated into the endothelium. (D) A DIC image of a blood vessel segment from a noninjected stage-matched normal embryo. Comparison of YFPϩ cells (C, arrows) and normal endothelial cells (D, arrows) demonstrates that the YFPϩ cells have a phenotype characteristic of normal endothelial cells intercalated into the vascular endothelium. Avascular space, lumen of the blood vessel and red blood cells (RBCs) within the lumen are labeled. Scale bars: (AÐD) 25 ␮m. copy, we have demonstrated that YFPϩ cells of blood island which extraembryonic and intraembryonic portions of the origin incorporate into the vascular endothelium of intraem- embryos were separated at a specific time point post-injec- bryonic blood vessels. The fact that the cells are integrated tion (Fig. 6). In addition, as all injections were conducted at into the endothelium of the blood vessels (Figs. 3 and 4) and the primitive streakÐ2-somite stage, a period in develop- the fact that these cells coexpress the markers QH1 and ment prior to generation of blood vessels, the possibility of TAL1 (Fig. 5), which were previously described to be immediate viral transport via the circulation was negated. expressed by avian endothelial cells (Drake et al., 1997, The time at which the embryos were injected was insuffi- 2000), indicate that these YFPϩ cells are endothelial cells. cient to allow free virons to have entered the embryo proper The blood island-origin of the YFPϩ cells detected and for the cells within the embryo proper to express YFP within the blood vessels of the embryo and the circulation as once the circulation is established (Fig. 7). Finally, to ensure a mechanism of their distribution is indicated by our find- that the YFPϩ cells detected were not the result of viral ings that alternative scenarios that could account for these recombination, we demonstrated a similar experimental out- cells (i.e., cell migration/viron diffusion, viron diffusion via come using SPF chick embryos (Fig. 8). Our contention that circulation, and/or viral recombination) are not operative. the YFPϩ endothelial cells that we observed intraembryoni- With regard to YFPϩ cell migration and viron diffusion, we cally are derived from cells of blood island origin that gain detect no YFPϩ cells either within the embryo proper or in access to the embryo via the circulation is consistent with a gradient toward the embryo proper in experiments in the fact that such cells are absent in areas that had not been 168 A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172

Fig. 5. YFPϩ endothelial cells express TAL1. LSCM images (ϳ4 ␮m thickness) of an intraembryonic lateral vascular network from a 12-somite stage retrovirally injected embryo showing QH1 immunolabeling (A), TAL1 immunolabeling (B) and immunofluorescence of retrovirally labeled cells (C). (D) YFPϩ endothelial cells (arrowheads) coexpress QH1 and TAL1, an expression pattern consistent with that previously described for endothelial cells. In addition, YFPϩ cells within the vascular lumen express TAL1 (arrow). Avascular space and lumen of the blood vessel are labeled. Scale bar, 50 ␮m. vascularized (i.e., areas adjacent to Henson’s node). Addi- cells suggests that their incorporation into the vascular en- tionally, heart extirpation, in which cardiac function was dothelium may not be random, for example no YFPϩ cells ablated, resulted in no intraembryonic YFPϩ cells. To- were incorporated into the dorsal aortae, it is unclear if gether, the above findings suggest that the YFPϩ endothelial circulating cells discriminate between arteries and veins. cells detected in intraembryonic regions are derived from While this is possible, the differences in the distribution extraembryonic blood islands and that these cells entered pattern may reflect the fact that our labeling strategy gen- the embryo via the circulation and participated in the vas- erates only finite numbers of labeled cells and that such cells culogenic process. While the pattern of YFPϩ endothelial entering the circulation are initially exposed to venous ves- A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172 169

Fig. 6. YFPϩ cells within the embryo do not result from cell migration or diffusion of unincorporated virons. This schematic shows the approach used to evaluate cell migration and viron diffusion. Blood island primordia were retrovirally infected and the embryo cultured for 30 min to 10 h. For half of the injected embryos (upper panels), the extraembryonic region was separated from the intraembryonic region and the two fragments cultured separately for an additional 16Ð22 h. Control embryos were cultured intact for 16Ð22 h. (lower panels). No YFPϩ cells were detected in the intraembryonic vasculature of dissected embryos (A,B), while YFPϩ cells were apparent in the extraembryonic fragments of dissected embryos (C, D) and the vasculature of intact embryos (E, F). Note: The contrast levels were increased in (A) and (B) to reveal structures. Scale bars: (A, C, E) 200 ␮m; (B, F) 100 ␮m; (D) 50 ␮m. sels (i.e., the sinus venosus). Alternatively, the difference in vessels and evidence that such cells gained access to the the distribution of YFPϩ cells may reflect whether the embryo proper via the circulation. In addition to YFPϩ cells vessel was established primarily by in situ vasculogenesis associated with blood vessels, YFPϩ cells were also ob- (i.e., the dorsal aortae). served within the connective tissue of the splanchnic meso- In addition to YFPϩ endothelial cells, we observed other derm. While the nature of these cells is unclear, there is populations of YFPϩ cells (Fig. 2). Prominent among these reason to believe that they may represent a population of were YFPϩ cells within the lumen of blood vessels. Given extravasated monocytes (macrophages) of blood island or- that the retroviral labeling experiments targeted the blood igin, as several studies suggest that the early embryo con- islands, some of these cells are likely of the hematopoietic tains yolk sac-derived macrophage-like cells (Cuadros et al., lineage (i.e., erythroid cells, myeloid cells). Others likely 1992; Martin-Partido et al., 1991). represent circulating endothelial cells or endothelial cell While our finding that circulating cells contribute to the progenitors. This later conclusion is based on our finding the neovascularization of the early embryo is in accord with YFPϩ endothelial cells incorporated into intraembryonic similar findings in the adult, it is at odds with other works 170 A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172

Fig. 7. Free virons do not gain access to the embryo proper via circulation. This schematic depicts experiments addressing the ability of free virons to infect resident intraembryonic endothelial cells via circulation. Retrovirus was injected into the blood island primordia at the primitive streakÐ2 somite stage and embryos allowed to culture for 15 h. During this period, the extra- and intraembryonic vasculatures anastamose and circulation begins, allowing either virally infected cells (upper panels) or free virons (lower panels) access to the embryo proper. Embryos were examined at ϳ13 somites by using LSCM, and YFPϩ cells were observed within the embryo proper. Given that the minimum time between injection and viral expression was determined to be 6.5 h and the initiation of cardiac function does not occur until the 10 somite stage, providing a maximum of 4.5 h between viron access via circulation and fixation of the embryos, it was concluded that YFPϩ cells were not derived from postanastamosis viral infection, but from an extraembryonic source. “s” indicates somite number. Scale bars, 50 ␮m. that have examined this potential in the embryo. In a study concluded that circulating cells did not contribute to in- closely related to our own, Cuadros et al. (1992) reported traembryonic vasculogenesis. This potential was investi- the presence of patches of QH1ϩ endothelial cells in blood gated in quail-chick chimeras by using peripheral blood vessel walls of chick embryos after quail yolk sac (contain- transplantation, bone marrow transplantation and parabiosis ing blood islands) transplantation. While we consider this experiments (quail and chick embryos cocultured such that observation as support for our findings, it must be noted that their chorioallantoic circulations become continuous). They the authors concluded that the yolk-sac/blood islands were found that regardless of the source of cells introduced into not a source of cells contributing to vasculogenesis based on the extraembryonic circulation, no donor endothelial cells the failure of the reciprocal experiment to produce endothe- were found as a part of the host embryonic blood vessels. lial cells of donor origin. Similarly, Kurz et al. (2001) While it is difficult to explain the conflicting results between

Fig. 8. Viral recombination does not contribute to YFPϩ expression in intraembryonic endothelial cells. Blood island primordia of both quail and SPF chick embryos were retrovirally labeled at the primitive streakÐ2 somite stage and embryos were allowed to culture 15 h. Arrows in (A) show YFPϩ cells (green) incorporated into QH1ϩ (red) quail intraembryonic blood vessels from the lateral networks. (B) (arrows) Similar results from a SPF chick embryo (YFPϩ cells are green, vessels are imaged by using DIC). Avascular space, lumen of the blood vessel and red blood cells (RBCs) within the lumen are labeled. Scale bars, 25 ␮m. A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172 171 the above studies and our own, possible explanations lie in References differences between the stages studied and the markers used. Our studies were conducted at significantly earlier Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C., Silver, M., stages and used retroviral markers to trace the fate of blood Kearne, M., Magner, M., Isner, J.M., 1999a. Bone marrow origin of island-derived cells rather than the expression of QH1. In endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ. Res. 85, 221Ð228. contrast to previous studies, the use of a retrovirus as a Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee, R., Li, T., lineage-tracing tool allows the cells to be followed in a Witzenbichler, B., Schatteman, G., Isner, J.M., 1997. Isolation of putative tissue independent process. progenitor endothelial cells for angiogenesis. Science 275, 964Ð967. Given the evidence supporting a common origin for the Asahara, T., Takahashi, T., Masuda, H., Kalka, C., Chen, D., Iwaguro, H., hematopoietic and endothelial cell lineages from a cell re- nai, Y., Silver, M., Isner, J.M., 1999b. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial ferred to as the hemangioblast, it is tempting to interpret our progenitor cells. EMBO J. 18, 3964Ð3972. findings as support for such a cell. However, our experi- Burns, J.C., Friedmann, T., Driever, W., Burrascano, M., Yee, J.K., 1993. ments do not distinguish whether endothelial cells and he- Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vec- matopoietic cells have a common or separate cell origin. tors: concentration to very high titer and efficient gene transfer into While the specific origin of cells within the circulation that mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, have endothelial cell potential is unclear, there is convincing 8033Ð8037. Cuadros, M.A., Coltey, P., Carmen Nieto, M., Martin, C., 1992. Demon- evidence that populations of both circulating endothelial stration of a phagocytic cell system belonging to the hemopoietic cell precursors (Ikpeazu et al., 2000; Lin et al., 2000; Shi et lineage and originating from the yolk sac in the early avian embryo. al., 1998, 1999; Takahashi et al., 1999) and so-called cir- Development 115, 157Ð168. culating endothelial cells in the adult (Grefte et al., 1993; deMello, D.E., Sawyer, D., Galvin, N., Reid, L.M., 1997. Early fetal Lin et al., 2000; Solovey et al., 1997) can contribute to development of lung vasculature. Am. J. Respir. Cell. Mol. Biol. 16, neovascularization. 568Ð581. Drake, C.J., Brandt, S.J., Trusk, T.C., Little, C.D., 1997. TAL1/SCL is The findings of our study are significant in two respects. expressed in endothelial progenitor cells/angioblasts and defines a First, the fact that two mechanisms for vasculogenesis are dorsal-to-ventral gradient of vasculogenesis. Dev. Biol. 192, 17Ð30. operative in embryos, in situ and circulating vasculogenesis, Drake, C.J., Davis, L.A., Little, C.D., 1992a. Antibodies to beta 1-integrins necessitates a reappraisal of previous results that attribute cause alterations of aortic vasculogenesis, in vivo. Dev. Dyn. 193, 83Ð91. experimental outcomes solely to the process of vasculogen- Drake, C.J., Fleming, P.A., 2000. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95, 1671Ð1679. esis or angiogenesis. Second, considering the importance of Drake, C.J., LaRue, A., Ferrara, N., Little, C.D., 2000. VEGF regulates cell understanding regulation of new blood vessel formation as behavior during vasculogenesis. Dev. Biol. 224, 178Ð188. it occurs in adult vasculogenesis, the development of model Drake, C.J., Little, C.D., 1995. Exogenous vascular endothelial growth systems to elucidate mechanisms controlling this process is factor induces malformed and hyperfused vessels during embryonic useful. The work presented herein suggests that the embryo neovascularization. Proc. Natl. Acad. Sci. USA 92, 7657Ð7661. may be a valuable experimental model to address many of Gebb, S.A., Shannon, J.M., 2000. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev. Dyn. 217, 159Ð169. the outstanding questions regarding the regulation of circu- Giloh, H., Sedat, J.W., 1982. Fluorescence microscopy: reduced photo- lating endothelial cell precursors and/or endothelial cells. bleaching of rhodamine and fluorescein protein conjugates by n-propyl For example, what controls the numbers of extraembryonic gallate. Science 217, 1252Ð1255. circulating cells that participate in vasculogenesis and what Grefte, A., van der Giessen, M., van Son, W., The, T.H., 1993. Circulating mechanisms regulate their homing and incorporation into cytomegalovirus (CMV)-infected endothelial cells in patients with an active CMV infection. J. Infect. Dis. 167, 270Ð277. the vascular endothelium? Does VEGF, a cytokine known Ikpeazu, C., Davidson, M.K., Halteman, D., Browning, P.J., Brandt, S.J., to be involved in both embryonic (Drake et al., 2000; Drake 2000. Donor origin of circulating endothelial progenitors after alloge- and Little, 1995) and adult (Asahara et al., 1999b) vascu- neic bone marrow transplantation. Biol. Blood Marrow Transplant 6, logenesis, play a role in this regulation? As the avian em- 301Ð308. bryo is easily manipulated, we feel that experiments ad- Kallianpur, A.R., Jordan, J.E., Brandt, S.J., 1994. The SCL/TAL-1 gene is dressing these and other questions are highly feasible and expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis. Blood 83, 1200Ð1208. should provide new insights into vasculogenesis occurring Kanda, T., Sullivan, K.F., Wahl, G.M., 1998. Histone-GFP fusion protein via circulating cells. enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377Ð385. Kurz, H., Korn, J., Eggli, P.S., Huang, R., Christ, B., 2001. Embryonic central nervous system angiogenesis does not involve bloodborne en- Acknowledgments dothelial progenitors. J. Comp. Neurol. 436, 263Ð274. Lin, Y., Weisdorf, D.J., Solovey, A., Hebbel, R.P., 2000. Origins of We thank Dr. Stephen J. Brandt for his generous gift of circulating endothelial cells and endothelial outgrowth from blood [see SCL/TAL-1 antibodies (Vanderbilt University and Veterans comments]. J. Clin. Invest. 105, 71Ð77. Martin-Partido, G., Cuadros, M.A., Martin, C., Coltey, P., Navascues, J., Affairs Medical Center, Nashville, TN). This work was 1991. Macrophage-like cells invading the suboptic necrotic centres of supported by NIH grants to C.J.D. (RO1 HL57375, DAMD the avian embryo diencephalon originate from haemopoietic precur- 17-00-1-0338, NIH HL52813). sors. J. Neurocytol. 20, 962Ð968. 172 A.C. LaRue et al. / Developmental Biology 262 (2003) 162Ð172

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